Enteekateeka z’okwerinda kw’ebimera zitari zimwe na zimwe niziha obuhabuzi bw’ebiteekateeko obukuru obw’okushoboorora enkora y’okuhindura eby’okurya omu bimera, kwonka okuteebereza kwabo okukuru nikuguma nikugyezibwa. Aha, tukakoresa okucondooza okutaine rubaju okwa tandem mass spectrometry (MS/MS) kucondooza omu muringo ogutebeekanisiibwe gye (metabolome) y’ebika by’etaabe ebiine amaani kuruga omu bimera by’omuntu kuhika aha bantu n’ebika ebiine akakwate ka haihi, kandi twakora aha mubaro muhango gw’ebiteekateeko by’emitwarize y’eby’obuhangwa ebirikwegamira aha nkora y’eby’obuhangwa ebiri omu makuru. Omutindo gw‘okugyezaho okuteebereza okukuru kw‘ebiteekateeko by‘okwerinda okuhikire (OD) n‘okutambura kw‘ekigyendererwa (MT). Ekicweka ky’amakuru eky’eby’okurya by’ebimera nikihikaana n’enteekateeka ya OD, kwonka nikihakanisa okuteebereza okukuru okwa MT theory aha nkora y’eby’okurya ebirikureetwa ebinyonyi ebirikurya ebishaka. Kuruga aha rulengo rwa micro kuhika aha macro evolutionary scale, akamanyiso ka jasmonate kakamanyirwa nk’akakuru akarikumanyisa OD, obwo akamanyiso ka ethylene karikuha okutereeza okurungi kw’okugarukamu kw’eby’okurya by’ebishaka okuhandiikirwe omu nkora ya MS/MS molecular network.
Ebirungo by’omutaano ebiine emiringo etari emwe n’emwe nibyo birikwejumba munonga omu kumanyiira eby’obuhangwa, namunonga omu kurinda abazigu (1). Okutaana kw'omutaano kw'eby'okurya eby'omutaano ebirikushangwa omu bimera kureetsireho emyaka mingi y'okucondooza okuri omunda aha bintu bingi eby'emirimo y'obuhangwa, kandi kwakora orunyiriri rureingwa rw'ebiteekateeko by'okwerinda kw'ebimera, ebiri eby'entunguuka n'entunguuka y'obuhangwa bw'ebimera n'ebikooko. Okucondooza okurikukorerwa aha bintu ebirikubaho nikuha obuhabuzi bukuru (2). Kwonka, ebiteekateeko ebi eby‘okwerinda ebimera tibikakuratiire omuhanda gw‘omutindo gw‘okuteekateeka okurikureetaho okuteekateeka (hypothetical deductive reasoning), omu kuteebereza okukuru kwabaire kuri aha rurengo rumwe rw‘okushwijuma (3) kandi kukagyezibwa omu kugyezesa kutunguura omuze ogurikukurataho ogw‘okutunguuka kw‘ebiteekateeko (4). Oburemeezi bw‘eby‘obuhangwa nibuzibira okuteerana amakuru aha miringo y‘eby‘okurya etari emwe n‘emwe kandi nibubingamu okweshwijuma okuhikire kw‘eby‘okurya by‘omutaano, n‘ahabw‘ekyo nikuzibira okugyeragyeranisa ahagati y‘ebicweka ebirikwetengyesa omu kutunguuka kw‘ebiteekateeko (5). Okubura amakuru g’eby’okurya ebihikire n’esente ezirikushushana ez’okugyeragyeranisa omuringo gw’okukora eby’okurya omu mwanya gw’eby’okurya ahagati y’ebicweka by’ebimera ebitarikushushana nikiremesa okukura kwa sayansi omu musiri.
Entunguuka ezihererukireyo omu by’okucondooza aha nkora ya tandem mass spectrometry (MS/MS) metabolomics nizibaasa kushoboorora gye empindahinduka z’eby’okurya omunda n’ahagati y’ebika by’enkora ya clade erikuheebwa, kandi nizibaasa kugambwaho n’emiringo y’okubara kubara okushushana kw’enkora ahagati y’ebintu ebi ebigumire. Okumanya okurikubanza aha bya kemikoro (5). Okuteerana kwa tekinologiya y’omurembe omu kucondooza n’okubara nikuha omutindo ogurikwetengyesa ogw’okugyezaho obwire buraingwa okuteebereza kwingi okukozirwe enteekateeka z’eby’obuhangwa n’entunguuka y’eby’okurya. Shannon (6) akatandikaho enteekateeka y’amakuru omurundi gw’okubanza omu kihandiiko kye eky’okubanza omu mwaka gwa 1948, yaataho omusingi gw’okushwijuma amakuru omu kubara, ekikoresiibwe omu miringo mingi etari y’okubanza. Omu bya genomics, enteekateeka y‘amakuru ekoresiibwe gye omu kugyeragyeranisa amakuru g‘okukuratana (7). Omu kucondooza kwa transcriptomics, enteekateeka y‘amakuru neeshwijuma empindahinduka zoona eziri omu bihandiiko (8). Omu kucondooza okuhwaire, tukakozesa omutindo gw’embariirira y’enteekateeka y’amakuru aha metabolomics kushoboorora obukugu bw’okuhindura eby’okurya omu rurengo rw’ebicweka by’omubiri omu bimera (9). Aha, nitugaita omuringo gw‘okukora ogurikugyendera aha MS/MS n‘omutindo gw‘embariirira gw‘enteekateeka y‘amakuru, ogurikumanyirwa aha kuhinduka kw‘eby‘okurya omu sente z‘abantu boona, kugyeragyeranisa okuteebereza okukuru kw‘enteekateeka y‘okwerinda kw‘ebimera ey‘enteekateeka y‘okurinda eby‘okurya (metabolome) erikureetwaho ebinyonyi ebirikurya ebishaka.
Enkora z‘ebiteekateeko z‘okwerinda kw‘ebimera nizikira kuba zirimu buri omwe kandi nizibaasa kugabwa omu miringo ebiri: ezo ezirikugyezaho kushoboorora oku ebimera birikugabwa kurugiirira aha mirimo y‘okwerinda, nk‘okwerinda okuhikire (OD) (10), ekigyendererwa ekirikugyendera (MT) (11) ) N‘okureebeka (12) oku selan changes of the mechanicals in, . okubaho nikukwata aha kukura kw’ebimera n’okuteerana kw’ebintu by’omutaano ebirikuhindura ebiriisa, nka kaboni: enteekateeka y’okugyeragyeranisa ebiriisa (13), enteekateeka y’omubaro gw’okukura (14 ), n’enteekateeka y’okugyeragyeranisa okukura n’okutaanisa (15). Ebicweka bibiri by‘ebiteekateeko biri aha rurengo rw‘okweshwijuma rutari rumwe (4). Kwonka, enteekateeka ibiri ezirimu emirimo y‘okwerinda aha rurengo rw‘emirimo nizitegyeka okugaaniira aha kwerinda kw‘ebimera okurikubaho n‘okutarikubaasa kureetaho: enteekateeka ya OD, erikuteekateeka ngu ebimera nibiteeka sente omu kwerinda kwabyo okw‘omuhendo okw‘eby‘emibazi kyonka kyaba nikyetengyesa, eky‘okureeberaho, kyaba nikinywa Obu ekinyamaishwa ky‘obunyaatsi kirikutaahirira, n‘ahabw‘ekyo, kurugiirira aha kubaasa kw‘okwerinda kw‘omu biro by‘omumaisho, kurugiirira aha kubaasa kw‘okwerinda kw‘omu biro by‘omumaisho. omurimo niguheebwa (10); enteekateeka ya MT neeteekateeka ngu tihaine axis y‘okuhindura kw‘eby‘okurya ebiri omu rubaju, kwonka eby‘okurya nibihinduka omu muringo gw‘okutooranamu, n‘ahabw‘ekyo nikireetaho okubaasa kw‘okuremesa eby‘okurya “ekigyendererwa ky‘okutambura” ky‘okutaahirira ebinyonyi ebirikurya ebishaka. Omu bindi bigambo, enteekateeka ezi zombi nizikora okuteebereza okurikuhakanisa aha kuhindura kw‘eby‘okurya okurikubaho bwanyima y‘okutahirirwa kw‘ebinyonyi ebirikurya ebishaka: akakwate akari ahagati y‘okuteerana kw‘eby‘okurya omu muringo gumwe n‘okukora kw‘okwerinda (OD) n‘empindahinduka z‘eby‘okurya ezitarikugyenderwaho (MT) (11 ).
Ebiteekateeko bya OD na MT tibirikutwariramu empindahinduka ezirikureetwaho omu nkora y‘okuhindura eby‘okurya byonka, kureka n‘ebirikuruga omu by‘obuhangwa n‘entunguuka y‘okuteerana kw‘ebintu ebi, nk‘esente z‘okuhindura n‘ebirungi by‘empindahinduka ezi z‘eby‘okurya omu mbeera y‘eby‘obuhangwa emwe (16). N‘obu ebiteekateeko byombi biraabe nibimanya omurimo gw‘okwerinda gw‘ebintu by‘omutaano ebirikuhindura eby‘okurya, ebirikubaasa kuba nari bitarikubaasa kuba eby‘obuguzi bwingi, okuteebereza okukuru okurikutaanisa ebiteekateeko bya OD na MT kuri omu rubaju rw‘empindahinduka z‘eby‘okurya ezirikureetwaho. Okuteebereza kw‘enteekateeka ya OD kutungire okugyezesa okurikukirayo kuhitsya hati. Ebigyezo ebi nibitwariramu okwega aha mirimo y’okwerinda butoosha nari etari butoosha ey’ebicweka by’omubiri ebitarikushushana eby’ebirungo by’omutaano omu bishengye by’okuriisa n’embeera z’obuhangwa, hamwe n’empindahinduka omu rurengo rw’okukura kw’ebimera (17-19). Kwonka, okuhitsya hati, ahabw‘okubura omuringo gw‘okukora n‘omutindo gw‘embariirira gw‘okushwijuma okuhikire okw‘ensi yoona okw‘entaaniso y‘eby‘okurya by‘ekihangirwe kyona, okuteebereza kw‘entaaniso mpango ahagati y‘ebiteekateeko bibiri (nikwo kugira ngu, orubaju rw‘empindahinduka z‘eby‘okurya) nikisigara kugyezibwa. Aha, nituha okweshwijuma nk’okwo.
Ekimwe aha bintu ebikuru ebirikumanyisa ebiriisa by’ebimera n’okutaana kwabyo okurikukirayo omu nkora aha rurengo rwona kuruga aha bimera bimwe, omubaro gw’abantu kuhika aha miringo erikushushana (20). Empindahinduka nyingi z‘omubaro omu bihingwa by‘omutaano nizibaasa kureebwa aha rurengo rw‘abantu, obwo entaaniso z‘amaani z‘omutindo zirikukira kuguma aha rurengo rw‘ebika (20). Nahabwekyo, okuhindura eby’okurya omu bimera nikyo kikuru omu kuhinduka kw’emirimo, ekirikworeka okubaasa kumanyiira emyanya etari emwe n’emwe, namunonga emyanya egyo erimu obushoboorozi bw’okutaahirira obutarikushushana ahabw’ebinyonyi eby’omutaano n’enyamaishwa ezirikurya ebishaka eza buriijo (21). Kuruga omu kihandiiko kya Fraenkel (22) ekyatandikireho aha nshonga z’okubaho kw’ebintu ebirikuhindura ebimera, okukoragana n’ebinyonyi ebitari bimwe na bimwe kukaba nikutwarwa nk’ebintu bikuru ebirikuteganisa okutoorana, kandi okukoragana oku nikuteekateekwaho kuba kwakozire ebimera omu bwire bw’okutunguuka. Omuhanda gw’okuhindura eby’okurya (23). Entaaniso ahagati y’ebika by’ebimera omu miringo etari emwe n’emwe y’ebintu ebirikuhindura ebiriisa by’omubiri eby’omutaano nibibaasa n’okworeka oburinganiza bw’omubiri oburikukwatagana n’okwerinda kw’ebimera okurikutegyeka n’okutarikubaasa kureetaho obukodyo bw’okurya ebishaka, nk’oku ebika byombi birikukira kuba biine akakwate karungi n’ebindi (24). N‘obu kiraabe nikibaasa kuba eky‘omugasho kuguma n‘okwerinda kurungi obwire bwona, empindahinduka z‘eby‘okurya omu bwire eziine akakwate n‘okwerinda niziha ebirungi ebirikureebwa gye omu kwikiriza ebimera kugaba eby‘obugaiga by‘omuhendo omu bindi biteekateeko by‘omubiri (19, 24), n‘okwetantara okwetenga kw‘okukwatanisa. Okushiisha kw’omusingo (25). Okwongyera ahari ekyo, okugaruka kutebeekanisa oku kw‘ebintu by‘omutaano ebirikureetwa ebinyonyi ebirikurya ebishaka nikubaasa kureetera okujanjaara okurikushiisha omu bantu (26), kandi nikubaasa kworeka okushoma okuhikire kw‘empindahinduka z‘obuhangwa z‘amaani omu kamanyiso ka jasmonic acid (JA), akarikubaasa kuguma omu bantu. Obumanyiso bwa JA obw‘ahaiguru n‘obw‘ahansi n‘okushuubuza ahagati y‘okwerinda ebinyonyi ebirikurya ebishaka n‘okuhakanisa n‘ebika by‘ebinyonyi ebimwe (27). Okwongyera ahari ekyo, emihanda y’omutaano ey’okukora ebiriisa omu mubiri neija kufeerwa juba kandi ehindukye omu bwire bw’okutunguuka, ekirikureetaho okugabwa kw’ebiriisa omu miringo y’ebihangirwe ebiine akakwate ka haihi (28). Ezo polymorphisms nizibaasa kutandikaho juba omu kugarukamu okuhinduka kw’emitwarize y’ebinyonyi ebirikurya ebishaka (29), ekirikumanyisa ngu okuhindukahinduka kw’ebyanga by’ebinyonyi ebirikurya ebishaka n’enshonga nkuru erikureetaho obutashushana bw’eby’okurya.
Aha, tukashoboorora buzima ebizibu ebi ahaifo. (I) Ekinyonyi ekirikurya ebishaka nikigaruka kita kuhindura omubiri gw’ebimera? (Ii) Ni bicweka ki eby‘amakuru ebikuru eby‘okuhindura eby‘okurya ebirikubaasa kugyezibwa okutebereza kw‘enteekateeka y‘okwerinda ey‘obwire buraingwa? (Iii) Yaaba okugaruka kutebeekanisa metabolome y’ebimera omu muringo gw’omutaano aha muntu orikuteera, kyaba kiri kityo, ni murimo ki ogu hormone y’ebimera erikukora omu kuteekateeka aha kugarukamu kw’eby’okurya okumanyirwe, kandi ni metabolites ki ezirikuhwera omu nkora y’okwerinda kw’ebika by’ebimera? (Iv) Ahabw‘okugira ngu okuteebereza okukozirwe enteekateeka nyingi z‘okwerinda nikubaasa kugambwaho aha mitwe yoona y‘ebicweka by‘omubiri ebiine amagara, tukabuuza oku okugarukamu kw‘okuhindura eby‘okurya okurikureetwaho kurikuhikaana kuruga omu kugyeragyeranisa kw‘omunda kuhika aha kugyeragyeranisa kw‘ebika by‘ebihangirwe? Okuhikiiriza eki, tukacondooza omu nkora aha nkora y’okuhindura amababi g’etaabe, ekimera ky’eky’okureeberaho ky’eby’obuhangwa ekiine enkora y’omutaano ey’obugaiga, kandi nikikora gye aha nkoko z’ebinyonyi bibiri eby’omu ihanga ebirikurya ebishaka, Lepidoptera Datura (Ms) ( Ebiine obushaarizi bwingi, ebirikukira kuriibwa) Aha bihingwa bya Solanaceae na Spodoliton a). “genus”, n’ebimera ebirikutwara ebimera bya Solanaceae n’ebindi bimera ebirikutwara ebindi bimera n’amaka Ebyokurya by’ebimera. Twashwijuma MS/MS metabolomics spectrum kandi twaihayo ebishoboorora by‘embariirira y‘enteekateeka y‘amakuru kugyeragyeranisa enteekateeka za OD na MT. Taho maapu z‘okwemanyiza kworeka okumanyisa kw‘ebintu ebikuru ebirikuhindura omubiri. Okweshwijuma kukagambwaho aha bantu b’obuzaarwa bwa N. nasi n’ebika by’etaabe ebiine akakwate ka haihi kwongyera kweshwijuma okutaana ahagati y’okumanyisa kw’ebiriisa by’ebimera n’okureetaho OD.
Okubaasa kukwata maapu yoona erikukwata aha buveera n’omutindo gw’ebibabi by’etaabe erikurya ebishaka, tukakoresa omuringo gw’okushwijuma n’okubara ebyabaire bikozirwe enyimaho kubaasa kushorooza omu bwijwire n’okushoboorora amakuru g’omutindo gw’ahaiguru agatarikwetongora MS/MS spectra kuruga omu bihingwa ebirugire omu bimera ( 9). Enkora egi etaine ntaaniso (erikwetwa MS/MS) neebaasa kwombeka ebishushani by‘ebirungo ebitarikugarukagaruka, ebirikubaasa kukoresibwa bwanyima omu kucondooza kwona okw‘omutindo gw‘ebirungo okushobooroirwe aha. Ebirungo ebi eby’ebimera ebitaine nkora n’eby’emiringo etari emwe n’emwe, birimu ebikumi kuhika aha nkumi z’ebirungo (nk’ebicweka 500-1000-s/MS/MS aha). Aha, nituteekateeka aha nkora y‘okuhindura eby‘okurya omu nkora y‘okuteekateeka aha makuru, kandi nitupima obutashushana n‘obukugu bw‘eby‘okurya kurugiirira aha Shannon entropy y‘okugabwa kw‘emirundi y‘okuhindura eby‘okurya. Okukozesa enkora eyateirweho enyima (8), tukabara ekibiina ky‘eby‘okureeberaho ebirikubaasa kukoresibwa kugyeragyeranisa obutashushana bw‘eby‘okurya (eby‘okureeberaho bya Hj), obumanyi bw‘eby‘okurya (eby‘okureeberaho bya δj) n‘eby‘okurya by‘omutaano eby‘eby‘okurya by‘omubiri (Si indicator) . Okwongyera ahari ekyo, tukakozesa omubaro gw’obutafaari bw’omubiri (RDPI) okupima obuhango bw’okuhindura eby’okurya omu nyamaishwa ezirikurya ebishaka (ekishushani 1A) (30). Omu nkora egi ey‘embariirira, nitutwara MS/MS spectrum nk‘ekicweka ky‘amakuru g‘omutaano, kandi nitukora aha bwingi bw‘ebicweka bya MS/MS omu maapu y‘okugabanisa emirundi, reero tukorese Shannon entropy kuteekateeka aha nkora ya metabolome kuruga ahariyo. Obukugu bwa metabolome nibupimwa omu nkora y‘obumanyiso bwa MS/MS emwe. Nahabwekyo, okweyongyera kw‘obungi bw‘ebicweka bimwe bya MS/MS bwanyima y‘okutaho eby‘okurya by‘ebishaka nikuhindurwa omu spectral inducibility, RDPI n‘okukora eby‘obukugu, nikwo kugira ngu okweyongyera kw‘omubaro gwa δj, ahabw‘okuba ebiriisa by‘omutaano ebirikukiraho nibikorwa kandi omubaro gwa Si gw‘ahaiguru nigukorwa. Okukyendeeza aha nkora ya Hj diversity index nikworeka ngu omubaro gw‘ebintu ebikozirwe MS/MS nigukyendeera, nari okugabwa kw‘emirundi y‘okucondooza nikuhinduka omu muringo ogutarikushushana, obwo kirikukyendeeza obutamanya bwakyo bwona. Kurabira omu kubara kwa Si index, nikibaasika kworeka MS/MS ezirikureetwaho ebinyonyi ebimwe ebirikurya ebishaka, okutaana n‘ekyo, ni MS/MS ki etarikugarukamu okureetaho, eki n‘eky‘okureeberaho kikuru eky‘okutaanisa okuteebereza kwa MT na OD.
(A) Ebishoboorora by‘embariirira ebirikukoresibwa aha by‘okurya by‘ebishaka (H1 kuhika Hx) MS/MS ebirikureetaho amakuru (RDPI), obutashushana (Hj index), obumanyi (δj index) n‘obumanyiso bw‘ebiriisa (Si index). Okwongyera aha rulengo rw’obukugu (δj) nikworeka ngu, omu mwanya, ebiriisa by’ebishaka bingi nibiija kurugamu, obwo okukyendeera kw’ebintu bitari bimwe na bimwe (Hj) nikworeka okukyendeera omu kureetaho ebiriisa nari okugabwa okutarikushushana kw’ebiriisa omu maapu y’okugabwa . Omuhendo gwa Si nigukyebera yaaba ekirungo ky‘omubiri kiri eky‘omutaano aha mbeera emwe (aha, ekirikurya ebishaka) nari ekirikuhinduka nikiguma aha rurengo rumwe. (B) Ekishushani ky‘ebiteekateeko ky‘okuteebereza kw‘enteekateeka y‘okwerinda orikukoresa enkora y‘enteekateeka y‘amakuru. Enteekateeka ya OD neeteekateeka ngu okutaahirira kw‘ebishaka nikwija kwongyera aha bihingwa by‘okwerinda, obwo kwongyera δj. Omu bwire nibwo bumwe, Hj neekyendeera ahabw‘okuba purofayiro neegaruka kutebeekanisibwa kuza omu kukyendeeza obutamanya bw‘amakuru g‘okuhindura eby‘okurya. Enteekateeka ya MT neeteekateeka ngu okutaahirira kw‘ebinyonyi ebirikurya ebishaka nikwija kureetera empindahinduka ezitari z‘orubaju omu nkora y‘okuhindura eby‘okurya, obwo nibwongyera Hj nk‘eky‘okureeberaho ky‘okwongyera aha kutamanya amakuru g‘eby‘okurya kandi kireeteho okugabwa kwa Si okutaine rubaju. Kandi tukateekateeka aha nkora y‘okutabura, MT erikukirayo oburungi, omu bimwe aha bihingwa ebiine emihendo y‘okwerinda y‘ahaiguru nibiija kweyongyera munonga (omuhendo gwa Si gw‘ahaiguru), obwo ebindi birikworeka okugarukamu okutaine rubaju (omuhendo gwa Si ogw‘ahansi).
Twakoresa abashoboorora enteekateeka y‘amakuru, nitushoboorora enteekateeka ya OD kuteebereza ngu empindahinduka z‘omutaano ezirikureetwaho eby‘okurya by‘ebishaka omu mbeera y‘omutaano etarikureetwa niziija kureetera (i) okweyongyera kw‘eby‘okurya (Si index) ebirikuvuga eby‘okurya by‘omutaano (δj index) Okwongyera kwa) guruupu z‘omutaano z‘eby‘okurya ezimwe ez‘omuhendo gw‘okwerinda gw‘ahaiguru ( de metaboloiH) . index) ahabw‘empindahinduka y‘okugabwa kw‘emirundi y‘okuhindura eby‘okurya kuza omu kugabwa kw‘omubiri okwa leptin kwingi. Aha rurengo rw‘ekirungo kimwe, okugabwa kwa Si okuragiirwe nikuteekateekwaho, ahu ekirungo kirayongyere aha muheendo gwa Si kurugiirira aha muheendo gwakyo gw‘okwerinda (ekishushani 1B). Omu murongooti ogu, nitushoboorora enteekateeka ya MT kuteebereza ngu okushemererwa nikwija kureetera (i) empindahinduka ezitari z‘orubaju omu bihingwa ebirikuhindura eby‘okurya ebirikurugamu okukyendeera kw‘omubaro gwa δj, na (ii) okweyongyera kw‘omubaro gwa Hj ahabw‘okwongyera aha kutamanya eby‘okurya. Nari randomness, erikubaasa kugyezesibwa Shannon entropy omu muringo gw‘obutashushana oburikugambwaho omu bwijwire. Aha bikwatiraine n’eby’okurya ebirimu ebiriisa, enteekateeka ya MT neija kuteebereza oku Si erikugabwa omu muringo gw’obutoosha. Okuteekateeka ngu ebiriisa ebimwe biri ahansi y‘embeera z‘omutaano ahansi y‘embeera z‘omutaano, kandi embeera ezindi tiziri ahansi y‘embeera z‘omutaano, kandi omuhendo gwazo gw‘okwerinda nigurugiirira aha mbeera, tukateekateeka n‘omuringo gw‘okwerinda ogutaine rubaju, omu δj na Hj nizigabwa omu bibiri aha rubaju rwa Si Okwongyera omu nshonga zoona, ebiriisa ebimwe byonka ebirikweyongyera aha guruupu z‘okwerinda, eziine Si . okugabanisa (Ekishushani 1B).
Okubaasa kugyezaho okuteebereza kw’enteekateeka y’okwerinda okugarukiremu kushoboororwa aha rubaju rw’okushoboorora enteekateeka y’amakuru, tukatunguura enkwanzi z’ebishaka (Ms) nari omuhangu (Sl) aha mababi ga Nepenthes pallens (ekishushani 2A). Twakozesa okucondooza kwa MS/MS, tukagaruraho 599 ebitarikugarukagaruka bya MS/MS spectra (data fairo S1) kuruga omu bihingwa bya methanol eby’ebicweka by’amababi ebyashohoziibwe bwanyima y’okuriisa ebinyonyi. Okukozesa RDPI, Hj, na δj indexes kureeba omu maisho okugaruka kutebeekanisa ebiri omu makuru omu fairo z‘okutebeekanisa MS/MS nikworeka enkora ezirikushemeza (ekishushani 2B). Omuze gwona nigugira ngu, nk‘oku kirikushoboororwa omushoboorozi w‘amakuru, nk‘oku ebinyonyi birikugumizamu nibirya amababi, omutindo gw‘okugaruka kutebeekanisa eby‘okurya byona nigweyongyera omu bwire: eshaaha 72 bwanyima y‘ekinyonyi ekirikurya ebishaka kurya, RDPI neeyeyongyera munonga. Kugyeragyeranisa n‘okutegyeka okutaine kabi koona, Hj ekakyendeera munonga, ekyabaireho ahabw‘okwongyera aha rurengo rw‘obukugu bw‘enkora y‘okuhindura eby‘okurya, ekyabaire nikibarwa n‘omubaro gwa δj. Omuze ogu ogurikureebeka nigukwatagana n‘okuteebereza kw‘enteekateeka ya OD, kwonka tigurikuhikaana n‘okuteebereza okukuru kw‘enteekateeka ya MT, erikwikiriza ngu empindahinduka z‘okutooranamu (ezitari z‘okuhabura) omu rurengo rwa metabolite nizikoresibwa nk‘okwehereera kw‘okwerinda (ekishushani 1B). N’obu ebiri omu kanwa (OS) elicitor content n’emitwarize y’okuriisa ey’ebinyonyi ebi bibiri ebirikurya ebishaka biraabe nibitaana, okuriisa kwabyo okuhikire kukarugamu empindahinduka zirikushushana omu nshonga za Hj na δj omu bwire bw’okugyesha obw’eshaaha 24 n’eshaaha 72. Entaaniso yonka ekabaho aha shaaha 72 za RDPI. Kugyeragyeranisa n‘ekyo ekirikureetwaho okuriisa Ms, okuhindura kw‘omubiri kwona okurikureetwaho okuriisa Sl kukaba kuri ahaiguru.
(A) Entebekanisa y‘okugyezesa: empunu eya buriijo (S1) nari abakugu (Ms) abarikurya ebishaka nibariisibwa amababi g‘ebimera by‘ebishaka agarimu omunyugunyu, obwo aha bimera ebirikurya ebishaka ebirikugyeragyeranisibwa, OS ya Ms (W + OSMs) neekoresibwa kukwaata okucumita kw‘emyanya y‘amababi erikugyenderwaho ekihuta. S1 (W + OSSl) enkwanzi nari amaizi (W + W). Okutegyeka (C) n’eibabi eritarikushiisha. (B) Okubaasa kureetaho (RDPI kugyeragyeranisa n‘ekipande ky‘okutegyeka), okutaana (Hj index) n‘obukugu (δj index) index ebariirwe ahabwa maapu y‘omutaano ey‘okuhindura ebiri omu mubiri (599 MS/MS; fairo y‘amakuru S1). Enyonyoozi nizooreka entaaniso mpango ahagati y‘okuriisa kw‘ebinyonyi ebirikurya ebishaka butoosha n‘ekibiina ky‘okukyebera (ekigyezo kya t ky‘omwegi n‘ekigyezo kya t ky‘ababiri, *P<0.05 na ***P<0.001). ns, ti kikuru. (C) Obwire bw‘okushoboorora indekisi y‘ebikuru (akabokisi ka bururu, amino asidi, asidi y‘ebihangwa na sukaari; fairo y‘ebihandiiko S2) n‘eby‘omutaano eby‘okuhindura ebiri omu mubiri (akabokisi akatukuriire 443 MS/MS; fairo y‘ebihandiiko S1) bwanyima y‘okujanjaba kw‘okurya ebishaka okurikugyeragyeranisibwa. Ekipande ky‘erangi nikimanyisa 95% y‘obwire bw‘okwesiga. Enyonyoozi neeyoreka entaaniso mpango ahagati y‘okujanjaba n‘okutegyeka [okucondooza kwa kwadratic okw‘okuhinduka (ANOVA), kukuratirwaho entaaniso mpango ya Tukey ey‘amazima (HSD) ahabw‘okugyeragyeranisa kw‘enyima y‘obwire, *P<0.05, **P<0.01 Na *** P <0.001]. (D) Okumanya eby‘okureeberaho by‘okujanjaaza n‘eby‘okureeberaho by‘eby‘okurya eby‘omutaano (okugarukamu eshampu n‘obujanjabi obutarikushushana).
Okucondooza yaaba okugaruka omu nkora okurikureetwaho eby’okurya by’ebishaka aha rurengo rwa metabolome nikworeka empindahinduka omu rurengo rw’eby’okurya by’omuntu, tukabanza twata omutima aha by’okurya ebyabaire byegyesiibwe enyima omu mababi ga Nepenthes pallens ebiine obutabaasa kugumira eby’okurya by’ebishaka ebihamibwe. Phenolic amides ni ebiriisa ebirikwerundanira omu bwire bw’okurya ebishaka by’ebinyonyi kandi nibimanyirwa aha kukyendeeza aha kukora kw’ebinyonyi (32). Twasherura ebirikubanza bya MS/MS ebirikuhikaana kandi twahandiika ebishushani byabyo eby‘okuteerana (ekishushani S1). Tikirikutangaaza, ebirikuruga omu phenol ebitarikwejumba butoosha omu kwerinda ebinyonyi ebirikurya ebishaka, nka chlorogenic acid (CGA) na rutin, nibikyendeera bwanyima y’okurya ebishaka. Okutaana n’ekyo, ebinyonyi ebirikurya ebishaka nibibaasa kukora phenol amides kuba ey’amaani maingi. Okuguma nibiriisa ebinyonyi bibiri ebirikurya ebishaka kukarugamu haihi okushemererwa kw’eby’okurya ebirimu ebiriisa, kandi omuringo ogu gukaba nigureebeka gye munonga aha de novo synthesis y’ebiriisa. Ekintu nikyo kimwe nikiija kureebeka baaba nibacondooza aha muhanda gw‘okukoresa ebiriisa by‘omubiri (17-HGL-DTGs), ebirikureetaho omubaro muhango gw‘ebiriisa by‘omubiri ebiine emirimo mirungi y‘okurwanisa ebihingwa ebirikurya ebishaka (33), ebi Ms Feeding yaahandiikireho n‘okushoboorora kwa SlFig tri1).
Ekizibu ekirikubaasa kubaho omu kugyezesa okuriisa kw’ebinyonyi ebirikurya ebishaka butoosha n’entaaniso eri omu mubaro gw’okurya amababi n’obwire bw’okuriisa ebinyonyi ebirikurya ebishaka, ekirikureetera kigumire kwihaho ebizibu by’ebinyonyi ebirikurya ebishaka ebirikureetwaho ebihuta n’ebinyonyi ebirikurya ebishaka. Okubaasa kushoboorora gye ebika by’ebinyonyi ebirikurya ebishaka eby’omutaano eby’okugarukamu kw’ebibabi ebireetsirweho, tukagyeragyeranisa okuriisa kw’ebinyonyi bya Ms na Sl turikutaho ahonaaho OS eyaabaire eherize kurundaanwa (OSM na OSS1) aha standard puncture W ey’emyanya y’ebibabi etarikuhinduka. Enkora egi neeyetwa obujanjabi bwa W + OS, kandi neegyeragyeranisa okutandikaho obwo erikutaho obwire obuhikire bw‘okutandika kw‘okugarukamu okurikureetwaho ekinyonyi ekirikurya ebishaka hatariho kureetaho ebirugiremu ebirikuteganisa eby‘entaaniso omu mubaro nari omu bwingi bw‘okufeerwa kw‘ebicweka by‘omubiri (ekishushani 2A) (34). Twakozesa omuhanda gw‘okushwijuma n‘okubara ogwa MS/MS, tukagaruraho ebishushani bya MS/MS 443 (data fairo S1), ebyabaire nibikwatagana n‘ebishushani ebyabaire biteeraine enyima kuruga omu kucondooza kw‘eby‘okurya ebihikire. Okushwijuma kw‘enteekateeka y‘amakuru g‘ekicweka eki ekya MS/MS kukooreka ngu okugaruka kuteekateeka aha metabolomes z‘omutaano z‘amababi barikugyeragyeranisa ebinyonyi ebirikurya ebishaka kukooreka okureetaho OS-specific inducements (ekishushani 2C). Okukira munonga, waagyeragyeranisa n‘obujanjabi bwa OSS1, OSM ekareetera okweyongyera kw‘obukugu bwa metabolome aha shaaha 4. Nikirungi kumanya ngu waagyeragyeranisa n‘ebihandiiko by‘okugyezesa eby‘okurya by‘ebishaka ebihikire, enkora y‘okuhindura eby‘okurya erikureebeka omu mwanya gw‘ebicweka bibiri barikukoresa Hj na δj nk‘enkoragana n‘okuhabura kw‘obukugu bw‘eby‘okurya omu kugarukamu okujanjaba kw‘ebishaka okurikugyeragyeranisibwa omu bwire Okwongyera aha nkora (ekishushani 2D). Omu bwire nibwo bumwe, tukapima ebiri omu amino acids, organic acids na shukaari (data fairo S2) kucondooza yaaba okweyongyera oku okugyendereirwe omu bukugu bwa metabolome nikureetwaho okugaruka omu nkora y‘okuhindura carbon eya rwagati omu kugarukamu eby‘okurya by‘ebishaka ebirikugyeragyeranisibwa ( Ekishushani S2). Okushoboorora gye enkora egi, tukagaruka twareeba enkora y‘okuteerana kw‘eby‘okurya omu nkora ya phenolamide na 17-HGL-DTG eyaagambirweho enyima. OS-specific induction y‘ebinyonyi ebirikurya ebishaka neehindurwa omu nkora y‘okugaruka kutebeekanisa ebirikutaana omu nkora y‘okuhindura eby‘okurya bya phenolamide (ekishushani S3). Phenolic amides ezirimu ebicweka bya coumarin na caffeoyl nizikira kureetaho OSS1, obwo OSMs zirikureetaho okutandika kw’omutaano kw’ebicweka bya ferulyl. Aha muhanda gwa 17-HGL-DTG, okureetaho OS etari emwe n‘emwe ahabw‘ebintu by‘okukyendeeza aha nkora ya malonylation na dimalonylation bikashangwa (ekishushani S3).
Ekindi, tukacondooza aha nkora ya OS-induced transcriptome plasticity turikukoresa ekicweka ky’amakuru eky’obwire, ekirikugyeragyeranisa okukoresa OSMs kujanjaba amababi g’ebimera bya rosette omu nyamaishwa ezirikurya ebishaka. Enkora y‘okutooranamu neekwatagana n‘enkora y‘okutooranamu erikukoresibwa omu kucondooza oku okwa metabolomics (35). Kugyeragyeranisa n‘okugaruka omu nkora ya metabolome omu kuhindura kw‘omubiri (metabolic plasticity) nikweyongyera munonga omu bwire, nitureeba okuhandiika kw‘obwire bukye omu mababi okurikureetwaho Ms, ahu okuhandiika kw‘obwire bukye (RDPI) n‘obukugu (δj) biri aha 1 Hakaba hariho okweyongyera kw‘amaani omu shaaha, kandi okutaana (Hj) okukyendeera munonga omu bwire obu obw‘obwire, the relation kukaba kuri aha BMP1. y‘obukugu bw‘okuhandiika (ekishushani S4). Amaka g‘ebicweka by‘omubiri ebirikuhindura eby‘okurya (nka P450, glycosyltransferase, na BAHD acyltransferase) nibeejumba omu nkora y‘okuteerana eby‘okurya by‘omutaano kuruga omu bicweka by‘omubiri ebirikuruga omu kuhindura eby‘okurya eby‘okubanza, kurugiirira aha nkora y‘okubanza ey‘obukugu bw‘ahaiguru egambirweho. Nk‘eky‘okureeberaho, omuhanda gw‘okukoresa phenylalanine gukakyeberwa. Okweshwijuma kukahamya ngu ebiriisa by’omutaano omu nkora ya phenolamide nibireetaho OS munonga omu bimera ebirikurya ebishaka waagyeragyeranisa n’ebimera ebitarikukwatwaho, kandi nibikwatagana munonga omu miringo y’okworeka kwabyo. Enshonga y‘okuhandiika MYB8 n‘ebicweka by‘omubiri ebirikukora PAL1, PAL2, C4H na 4CL omu rubaju rw‘ahaiguru rw‘omuhanda ogu bikooreka okutandika kw‘okuhandiika juba. Acyltransferases ezirikukora omurimo omu kuteerana kw‘aha muheru okwa phenolamide, nka AT1, DH29, na CV86, nizooreka omuringo gw‘okutunguuka ogurikumara obwire buraingwa (ekishushani S4). Ebireebire aharuguru nibyoreka ngu okutandika juba kw‘obukugu bw‘okuhandiika n‘okutunguuka kw‘obukugu bw‘okuhandiika eby‘okurya bwanyima n‘omuringo gw‘okuteerana, ogurikubaasa kuba nigureetwaho enkora y‘okutegyeka erikushushana erikutandikaho okugarukamu kw‘amaani okw‘okwerinda.
Okugaruka omu nkora omu kumanyisa kw’ebiriisa by’ebimera nikukora nk’omutindo gw’okutegyeka ogurikugaita amakuru g’ebimera ebirikurya ebishaka kugaruka kuteekateeka aha nkora y’ebimera. Bwanyima y‘okugyeragyeranisa eby‘okurya by‘ebishaka, tukapima entunguuka y‘okuteerana kw‘ebicweka by‘ebimera ebikuru kandi twareeba omu maisho okuteerana kw‘obwire ahagati yaabyo [Pearson correlation coefficient (PCC)> 0.4] (Ekishushani 3A). Nkoku kirikuteekateekwaho, obutafaari bw’ebimera oburikukwatagana n’okukora eby’okurya nibukwatanisibwa omu nkora y’okumanyisa obutafaari bw’ebimera. Okwongyera ahari ekyo, okuhindura eby‘okurya (Si index) nikuteebwa aha maapu aha nkora egi kworeka obutafaari bw‘ebimera oburikureetwaho obujanjabi obutari bumwe na bumwe. Emyanya ebiri emikuru ey‘okugarukamu kw‘omutaano kw‘ebihingwa ebirikurya ebishaka neekwatwa: kimwe kiri omu kicweka kya JA, ahu JA (omuringo gwayo ogurikukora omu by‘amagara JA-Ile) n‘ebindi ebirugire omu JA birikworeka obubonero bwa Si oburikukirayo; ekindi n’ekirungo kya etileni (ET). Gibberellin ekooreka okweyongyera kw’omutaano omu nkora y’okurya ebishaka, kandi ebindi bihingwa ebirikukora nk’ebiriisa, nka cytokinin, auxin, na abscisic acid, bikaba biine obumanyiso bukye bw’okureetaho ebiriisa. Kugyeragyeranisa n‘okukoresa W + W yonka, okutunguuka kw‘omuhendo gw‘ahaiguru gw‘ebirugire omu JA kurabira omu kukozesa OS (W + OS) omu kubanza nikubaasa kuhindurwa kuba eky‘okureeberaho ky‘amaani eky‘omutaano kya JAs. Tikirikuteekateekwaho, OSM na OSS1 eziine elicitor ezitarikushushana nizimanyirwa aha kureetera okuteerana kwa JA na JA-Ile okurikushushana. Okutaana na OSS1, OSM neereetaho OSMs omu muringo gw‘omutaano kandi gw‘amaani, obwo OSS1 etarikutunguura okugarukamu kw‘ebihuta by‘ahansi (ekishushani 3B).
(A) Okushwijuma omukura gw’okumanyisa hamwe okurugiirira aha kubara kwa PCC okw’okugyeragyeranisa kw’okuteerana kw’ebiriisa by’ebimera ebirikureetwa eby’okurya by’ebishaka. Enkora neejwekyera ekirungo kimwe ky’ekimera, kandi obuhango bw’enyuguta nibujwekyera Si index erikumanyisa ekirungo ky’ebimera ahagati y’okujanjaba. (B) Okuteerana kwa JA, JA-Ile na ET omu mababi okurikureetwaho obujanjabi obutari bumwe na bumwe oburikworekwa erangi zitari zimwe na zimwe: apricot, W + OSM; bururu, W + OSSl; omujungu, W + W; enzirugavu, C (okutegyeka) . Enyonyoozi nizooreka entaaniso mpango ahagati y‘okujanjaba n‘okutegyeka (ANOVA y‘emiringo ebiri erikukuratirwa okugyeragyeranisa kwa Tukey HSD post hoc emirundi mingi, *** P <0.001). Okushwijuma enteekateeka y‘amakuru ya (C)697 MS/MS (fayiro y‘amakuru S1) omu JA biosynthesis n‘okuburabura kw‘okuteekateeka (irAOC na irCOI1) na (D)585 MS/MS (fairo y‘amakuru S1) omu ETR1 n‘okuburabura kw‘akamanyiso ka ET Enteekateeka ibiri z‘okukyebera eby‘okurya by‘ebishaka n‘entebeekanisa y‘okukyebera eveclet ekareetaho. Enyonyoozi nizooreka entaaniso mpango ahagati y‘okujanjaba kwa W+OS n‘okutegyeka okutaine kabi koona (ANOVA y‘emiringo ebiri erikukuratirwaho okugyeragyeranisa kwa Tukey HSD bwanyima y‘okukora emirundi mingi, *P<0.05, **P<0.01 na ***P<0.001). (E) Ebishushani ebijanjaire eby‘okuhakanisa okujanjaire aha kumanyiira. Erangi nizijwekyera ebika by’ebimera ebitarikushushana ebihindwirwemu; obubonero nibujwekyera emiringo y‘okujanjaba etari emwe n‘emwe: enkokora ishatu, W + OSS1; ekicweka ky’omubiri, W + OSM; Okwetoorora C
Ekindi, nitukoresa oruganda oruhindwirwemu omu nkora y‘obuzaarwa bwa Nepenthes ezikyendeire (irCOI1 na sETR1) omu matembezo makuru g‘okukora JA na ET (irAOC na irACO) n‘okuteekateeka (irCOI1 na sETR1) kucondooza aha nkora y‘okuhindura eby‘okurya by‘ebimera ebi bibiri aha nkora y‘okugaruzaho puroguraamu y‘ebinyonyi ebirikurya ebishaka The relative. Okurugiirira aha kucondooza okwabandize, tukahamya okutaho herbivore-OS omu bimera ebirikutwara busha (EV) (ekishushani 3, C kuhika D) n’okukyendeera kwona omu nkora ya Hj erikureetwa OSM, obwo enkora ya δj erikweyongyera. Okugarukamu nikworeka munonga kukira okugarukamu okurikureetwaho OSS1. Giraafu y‘enyuguta ibiri erikukoresa Hj na δj nk‘enkoragana neeyoreka okutaho ebiragiro eby‘omutaano (ekishushani 3E). Ekirikukirayo kureebwa n‘okugira ngu omu miringo etaine kamanyiso ka JA, empindahinduka omu nkora ya metabolome n‘obukugu oburikureetwa ebinyonyi ebirikurya ebishaka haihi nibihwaho (ekishushani 3C). Okutaana n‘ekyo, okuteekateeka kwa ET okutarimu kugamba omu bimera bya sETR1, n‘obu okukwatwaho kwona aha mpindahinduka omu nkora y‘okurya eby‘okurya by‘ebishaka kuri ahansi munonga kukira okw‘okumanyisa kwa JA, nikukyendeeza entaaniso omu Hj na δj indices ahagati y‘okushemererwa kwa OSM na OSS1 (ekishushani 3D n‘ekishushani S5). . Eki nikyoreka ngu okwiha aha murimo mukuru gw‘okuhindura obumanyiso bwa JA, okuhindura obumanyiso bwa ET nakwo nikukora nk‘okutereeza gye okugarukamu kw‘eby‘okurya by‘ebika by‘ebinyonyi ebirikurya ebishaka. Okuhikaana n’omurimo ogu ogw’okuboneza gye, hakaba hatariho mpindahinduka omu kureetaho metabolome yoona omu bimera bya sETR1. Aha rubaju orundi, kugyeragyeranisa n‘ebimera bya sETR1, ebimera bya irACO bikareetaho empindahinduka z‘omubiri ezirikushushana omu bwijwire ezirikureetwa ebinyonyi ebirikurya ebishaka, kwonka byorekire obubonero bwa Hj na δj obutarikushushana munonga ahagati y‘ebizibu bya OSM na OSS1 (ekishushani S5).
Okubaasa kumanya ebiriisa by‘omutaano ebiine ebikuru ebirikuhwera omu kugarukamu kw‘ebika by‘ebinyonyi ebirikurya ebishaka n‘okuboneza oku birikukora kurabira omu bumanyiso bwa ET, tukakoresa omuringo gw‘obuhangwa ogwa MS/MS ogwabaire gukozirwe enyima. Enkora egi neeyesiga enkora ya bi-clustering kugaruka kushoboorora eka y‘eby‘okurya kuruga omu bicweka bya MS/MS [normalized dot product (NDP)] n‘obubonero bw‘okushushana kurugiirira aha kufeerwa okutaine rubaju (NL). Ekicweka kya MS/MS ekyayombekirwe kurabira omu kucondooza aha nkora za ET transgenic kikareetaho 585 MS/MS (fayiro y‘ebihandiiko S1), ekyashoboororwa barikubiteerana omu bicweka mushanju ebikuru ebya MS/MS (M) (Ekishushani 4A) . Ebimwe aha bicweka ebi nibipakira munonga ebiriisa by‘omutaano ebyabaire bimanyirwe enyima: eky‘okureeberaho, M1, M2, M3, M4 na M7 biine eby‘obugaiga by‘ebintu bitari bimwe na bimwe ebirikuruga omu phenol (M1), ebiriisa by‘omubiri (M2), shukaari z‘omubiri (M3 Na M4), na 17-HGL-DTG (M7). Okwongyera ahari ekyo, amakuru g‘eby‘okurya (Si index) g‘eby‘okurya bimwe omu buri kicweka nigabarwa, kandi okugabwa kwa Si kwakyo nikubaasa kureebwa omu muringo gw‘okwemanyiza. Omu bugufu, MS/MS spectra ezirikworeka ebiriisa by’ebishaka n’eby’obuhangwa ebiri ahaiguru nibimanyirwa aha mihendo ya Si y’ahaiguru, kandi emibaro ya kurtosis neeyoreka okugabwa kw’ebyoya aha rubaju rwa buryo rw’omukira. Omwe aha kugabwa kwa lean colloid nk’okwo kukashangwa omu M1, omu phenol amide eyorekire ekicweka kya Si ekirikukirayo (ekishushani 4B). Eby‘okurya by‘ebishaka ebigambirweho enyima 17-HGL-DTG omu M7 bikooreka obubonero bwa Si obw‘omutindo gw‘ahaiguru, ekirikworeka obubonero bw‘omutindo gw‘ahaiguru bw‘okutegyeka entaaniso ahagati y‘ebika bibiri bya OS. Okutaana n‘ekyo, ebiriisa by‘omutaano ebirikuruga omu nkora y‘okuhindura ebiriisa, nka rutin, CGA, na acyl sugars, biri omu bihingwa ebiri ahansi y‘obubonero bwa Si. Okubaasa kucondooza gye aha nkora y‘okugumizamu n‘okugabwa kwa Si ahagati y‘ebintu by‘omutaano ebirikuhindura ebiriisa, omukura gw‘ebicweka by‘omubiri gukakorwa ahari buri kicweka (ekishushani 4B). Okuteebereza okukuru kw‘enteekateeka ya OD (okufunzire omu kishushani 1B) n‘okugaruka kutebeekanisa ebiriisa by‘omutaano bwanyima y‘okurya ebishaka biine kureetera empindahinduka z‘omuringo gumwe omu biriisa ebiine omuhendo gw‘okwerinda gw‘ahaiguru, namunonga omu kwongyera aha bumanyiso bwabyo (okutaana n‘okugabwa omu muringo gw‘okutooranamu) Omuringo) Ebirungo by‘okwerinda ebitebereziibwe enteekateeka ya MT. Ebirugire omu phenol ebingi ebirundainwe omu M1 nibikwatagana n’emirimo n’okukyendeera kw’okukora kw’ebinyonyi (32). Twagyeragyeranisa emihendo ya Si omu bihingwa bya M1 ahagati y’ebibabi ebiine akakwate n’ebihingwa ebirikutegyeka EV aha shaaha 24, tukareeba ngu obutashushana bw’ebihingwa bingi bwanyima y’ebinyonyi ebirikurya ebishaka biine omuze gw’okwongyera munonga (ekishushani 4C). Okwongyezibwaho kw‘omutaano omu muheendo gwa Si kukashangwa omu phenolamides z‘okwerinda zonka, kwonka tihaine kweyongyera kwa Si okwashangirwe omu phenols ezindi n‘ebintu ebitarikumanywa ebirikutuura hamwe omu kicweka eki. Eki n‘eky‘okureeberaho ky‘omutaano, ekiine akakwate n‘enteekateeka ya OD. Ebiteberezo ebikuru eby’empindahinduka z’eby’okurya ebirikureetwa ebinyonyi ebirikurya ebishaka nibishushana. Okubaasa kugyezaho yaaba obutashushana obu obw‘omuringo gw‘ebiriisa bya phenolamide bukareetwa OS-specific ET, tukakora puroguraamu ya metabolite Si index kandi twareetera omuhendo gw‘okworeka entaaniso ahagati ya OSM na OSS1 omu EV na sETR1 genotypes (Ekishushani 4D ). Omuri sETR1, entaaniso erikureetwa phenamide ahagati ya OSM na OSS1 ekakyeera munonga. Enkora ya bi-clustering ekakoresibwa n‘aha makuru ga MS/MS agaarundainwe omu miringo erimu JA etarikumara kubaasa kushoboorora ebicweka bya MS/MS ebikuru ebiine akakwate n‘obukugu bw‘okuhindura eby‘okurya oburikutegyekwa JA (ekishushani S6).
(A) Ebirugire omu kuteerana kw‘ebicweka bya 585 MS/MS kurugiirira aha kicweka ekirikugabwa (okushushana kwa NDP) n‘okufeerwa okutaine rubaju okurikugabwa (okushushana kwa NL) nibirugamu omushomo (M) kuba nigukwatagana n‘eka y‘ebirungo emanyirwe, nari ahabw‘ekirungo kya Metabolite ekitarikumanywa nari ekitarikuhindurwa gye. Aha rubaju rwa buri kicweka, okugabwa kw‘eby‘okurya (MS/MS) eby‘omutaano (Si) nikworekwa. (B) Omukutu gw‘ebicweka by‘omubiri: Enkora nizijwekyera MS/MS n‘empimbi, NDP (etukutuku) na NL (bururu) obubonero bwa MS/MS (okusharaho,> 0.6). Ekipimo ky‘okumanyisa ebiri omu mubiri (Si) ekiine erangi kurugiirira aha kicweka (aha rubaju rwa bumosho) kandi kihandiikirwe aha maapu aha nkora y‘ebicweka by‘omubiri (aha rubaju rwa buryo). (C) Module M1 y‘ekimera kya EV omu mbeera y‘okutegyeka (okutegyeka) n‘okureetaho (okugyezesa ebirikurya ebishaka) aha shaaha 24: ekishushani ky‘omutimbagano gw‘ebicweka by‘omubiri (omuhendo gwa Si n‘obuhango bw‘enyuguta, phenolamide y‘okwerinda neeyorekwa omu bururu). (D) Ekishushani ky‘omutimbagano gwa molekyu za M1 eky‘omubaro gw‘eby‘okureeberaho sETR1 n‘okuteekateeka kwa EV na ET okutaine mugasho: ekirungo kya phenolic ekirikujwekyerwa enkora y‘enzindo ya kinyasi, n‘entaaniso mpango (omuhendo gwa P) ahagati y‘obujanjabi bwa W + OSM na W + OSS1 nk‘obuhango bw‘enkora. CP, N-kafeoyili-tirosini; CS, N-kafeoyili-ekirungo ky’omubiri; FP, asidi ya N-feruliki ey’omubiri; FS, N-omubazi gw’okusheeshasheesha; CoP, N’, N “-Omubazi gw’okukyebera; CFS, N’, N”-kafeoyili, feruloyili-spermidini;
Twongyeire twayongyera aha kucondooza kuruga aha genotype emwe eya Nepenthes erikukyendeeza amaani kuhika aha bantu b’obuhangwa, ahu empindahinduka z’amaani omu nkora ya JA ey’ebishaka n’emiringo y’eby’okurya ebirimu ebiriisa by’omubiri ebyabaire bishobooroirwe enyima omu bantu b’obuhangwa (26). Kozesa data set egi kushoborora obukooko 43. Obukooko obu burimu ebika 123 by’ebimera kuruga omu N. pallens. Ebimera ebi bikaruga omu mbuto ezaabaire ziteeraniirwe omu myanya y’obuzaarwa etari emwe n’emwe omuri Utah, Nevada, Arizona, na California (ekishushani S7), tukabara obutashushana bw’ebiriisa (aha nibwetwa omutindo gw’abantu) obutashushana bwa β) n’obukugu oburikureetwaho OSM. Okurugiirira aha kucondooza okukozirwe enyima, tukareeba empindahinduka nyingi omu nkora y‘okuhindura eby‘okurya aha rubaju rwa Hj na δj axes, ekirikworeka ngu obukooko bw‘ensiri bwine entaaniso mpango omu nkora y‘okuhindura eby‘okurya byazo aha nyamaishwa ezirikurya ebishaka (ekishushani S7). Ekitongore eki nikitwijutsya ebyabaireho enyima aha mpindahinduka z‘amaani eza JAs ezirikureetwaho ebinyonyi ebirikurya ebishaka, kandi kigumire kiine omuhendo gw‘ahaiguru munonga omu bantu bamwe (26, 36). Omu kukoresa JA na JA-Ile kugyezaho enkoragana y‘omutindo gwona ahagati ya Hj na δj, tukashanga ngu hariho enkoragana nungi y‘amaani ahagati ya JA na metabolome β diversity and specialization index (ekishushani S7). Eki nikimanyisa ngu obutashushana oburikureetwaho eby’okurya by’ebishaka omu kureetaho JAs obushangirwe aha rurengo rw’abantu nibubaasa kuba nibureetwaho okuhinduka kw’eby’okurya ebikuru okurikureetwaho okutoorana kuruga omu by’okurya by’ebishaka eby’ebinyonyi.
Okucondooza okukozirwe enyima kworekire ngu ebika by‘etaabe nibitaana munonga omu kika n‘okurugiirira aha nkora y‘okwerinda kw‘eby‘okurya ebirikureetaho n‘ebirikukora. Nikiteekateekwaho ngu empindahinduka ezi omu kuhindura obumanyiso oburikurwanisa eby’okurya by’ebishaka n’obushoboorozi bw’okwerinda niziteebwaho okugyemeserezibwa kw’omubaro gw’ebinyonyi, amagara g’ebimera, n’esente z’okukora eby’okwerinda omu mwanya ahu ekika ekimwe kirikukura. Twacondooza aha kuhinduka kw’eby’okurya by’amababi okurikureetwaho ebinyonyi ebirikurya ebishaka eby’ebika mukaaga bya Nicotiana ebirikuruga omu bukiizi-bwa-bumosho bwa Amerika n’omu bukiizi-bwa-bumosho bwa Amerika. Ebika ebi nibikwatagana munonga n’ebimera bya Nepenthes eby’omu bukiizi-bwa-bumosho bwa Amerika, ni Nicolas Bociflo. La, N. ebiriisa by’omubiri, ebiriisa by’omubiri n. obunyaatsi obukye, etaabe y’omurongooti, ​​etaabe (Nicotiana spegazzinii) n’etaabe y’amababi g’etaabe (Ekishushani 5A) (37). Ebika mukaaga ahari ebi, otwariremu n’ebika ebimanyirwe gye N. please, n’ebimera ebirikumera buri mwaka eby’ekika kya petunia, kandi obtusifolia N. n’ebimera ebitarikuhwaho eby’ekika kya munyaanya Trigonophyllae (38). Okurugirira ahari ekyo, W + W, W + OSM na W + OSS1 induction zikakorwa aha miringo egi mushanju kwega okugaruka kutebeekanisa eby’okurya by’ebinyonyi aha rurengo rw’ebika.
(A) Omuti gwa bootstrap phylogenetic ogurikurugiirira aha kubaasa okurikukirayo [ahabw’okukora glutamine y’omubiri (38)] n’okugabwa kw’ebicweka mushanju eby’ebika bya Nicotiana ebiine akakwate ka haihi (erangi zitari zimwe na zimwe) (37). (B) Ekishushani ky’okujanjaaza eby’omutaano eby’omutaano ahabw’eby’okurya by’ebika mushanju bya Nicotiana (939 MS/MS; fairo y’amakuru S1). Aha rurengo rw’ebika, okutaana kw’ebiriisa (metabolome) nikukwatagana kubi n’omutindo gw’obukugu. Okushwijuma enkoragana y‘omutindo gw‘ebika ahagati y‘okuhindura eby‘okurya n‘okumanya n‘okuteerana kwa JA nikworekwa omu kishushani 2. S9. Erangi, ebika bitarikushushana; enkokora ishatu, W + OSS1; ekicweka ky’omubiri, W + OSM; (C) Nicotiana JA na JA-Ile ebirikuhinduka nibiteebwa omu mwanya kurugiirira aha nkora y‘okushemererwa kwa OS (ANOVA y‘emiringo ebiri na Tukey HSD bwanyima y‘okugyeragyeranisa emirundi mingi, * P <0.05, ** P <0.01 na * ** Ahabw‘okugyeragyeranisa W + OS na W + W, P <0.001). Box plot ya (D) obutashushana na (E) obumanyi bwa buri kika bwanyima y’okugyeragyeranisa eby’okurya by’ebishaka n’eby’okurya bya methyl JA (MeJA). Enyonyoozi neeyoreka entaaniso mpango ahagati ya W + OS na W + W nari lanolin hamwe na W (Lan + W) nari Lan hamwe na MeJA (Lan + MeJa) n‘okutegyeka Lan (okushwijuma kw‘emiringo ebiri kw‘okuhinduka, kukuratirwaho okugyeragyeranisa kwa Tukey okwa HSD bwanyima y‘obwire bw‘obwire , *P<0.05, **P<0.01 na).
Twakoresa omuringo gw‘okuteerana ebiri, tukamanya ebicweka 9 bya 939 MS/MS (fairo y‘ebihandiiko S1). Ebirimu MS/MS ebigarukiremu kutebeekanisibwa obujanjabi obutarikushushana nibitaana munonga omu bicweka bitarikushushana ahagati y‘ebika (ekishushani S8). Okureeba omu maisho Hj (ekirikugambwaho aha nka species-level γ-diversity) na δj nikworeka ngu ebika bitarikushushana nibiteerana omu guruupu zitari zimwe na zimwe munonga omu mwanya gw‘okuhindura eby‘okurya, ahu okugabanisa aha rurengo rw‘ebika kurikukira kuba nikureebeka munonga kukira okushemererwa. Okwihaho N. linear na N. obliquus, nizooreka ebirikuhinduka bingi eby’okureetaho (ekishushani 5B). Okutaana n’ekyo, ebika nka N. purpurea na N. obtusifolia biine okugarukamu kw’omubiri okutarikureebwa gye aha kujanjaba, kwonka metabolome neekira kuba ey’emiringo mingi. Okugabwa kw‘ebika by‘ebimera eby‘okugarukamu kw‘eby‘okurya ebireetsirweho kukarugamu akakwate karungi akatari karungi ahagati y‘okwemanyiza n‘okutaana kwa gamma (PCC = -0.46, P = 4.9×10-8). Empindahinduka ezirikureetwaho OS omu rurengo rwa JAs nizikwatagana gye n‘obukugu bwa metabolome, kandi ziine akakwate kabi n‘obutashushana bwa metabolic gamma oburikworekwa buri kika (ekishushani 5B n‘ekishushani S9). Nikirungi kumanya ngu ebika ebirikugambwaho omu rurimi rw’abantu boona nk’ebika bya “okugarukamu kw’obumanyiso” omu kishushani 5C, nk’ebinyonyi ebirikwetwa Nepenthes nematodes, Nepenthes nepenthes, Nepenthes acute, na Nepenthes attenuated, bikareetera obumanyiso bw’amaani omu dakiika 30. Oburwaire bwa JA na JA-Ile oburikukwata aha OS obuhererukireyo, obwo obukooko obundi oburikwetwa “obumanyiso obutarikugarukamu”, nk‘ebyoma bya Nepenthes, Nepenthes powdery na N. obtusifolia nibworeka okutandika kwa JA-Ile Edge kwonka hatariho OS yoona (ekishushani 5C). Aha rurengo rw‘okuhindura eby‘okurya, nk‘oku kyagambirweho ahaiguru, ahabwa Nepenthes ezikyeenirwe, ebintu ebirikugarukamu obumanyiso bikooreka OS specificity kandi byayongyera munonga δj, obwo birikukyendeeza Hj. Eki OS-specific priming effect tikikashangirwe omu miringo erikushoroorwa nk‘ebika ebitarikukora aha bumanyiso (ekishushani 5, D na E). OS-specific metabolites nizigabwa munonga ahagati y‘ebika ebirikugarukamu obumanyiso, kandi ebirundo by‘obumanyiso ebi nibiteerana n‘ebika ebiine okugarukamu kw‘obumanyiso okukye, obwo ebika ebiine okugarukamu kw‘obumanyiso okukye nibyoreka okwesigagana kukye (Ekishushani S8 ). Ekirugiremu eki nikyoreka ngu OS-specific induction ya JAs n‘okugarukaho kwa OS-specific okwa metabolome y‘ahansi nibikwatanisibwa aha rurengo rw‘ebika.
Ekindi, tukakozesa omubazi gwa lanolin ogurimu methyl JA (MeJA) kujanjaba ebimera kucondooza yaaba emiringo egi ey’okuteerana neezibirwa ahabw’okugira ngu hariho JA erikukoresibwa exogenous JA, erikuza kuba eri omu cytoplasm y’ebimera. Okukyendeeza aha nshaho omu bwangu ni JA. Twashanga omuze nigwo gumwe gw‘empindahinduka mporampora kuruga aha kika ekirikugarukamu obumanyiso kuza aha kika ekitarikugarukamu obumanyiso ekirikureetwaho okuguma nizitunga JA (ekishushani 5, D na E). Omu bugufu, obujanjabi bwa MeJA bukagaruka bwatebeekanisa n’amaani ebiriisa by’omubiri eby’ebinyonyi ebiri omu nkora, N. obliquus, N. aquaticus, N. pallens, na N. mikimotoi, ekyarugiremu okweyongyera kw’amaani omu δj n’okukyendeera kwa Hj. N. purpurea ekooreka okweyongyera kwa δj kwonka, kwonka ti Hj. N. obtusifolia, eyaabaire neeyorekwa enyima ngu neeteerana JAs eziri ahansi munonga, nayo neegarukamu kubi obujanjabi bwa MeJA omu kureeba okugaruka omu nkora ya metabolome. Ebirugiremu ebi nibyoreka ngu okukora JA nari okuhindura obumanyiso nikuzibirwa omu mubiri omu miringo etarikugarukamu obumanyiso. Okugyezaho enteekateeka egi, tukacondooza aha miringo ena (N. pallens, N. mills, N. pink na N. microphylla) ezirikureetwaho W + W, W + OSMs na W + OSS1 Transcriptome (39). Okurugiirira aha nkora y’okugaruzaho metabolome, ebika nibitaanisibwa gye omu mwanya gw’okuhandiika, omuri ebyo N. attenuated ekooreka RDPI erikureetwa OS erikukirayo, obwo N. gracilis ekaba eine erikukirayo ahansi (Ekishushani 6A). Kwonka kikashangwa ngu obutashushana bw’ebihandiiko (transcriptome diversity) oburikureetwa N. oblonga bukaba buri ahansi omu miringo ena, okutaana n’obutashushana bw’ebika bya metabonomic ebyabaire biri ahaiguru munonga ebya N. oblonga ebyabaire nibyorekwa enyima omu miringo mushanju. Okucondooza okukozirwe enyima kworekire ngu ekibiina ky‘ebicweka by‘omubiri ebiine akakwate n‘obumanyiso bw‘okwerinda kwa kare, obariiremu n‘obumanyiso bwa JA, nibishoboorora obumanyiso bw‘okwerinda kwa kare oburikureetwaho elicitors eziine akakwate n‘ebiriisa by‘ebishaka omu miringo ya Nicotiana (39). Okugyeragyeranisa emihanda y‘okumanyisa JA ahagati y‘ebika ebi bina kukashohoza enkora erikushemeza (ekishushani 6B). Ebicweka by’omubiri ebingi ebiri omu muhanda ogu, nka AOC, OPR3, ACX na COI1, bikooreka omutindo gw’ahaiguru munonga gw’okutaaha omu nkora omu miringo egi ena. Kwonka, akazaano akakuru, JAR4, nikahindura JA omu muringo gwayo ogurikukora omu by’amagara ogw’ebihandiiko bya JA-Ile ebirundainwe, kandi omutindo gwayo gw’okuhandiika nigukye munonga, namunonga omu N. mills, Nepenthes pieris na N. microphylla. Okwongyera ahari ekyo, ekihandiiko kyonka eky’omuringo ogundi ogw’obuzaarwa AOS tikirashangirwe omu N. bifidum. Empindahinduka ezi omu kumanyisa ebiri omu nkora y‘obuzaarwa nizibaasa kuba nizo zirikureetaho ebiriisa by‘omubiri ebirengire ebirikureetwaho okureetaho JA nkye omu miringo y‘obumanyiso obutarikukora gye n‘okureetaho N. gracilis.
(A) Okushwijuma enteekateeka y‘amakuru g‘okugaruka kutebeekanisa eby‘okugarukamu by‘okuhandiika eby‘okubanza eby‘ebika bina by‘etaabe ebiine akakwate ka haihi ebyatooranwa edakiika 30 bwanyima y‘okutandika kw‘okurya ebishaka. RDPI neebarwa orikugyeragyeranisa ebibabi ebirikuruga omu OS y‘okurya ebishaka n‘okuzibira ebihuta. Erangi nizooreka ebika bitarikushushana, kandi obubonero nibworeka emiringo y’okujanjaba etarikushushana. (B) Okushwijuma okureebeka kw’obuzaarwa omu mihanda y’okumanyisa JA omu miringo ena. Omuhanda gwa JA ogwanguhize nigworeka haihi n‘ekipande ky‘akabokisi. Erangi zitari zimwe na zimwe nizooreka emiringo etari emwe n’emwe ey’okukora ebintu. Enyonyoozi neeyoreka ngu hariho entaaniso mpango ahagati y‘obujanjabi bwa W + OS n‘okutegyeka kwa W + W (ahabw‘ekigyezo kya t-test ky‘omwegi ahabw‘entaaniso z‘ababiri, *P<0.05, **P<0.01 na ***P<0.001). OPDA, asidi y’ebihimba 12; OPC-8: 0,3-oxo-2(2′(Z)-penteni)-cyclopentain-1-oktanoiki asidi.
Omu kicweka ekirikuhererukayo, tukashoma oku okugaruzaho kw’ebika by’ebinyonyi eby’omutaano omu metabolome y’ebika by’ebimera ebitarikushushana kurikubaasa kugumira ebinyonyi ebirikurya ebishaka. Okucondooza okwabandize kukaba kuta omutima aha kika kya Nicotiana. Okuzibira kwazo Ms n’enzigye nikutaana munonga (40). Aha, tukashoma aha nkoragana eri ahagati y‘omuringo ogu n‘obutafaari bwabo obw‘okuhindura eby‘okurya. Okukozesa ebika bina by’etaabe ebiri aharuguru, n’okugyezaho akakwate akari ahagati y’okutaana n’obukugu bw’eby’okurya ebirikureetwa ebihingwa ebirikurya ebishaka n’okuzibira ebimera ahari Ms na Sl, tukashanga ngu okuzibira, okutaana n’obukugu aha mubazi gw’omutaano Sl Byona biine akakwate karungi, obwo akakwate akari ahagati y’okugumisiriza obutashushana bw’abakazi abakugu ti, Fig na Fig. S10). Aha bikwatiraine n’okuzibira S1, byombi N. chinensis na N. gracilis ebyabaire bikye, ebyabaire byorekwa enyima ngu nibyoreka emiringo y’okuhindura obumanyiso bwa JA n’obutafaari bwa metabolome, bikaba biine eby’okugarukamu bitarikushushana munonga aha kureetaho eby’okurya by’ebishaka, kandi nabyo bikaba nibyoreka okuzibira kw’ahaiguru okurikushushana. Okuteerana.
Omu myaka makumi mukaaga ehweire, enteekateeka y’okwerinda kw’ebimera ekaha omutindo gw’enteekateeka, orikwegamira aha bacondoozi okutebereza omubaro muhango gw’okutunguuka n’emirimo y’ebimera eby’omutaano ebirikuhindura omubiri. Ebiteekateeko ebi ebingi tibirikukuratira enkora eya butoosha ey‘okushoboorora eby‘amaani (41). Nibaha enteekateeka z‘okuteebereza okukuru (3) aha rurengo rumwe rw‘okweshwijuma. Okugyezibwa kw‘okuteebereza okukuru kwaba nikwikiriza enteekateeka z‘omutaano kushwijumwa, eki nikiija kureetera Omusiri niguza omu maisho. Oshagikwe, kwonka oyangye abandi (42). Omu mwanya gw‘ekyo, enteekateeka ensya neekora okuteebereza aha mitwe y‘okweshwijuma etari emwe n‘emwe kandi eyongyera aha nkora nsya y‘okuteekateeka aha kushoboorora (42). Kwonka, enteekateeka ibiri eziteirweho aha rurengo rw‘emirimo, MT na OD, nizibaasa kushoboororwa omu muringo gworobi nk‘okuteebereza okukuru okw‘empindahinduka z‘omutaano z‘eby‘okurya ezirikureetwaho ebinyonyi ebirikurya ebishaka: enteekateeka ya OD neeikiriza ngu empindahinduka omu by‘okurya by‘omutaano “omwanya” nizihabura munonga . Enteekateeka ya MT neeteekateeka ngu empindahinduka ezi niziija kuba zitari z‘okuhabura kandi ziri omu mwanya gw‘okuhindura eby‘okurya, kandi nizikira kugira eby‘okurya by‘omuhendo gw‘okwerinda gw‘ahaiguru. Ebigyezo eby‘enyima eby‘okuteebereza kwa OD na MT bikagyezibwa barikukoresa ekicweka kikye ky‘ebirungo bya priori “okwerinda”. Ebigyezo ebi ebirikuta omutima aha metabolite nibizibira obushoboorozi bw’okushwijuma obuhango n’omuhanda gw’okugaruka kuhindura metabolome omu bwire bw’okurya ebishaka, kandi tibirikwikiriza okukyebera omu nkora y’embariirira etarikuhinduka kwetenga okuteebereza okukuru okurikubaasa kuteekateekwaho nk’ekintu kyona Quantify empindahinduka omu metabolome y’ebimera. Aha, tukakoresa tekinologiya y‘obuhanguzi omu metabolomics erikwegamira aha MS y‘okubara kandi twakora okucondooza kwa deconvolution MS omu sente z‘omugasho z‘okushoboorora enteekateeka y‘amakuru kugyezaho entaaniso eri ahagati y‘ebibiri ebiteirweho aha rurengo rwa metabolomics rw‘ensi yoona. Okuteebereza okukuru okw’enteekateeka egi. Enteekateeka y‘amakuru ekozesiibwe omu miringo mingi, namunonga omu nkora y‘okucondooza aha bihangirwe n‘okugyendera kw‘ebiriisa (43). Kwonka nk’oku turikumanya, egi niyo nkora y’okubanza erikukoresibwa kushoboorora omwanya gw’amakuru g’eby’okurya omu bimera n’okushoboorora oburemeezi bw’eby’obuhangwa oburikukwata aha mpindahinduka z’eby’obuhangwa ez’akaire kakye omu kugarukamu ebishushani by’eby’obuhangwa. Okukira munonga, obushoboorozi bw’omuringo ogu buri omu kubaasa kwayo kugyeragyeranisa enkora omunda n’ahagati y’ebika by’ebimera kukyebera oku ebinyonyi ebirikurya ebishaka byahindukire kuruga omu miringo etari emwe n’emwe kuza omu nkora z’okutunguuka kw’ebika by’ebimera ahagati y’ebika aha mitwe y’okutunguuka etari emwe n’emwe. Okuhindura eby’okurya omu mubiri.
Okushwijuma ebicweka ebikuru (PCA) nikuhindura ebihandiiko by‘ebintu bingi omu mwanya gw‘okukyendeeza aha buhango bw‘ebintu kugira ngu omuze mukuru gw‘ebihandiiko gushoboororwe, n‘ahabw‘ekyo nigukira kukoresibwa nk‘omuringo gw‘okucondooza okushwijuma ebihandiiko by‘ebintu, nk‘oku deconvolution metabolome. Kwonka, okukyendeeza aha buhango nikwija kufeerwa ekicweka ky‘amakuru agari omu nkora y‘okuhandiika, kandi PCA terikubaasa kuha amakuru g‘omubaro aha mitwarize erikukwatagana munonga n‘enteekateeka y‘eby‘obuhangwa, nka: oku ebinyonyi ebirikurya ebishaka birikugaruka kutebeekanisa obutashushana omu myanya y‘omutaano (eky‘okureeberaho, obugaiga, okujanjaara) N‘obungi) ebirikuhindura ebiriisa? Ni metabolites ki ezirikutebereza embeera erikureetwaho ey‘ekihangirwe ekirikurya ebishaka ekihairwe? Okurugirira aha kureeba kw’omutaano, okutashushana n’okutabaasa kureetaho, amakuru agari omu nkora y’okuhindura ebiri omu mababi nigavunda, kandi nikishangwa ngu okurya ebinyonyi ebirikurya ebishaka nikubaasa kureetaho okuhindura kw’omutaano. Titurikuteekateeka, tukareeba ngu, nk‘oku kyashoboororwa omu by‘okureeberaho by‘enteekateeka y‘amakuru ebiteirwe omu nkora, embeera y‘okuhindura eby‘okurya erikurugamu eine okutaana kwingi bwanyima y‘okutahirirwa kw‘ebinyonyi bibiri ebirikurya ebishaka (omuhangu omu by‘okurya by‘ekiro Sl) n‘omuhangu omu bya Solanaceae Ms. N‘obu emitwarize yaabo y‘okurya n‘okuta omutima aha kintu biraabe nibitaana munonga. Omutandikyi w‘ebishaju-asidi z‘omubiri (FAC) omu OS (31). Omu kukozesa herbivore OS kujanjaba ebihuta by‘okucumita ebitebeekanisiibwe, okujanjaba kw‘ebishaka okurikugyeragyeranisibwa nakwo kukooreka omuze nigwo gumwe. Enkora egi erimu omutindo gw’okugyeragyeranisa oku ebimera birikugarukamu okutaahirira kw’ebinyonyi ebirikurya ebishaka neemaraho enshonga ezirikuteganisa ezirikureetwaho empindahinduka omu mitwarize y’okurya ebinyonyi ebirikurya ebishaka, ebirikureetaho okusiisikara kw’emiringo etari emwe n’emwe omu bwire butarikushushana (34). FAC, emanyirwe nk‘ekikuru ekirikureetaho OSM, neekyendeeza aha kugarukamu kwa JAS n‘ebindi bihingwa by‘omubiri omu OSS1, obwo OSS1 erikukyendeeza emirundi mingi (31). Kwonka OSS1 ekareetera emiringo erikushushana y‘okuteerana kwa JA kugyeragyeranisa na OSM. Kikagambwaho enyima ngu okugarukamu kwa JA omu Nepenthes ezikyeire nikukwatwaho munonga OSM, ahu FAC erikubaasa kuguma n‘emirimo yaayo n‘obu yaakuba erimu amaizi 1:1000 (44). Nahabwekyo, waagyeragyeranisa na OSM, n‘obu FAC omu OSS1 eraabe eri ahansi munonga, neemara kureetaho okujanjaara kwa JA okurikumara. Okucondooza okukozirwe enyima kworekire ngu puroteyini eziri nk’eza porin (45) na oligosaccharides (46) nizibaasa kukoresibwa nk’obumanyiso bw’ebicweka by’omubiri kureetaho okugarukamu kw’okwerinda kw’ebimera omu OSS1. Kwonka, tikikamanyirwe gye yaaba elicitors ezi omu OSS1 nizo zirikureetaho okurundaana kwa JA okureebirwe omu kucondooza okuriho hati.
N‘obu haraabe hariho okucondooza kukye okurikushoboorora aha ntaaniso y‘enkumu z‘okuhindura eby‘okurya ezirikureetwaho okukoresa eby‘okurya by‘ebishaka ebitarikushushana nari exogenous JA nari SA (salicylic acid) (47), tihaine owaateganiise okutabura kw‘ebika by‘ebishaka eby‘omutaano omu nkora y‘ebishaka by‘ebimera n‘ebirikurugamu aha nkora y‘okuhindura eby‘okurya eby‘omutaano The overall is. amakuru g’omuntu. Okweshwijuma oku kukagaruka kwahamya ngu omukago gw’omunda gw’ebiriisa n’ebiriisa ebindi eby’ebimera ebitari bya JAs nibibumba obumanyiso bw’okugaruka kutebeekanisa eby’okurya ebirikureetwa ebinyonyi ebirikurya ebishaka. Okukira munonga, tukashanga ngu ET eyareetsirweho OSM ekaba eri mpango munonga kukira eyareetsirweho OSS1. Omuringo ogu nigukwatagana n‘ebiri omu FAC bingi omu OSM, ekirikwetengyesa kandi ekirikumara kureetaho okuturika kwa ET (48). Omu nkora y‘okukoragana ahagati y‘ebimera n‘ebirikurya ebishaka, omurimo gw‘okumanyisa ogwa ET aha nkora y‘okuhindura eby‘okurya omu bimera n‘obunaku obu gukye kandi niguta omutima aha guruupu emwe y‘ebirungo byonka. Okwongyera ahari ekyo, okucondooza okurikukirayo kukozeise okukoresa ET kuruga aheeru nari ebirikugibanzaho nari ebizibu bitari bimwe na bimwe kwega aha nkora ya ET, omuri ebyo okukoresa emibazi egi ey‘aheeru nikwija kurugamu ebizibu bingi ebitari by‘omutaano. Okurugiirira aha kumanya kwaitu, okucondooza oku nikujwekyera okushwijuma kw’okubanza okw’omutindo muhango okw’omurimo gwa ET omu kukoresa ET kureetaho n’okuteekateeka aha bimera ebihindwirwemu ebiine oburemeezi kubaasa kukwatisa entunguuka y’ebimera. Okureetaho ET ey‘omutaano aha by‘okurya by‘ebishaka nikubaasa kuhindura okugarukamu kwa metabolome aha muheru. Ekikuru munonga n’okuhindura ebiri omu nkora ya ET biosynthesis (ACO) n’okuteekateeka (ETR1) ebyayorekire okuteerana kw’eby’okurya ebirimu ebiriisa by’ebishaka (phenolamides de novo). Kikagambwaho enyima ngu ET neebaasa kuboneza okuteerana kw‘ekiriisa kya nikotini ekirikureetwa JA obwo erikutegyeka putrescine N-methyltransferase (49). Kwonka, kuruga omu kureeba kw‘ebyoma, tikirikumanywa gye oku ET erikukora gye okutaho phenamide. Oyihireho omurimo gw‘okuhindura obumanyiso bwa ET, omubazi gw‘okuhindura eby‘okurya nigubaasa n‘okuhindurwa kuza omu S-adenosyl-1-methionine kuhabura okuta sente omu amides za polyaminophenol. S-adenosyl-1-methionine ni ET kandi eri ahagati y‘omuhanda gw‘okukora ebiriisa by‘omubiri. Enkora ei ET signal erikutegyeka omutindo gwa phenolamide neeyetenga kwongyerwa kwega.
Kumara obwire buraingwa, ahabw‘omubaro muhango gw‘ebintu by‘omutaano eby‘omubiri ebitarikumanywa, okuta omutima munonga aha miringo y‘ebintu ebitari bimwe na bimwe kubaire kutarikubaasa kukyebera gye empindahinduka z‘obwire z‘ebintu by‘omubiri ebitari bimwe na bimwe bwanyima y‘okukoragana kw‘ebintu ebiine amagara. Obwahati, kurugiirira aha kucondooza kw’enteekateeka y’amakuru, ekikuru ekirugire omu kutunga MS/MS spectrum kurugiirira aha bihingwa ebitari by’orubaju n’okugira ngu ebihingwa ebirikurya nari ebirikugyeragyeranisa ebihingwa nibigumizamu kukyendeeza aha nkora y’eby’okurya yoona ey’ebihingwa by’amababi obwo birikweyongyera aha rurengo rwabyo rw’obukugu. Okwongyezibwaho oku okw‘akaire kakye omu nkora ya metabolome okurikureetwa ebinyonyi ebirikurya ebishaka nikukwatagana n‘okwongyezibwaho kw‘okukoragana omu nkora ya transcriptome. Ekintu ekirikuhwera munonga omu kumanyiira oku okuhango okwa metabolome (okugira omuhendo gwa Si gw’ahaiguru) n’ekirungo ky’omutaano ekiine omurimo gw’okurya ebishaka ogwabaire gumanyirwe enyima. Omuringo ogu nigukwatagana n‘okuteebereza kwa OD theory, kwonka okuteebereza kwa MT okurikukwata aha randomness y‘okugaruka kuteekateeka aha metabolome tikurikuhikaana. Kwonka, ebihandiiko ebi nabyo nibikwatagana n‘okuteebereza kw‘omuringo gw‘okutabura (MT erikukirayo oburungi; ekishushani 1B), ahabw‘okuba ebindi bihingwa ebitaine mitwarize ebiine emirimo y‘okwerinda etarikumanywa nibibaasa kuguma nibikuratira okugabwa kwa Si okutairweho.
Enkora y‘okumanya eyongyeire kukwatwa okucondooza oku n‘okugira ngu kuruga aha rurengo rw‘okutunguuka okukye (ekimera kimwe n‘omubaro gw‘etaabe) kuhika aha rurengo rw‘okutunguuka oruhango (ebika by‘etaabe ebiine akakwate ka haihi), emiringo etari emwe n‘emwe y‘entebeekanisa y‘okutunguuka eri omu “kwerinda okurungi” Hariho entaaniso mpango omu busobozi bw‘ebinyonyi ebirikurya ebishaka. Moore n’abandi. (20) na Kessler na Kalske (1) omu bwijwire bakateekateeka kuhindura emiringo eshatu y‘emirimo y‘ebintu ebiine amagara ebyabandize kutaanisibwa Whittaker (50) omu mpinduka z‘obwire ezirikureetaho n‘ezirikureetaho eby‘obuhangwa bw‘ebintu; abahandiiki aba tibarikufunza Emiringo y‘okushoroora amakuru g‘eby‘okurya bingi nayo terikushoboorora oku orikubaasa kubara obutashushana bw‘eby‘okurya kuruga omu makuru aga. Omu kucondooza oku, okuhindura okukye omu kushoroora kw‘emirimo kwa Whittaker nikwija kuteekateeka aha nkora ya α-metabolic nk‘entaaniso ya MS/MS spectra omu kimera ekihairwe, na β-metabolic entaaniso nk‘entaaniso y‘omutaano ey‘omunda y‘ekika ky‘abantu Omwanya, kandi γ-metabolic entaaniso neija kuba okwongyezibwaho kw‘entaaniso y‘eby‘okurya ebirikushushana.
Akamanyiso ka JA nikyetengyesa munonga aha kugarukamu kw‘eby‘okurya by‘ebishaka bingi. Kwonka, hariho okubura okukyebera okugumire okw’omubaro gw’obuhwezi bw’okutegyeka kw’omutaano okwa JA biosynthesis aha kuhinduka kw’eby’okurya, kandi yaaba akamanyiso ka JA n’omwanya gw’omutaano gw’okuhinduka kw’eby’okurya okurikureetwaho okuteganisibwa aha rurengo rw’ahaiguru rw’okutunguuka kw’ebintu n’obunaku obu tikirikwetegyerezibwa. Twareeba ngu obuhangwa bw’okurya ebishaka bwa Nepenthes herbivorous nibureetaho okumanyiira kwa metabolome kandi okuhinduka kw’obumanyi bwa metabolome omu bantu b’ebika bya Nicotiana n’omu bika bya Nicotiana ebiine akakwate ka haihi nibikwatagana gye n’okumanyisa kwa JA. Okwongyera ahari ekyo, akamanyiso ka JA kaaba kakye, okuhindura kw‘eby‘okurya okurikureetwaho ekihangirwe kimwe eky‘okurya ebishaka nikwija kusazibwamu (ekishushani 3, C na E). Ahabw‘okugira ngu empindahinduka omu nkora y‘okuhindura eby‘okurya omu nkora y‘obuhangwa bw‘abantu ba Nepenthes ezirikukyendeera omu buhangwa nizikira kuba ez‘omubaro, empindahinduka omu nkora y‘okuhindura eby‘okurya β n‘okwemanyiza omu kucondooza oku nizibaasa kuba nizireetwa munonga okushemererwa kw‘amaani kw‘ebicweka by‘ebirungo ebirimu ebiriisa bingi. Ebicweka ebi eby‘ebirungo nibitegyeka ekicweka ky‘enkora ya metabolome kandi nibireetaho akakwate karungi n‘obumanyiso bwa JA.
Ahabw‘okugira ngu emiringo y‘ebihangwa by‘omubiri ey‘ebika by‘etaabe ebiine akakwate ka haihi nayo neetaanira kimwe, ebiriisa ebiri omu mubiri nibimanyirwaho omu muringo gw‘omutindo, n‘ahabw‘ekyo nibikira kuba eby‘okushwijuma. Enteekateeka y‘amakuru ey‘okukora aha nkora y‘okuhindura eby‘okurya ekwatsirwe neeyoreka ngu okutandika kw‘okurya ebishaka nikuyongyera aha kushuubuza ahagati y‘eby‘okurya bya gamma n‘okumanya. Akamanyiso ka JA nikakora omurimo muhango omu kushuubuza oku. Okwongyezibwaho omu kumanyiira kwa metabolome nikuhikaana n‘okuteebereza kwa OD okukuru kandi nikukwatagana gye n‘akamanyiso ka JA, obwo akamanyiso ka JA kaine akakwate kabi n‘okutaana kw‘eby‘okurya bya gamma. Eby’okureeberaho ebi nibyoreka ngu obushoboorozi bwa OD bw’ebimera nibukira kumanyirwa aha buveera bwa JA, yaaba aha rulengo rw’entunguuka nkye nari aha rurengo rw’entunguuka mpango. Okucondooza kw’okukoresa JA ey’aheeru y’omubiri okurikutambura oburemeezi bwa JA biosynthesis nikwongera kworeka ngu ebika by’etaabe ebiine akakwate ka haihi nibibaasa kutaanisibwamu ebika ebirikugarukamu obumanyiso n’ebitarikugarukamu obumanyiso, nk’oku omuringo gwabyo gwa JA n’obutafaari bwa metabolome oburikureetwaho ebinyonyi ebirikurya ebishaka. Ebika by‘ebihangirwe ebitarikugarukamu eby‘obumanyiso tibirikubaasa kugarukamu ahabw‘okuremwa kwabyo kureetaho JA y‘omunda n‘ahabw‘ekyo nibibaasa kukwatwaho oburemeezi bw‘omubiri. Eki nikibaasa kureetwaho okuhinduka omu bimwe aha bikuru omu nkora y‘okumanyisa JA (AOS na JAR4 omu N. crescens) ya. Ekirugiremu eki nikyoreka ngu enkora ezi z‘okutunguuka kw‘ebika by‘ebihangirwe nizibaasa kuba nizireetwa munonga empindahinduka omu kuteekateeka n‘okugarukamu kw‘eby‘omubiri eby‘omunda.
Okwongyera aha nkoragana ahagati y’ebimera n’ebirikurya ebishaka, okucondooza aha nkora y’okuhindura eby’okurya omu mubiri nikukwatagana n’entunguuka zoona ez’omugasho omu kucondooza aha kuhindura kw’ebihangirwe omu mwanya n’okutunguuka kw’emitwarize y’eby’obuhangwa egumire. Ahabw‘okwongyera aha mubaro gw‘ebihandiiko ebitungirwe ebikwato bya MS eby‘obunaku obu, okugyezaho kw‘ebiteekateeko aha nkora y‘okuhindura eby‘okurya hati nikubaasa kurenga aha ntaaniso z‘eby‘okurya by‘omuntu/ekicweka kandi kukora okucondooza kw‘ensi yoona kworeka enkora ezitarikuteekateekwaho. Omu nkora y‘okushwijuma aha rurengo rwahaiguru, enfumu nkuru n‘ekiteekateeko ky‘okuteekateeka maapu z‘amakuru ezirikubaasa kukoresibwa kucondooza aha makuru. Nahabwekyo, ekikuru ekirugire omu kugaita okuriho hati okwa MS/MS metabolomics okutaine rubaju n‘enteekateeka y‘amakuru n‘okugira ngu neeha omubaro gworobi ogurikubaasa kukoresibwa kwombeka maapu z‘okucondooza aha nkora y‘okuhindura eby‘okurya aha minzaani y‘okushoroora etari emwe n‘emwe. Nikyo kikuru ekirikwetengwa omu muringo ogu. Okwega aha nkora y‘okutunguuka kw‘ebintu ebikye/ebihango n‘eby‘obuhangwa bw‘ekyanga.
Aha rurengo rw’entunguuka mpango, omusingi gw’enteekateeka y’entunguuka y’ebimera n’ebinyonyi hamwe eya Ehrlich na Raven (51) n’okuteebereza ngu okuhinduka kw’ebintu ebiri omu nkora y’ebika by’ebimera nikyo kirikureetaho okuhinduka kw’enganda z’ebimera. Kwonka omu myaka makumi ataano kuruga obu ekitabo eki eky’omutaano kyashohozibwa, enteekateeka egi ekaba etarikukira kugyezibwa (52). Eki nikireetwaho munonga obumanyiso bwa phylogenetic obw’okugyeragyeranisa obumanyiso bw’okuhindura eby’okurya omu nkora z’ebimera ebiri hare munonga. Obukye nibubaasa kukoresibwa kuhamya emiringo y‘okushwijuma ebigyendererwa. Omuringo gw‘okukora ogwa MS/MS oguriho hati ogurikukorwaho enteekateeka y‘amakuru nigupima okushushana kw‘enkora ya MS/MS ey‘ebintu ebitarikumanywa (hatariho kubanza kutoorana ebiriisa) kandi niguhindura MS/MS ezi omu nkora ya MS/MS, n‘ahabw‘ekyo omu nkora y‘obukugu Eby‘okureeberaho ebi eby‘entunguuka mpango nibigyeragyeranisibwa omu rulengo rw‘okushoroora. Ebirikumanyisa eby’embariirira ebyanguhi. Enkora neeshushana n’okushwijuma phylogenetic, okurikubaasa kukoresa okukuratana kw’ebintu kugyeragyeranisa omubaro gw’okuhinduka nari okuhinduka kw’emitwarize hatariho kubanza kuteebereza.
Aha rurengo rw‘eby‘amagara, enteekateeka y‘okushwijuma eya Firn na Jones (53) neeyoreka ngu okuhindura kw‘eby‘okurya nikugumaho aha rurengo rutari rumwe kuha ebintu ebikuru eby‘okukoresa emirimo y‘eby‘amagara ey‘eby‘okurya ebyabaire bitaine kakwate nari ebirikuhindurwa. Enkora z‘okuteekateeka aha makuru niziha omutindo ogu empindahinduka ezi z‘entunguuka z‘eby‘okurya ezirikubaho omu bwire bw‘okumanyisa eby‘okurya zirikubaasa kubarwa nk‘ekicweka ky‘enkora y‘okushwijuma entunguuka etebekanisiibwe: okuhindura kw‘eby‘amagara kuruga aha nkora y‘ahansi kuza aha nkora y‘ahaiguru Ebintu ebirikuhindura ebiri omu nkora ebirikuzibirwa eby‘omwanya oguhairwe.
Omubyona, omu biro by’okubanza eby’obuhangwa bw’ebimera, ebiteekateeko bikuru eby’okwerinda ebimera bikakorwa, kandi emiringo erikwebemberwa ebiteekateeko by’okucondooza (deductive hypothesis-driven methods) neetwarwa munonga nk’omuringo gwonka gw’okutunguuka omu bya sayansi. Eki nikireetwaho munonga oburemeezi bw‘eby‘obuhangwa bw‘okupima metabolome yoona. N‘obu emiringo erikwebemberwa ebiteekateeko eraabe eri ey‘omugasho munonga omu kutoorana emiringo endiijo y‘enshonga, obushoboorozi bwayo bw‘okutunguura okwetegyereza kwaitu aha nkora z‘eby‘amagara nibukye munonga kukira emiringo y‘okubara eriho hati omu sayansi y‘obunaku obu erikwejunisa amakuru maingi. Nahabwekyo, enteekateeka ezitarikubaasa kuteberezibwa nizikira munonga aha nkora y‘ebihandiiko ebiriho, n‘ahabw‘ekyo enkora y‘okuteekateeka/okugyezibwa kw‘entunguuka omu mwanya gw‘okucondooza terikubaasa kwihwaho (4). Nitureeba ngu omuringo gw‘okukora ogw‘okubara ogwa metabolomics ogwatandikirweho aha nigubaasa kugaruka kureetaho okukunda enshonga ezihererukireyo (oku) n‘ez‘aha muheru (ahabwenki) z‘okuhindura eby‘okurya, kandi guhwere omu bwire busya bwa sayansi y‘amakuru erikuhaburwa omu nteekateeka. Omurembe gukagaruka gwashwijuma enteekateeka nkuru ezaabaire nizihiga eminyeeto eyahingwireho.
Okuriisa kw’ebinyonyi ebirikurya ebishaka okuhikire nikukorwa omu kukuza enkwanzi ya kabiri eya instar nari enkwanzi ya Sl aha ibabi rimwe ry’ekimera ky’erangi erikutukura (Pale-colored pitcher plant) ry’ekimera kimwe ekirikumera ebimuri bya rose, n’ebimera 10 ebirikugarukagaruka ahari buri kimera. Enkwanzi z’ebinyonyi zikaba zikwatsirwe n’ebikwato, kandi enyama z’amababi ezaabaire zisigaireho zikaba zitorotoorwamu bwanyima y’eshaaha 24 na 72 z’okukwatwa endwara kandi zikaba zifukiirwa ahonaaho, kandi ebiriisa by’omubiri bikaihwaho.
Yegyeragyeranisa obujanjabi bw‘ebishaka omu muringo gw‘okuhikaana munonga. Omuringo n’okukozesa enziga z’omuringo gw’ebijwaro kucumita emirongooti eshatu y’amahwa ahari buri rubaju rw’omugati gw’ebibabi bishatu ebihikire eby’ekimera omu bwire bw’okukura kw’ekirungo ky’ebijwaro, kandi ahonaaho oteho 1:5 Diluted Ms. Nari okozese enkumu eziine giraavu kucumita S1 OS omu kucumita. Oshaaruure kandi okore eibabi nk’oku kyashoboororwa ahaiguru. Kozesa omuringo ogwashoboororwa enyima okushohoza ebiriisa by‘omubiri ebikuru n‘ebiriisa by‘ebimera (54).
Aha kukozesa JA ey’aheeru, amababi ashatu g’ebihimba bya buri kimwe aha bimera mukaaga ebirikumera ebimuri bya rose ebya buri kika nigajanjabibwa na 20μl y’omubazi gwa lanolin ogurimu 150μg MeJA (Lan + MeJA), na 20μl ya lanolin hamwe n’okujanjaba okukyebera ebihuta (Lan + la2), nari kukoresa purμl y’okukyebera ebihuta (Lan + la2). Amababi gakasharurwa bwanyima y’eshaaha 72 z’okugajanjaba, gakateebwa omu nkora ya snap-frozen omu liquid nitrogen, kandi gakabiikwa aha -80°C kuhitsya obu garikukoresibwa.
Emiringo ena ey’okuhindura ebiriisa bya JA na ET, niyo irAOC (36), irCOI1 (55), irACO na sETR1 (48), emanyirwe omu guruupu yaitu ey’okucondooza. irAOC ekooreka munonga okukyendeera omu rurengo rwa JA na JA-Ile, obwo irCOI1 etarikuhurira JAs. Kugyeragyeranisa na EV, okuteerana kwa JA-Ile kukeeyongyera. Omu muringo nigwo gumwe, irACO neija kukyendeeza aha kureetaho ET, kandi waagyeragyeranisa na EV, sETR1, etarikuhurira ET, neija kwongyera aha kureetaho ET.
Ekikwato ky‘okupima ebiri omu mubiri (Sensor Sense ETD-300 omu bwire bw‘amazima ET sensor) nikikoresibwa kukora okupima kwa ET hatariho kukwatwaho. Ahonaaho bwanyima y’okujanjaba, ekicweka kimwe kya kabiri ky’ebibabi bikasharaho kandi byatwarwa omu kikopo ky’endabirwamu ekikingire ekya miriyoni 4, kandi omwanya gw’omutwe gukaikirizibwa kwerundaana omu shaaha 5. Omu bwire bw‘okupima, buri nshaho ekaba eshemezibwa n‘omugyera gwa lita 2/eshaaha gw‘omuyaga ogushemeire kumara edakiika 8, ogwabaire guherize kuraba omu kikwato ekirikureetaho okukyendeeza aha nkora ekyaheebwayo Sensor Sense kwihaho CO2 n‘amaizi.
Ebihandiiko bya microarray bikabanza byashohozibwa omu (35) kandi byabiikwa omu kitongore ky’eihanga eky’amakuru g’eby’obuhangwa (NCBI) eky’okumanyisa ebiri omu nkora y’obuzaarwa (enamba y’okutunga GSE30287). Ebihandiiko ebirikuhikaana n’ebibabi ebyareetsirweho obujanjabi bwa W + OSMs n’okurinda okutaine kabi bikaihwa omu kucondooza oku. Obuhango bw’ebihimba ni log2. Okushwijuma kw‘embariirira kutakabaireho, omubaro gw‘okubanza gukahindurwa kandi gwahindurwa gwaba ogwa buriijo kuhika aha kicweka kyagwo kya 75 ahari igana barikukoresa puroguraamu ya R.
Ebihandiiko by’okubanza eby’okukuratana kwa RNA (RNA-seq) eby’ebika bya Nicotiana bikaruga omu bihandiiko bya NCBI eby’okushoma ebikye (SRA), enamba ya purojekiti ni PRJNA301787, eyaagambirweho Zhou na bagyenzi be. (39) kandi ogumizemu nk’oku kirikushoboororwa omu (56). Ebihandiiko ebitari birungi ebyakozirwe W + W, W + OSM na W + OSS1 ebirikuhikaana n’ebika bya Nicotiana bikatooranwa kukyeberwa omu kucondooza oku, kandi byakorwa omu muringo ogu: Eky’okubanza, okushoma kwa RNA-seq okutaine bugarukiro kukahindurwa omu muringo gwa FASTQ. HISAT2 neehindura FASTQ kuba SAM, kandi SAMtools neehindura fairo za SAM kuba fairo za BAM ezitebeekanisiibwe. StringTie neekoresibwa kubara okureebeka kw’obuzaarwa, kandi omuringo gwayo gw’okushohoza n’okugira ngu hariho ebicweka ahari buri rukumi rw’ebicweka by’omusingi ahari buri miriyoni y’ebicweka by’okuhandiika ebirikukuratana.
Enyomyo y’okukyebera erangi ya Acclaim (150 mm x 2.1 mm; obuhango bw’ebicweka 2.2μm) eyaakozesiibwe omu kucondooza n’enyomyo y’okurinda eya mm 4 x 4 mm erimu ekintu kimwe. Ekicweka eki nikikoresibwa omu nkora ya Dionex UltiMate 3000 Ultra Performance Liquid Chromatography (UHPLC): edakiika 0 kuhika ahari 0.5, isocratic 90% A [amaizi agatarimu ayoni, 0.1% (v/v) asetonitrile na B%toni acid na 0.05% ahabwa] 0.05% asidi y’omubazi); Edakiika 0.5 kuhika 23.5, omurundi gw‘okutunguuka ni 10% A na 90% B, nk‘oku biri; Eddakiika 23.5 kuhika 25, isocratic 10% A na 90% B. Omubaro gw‘okusheeshasheesha ni 400μl/edakiika. Aha kucondooza kwa MS kwona, ta omu nkora y‘okushwijuma ebiri omu nkora ya quadrupole n‘obwire bw‘okuguruka (qTOF) erimu enshonga y‘okufuuhirira amashanyarazi erikukora omu muringo gw‘okushemeza (amaani g‘okusheeshasheesha, 4500 V; okuruga kw‘okusheeshasheesha 130 V ; Obutagatsi bw‘okwoma 200 min aiters d10).
Kora okucondooza kw‘ebicweka bya MS / MS (aha muheru nikwetwa MS / MS) okutaine mugasho nari okutarikubaasa kutaanisibwa n‘ebihandiiko kutunga amakuru g‘enkora aha nkora y‘okuhindura eby‘okurya omu bwijwire erikureebeka. Enteekateeka y‘omuringo gwa MS/MS ogutarikushoroora neeyesiga aha mazima ngu quadrupole eine edirisa mpango munonga y‘okwetaanisa aha buremeezi [n‘ahabw‘ekyo, teekateeka aha bumanyiso bwona obw‘omubaro gw‘oburemeezi (m/z) nk‘ebicweka]. Ahabw‘enshonga egi, ahabw‘okugira ngu ekikwato kya Impact II kikaba kitarikubaasa kuhangaho okushendashenda kwa CE, okweshwijuma kw‘okwepimira kwingi kukakorwa barikukoresa okweyongyera kw‘okuteerana okurikureetwaho okuteerana kw‘amaani g‘okuteerana (CE). Omu bugufu, banza oshwijume eky‘okureeberaho orikukoresa UHPLC-electrospray ionization/qTOF-MS orikukoresa omuringo gw‘okupima obuhango bw‘ebintu (embeera z‘okucwanyagura eziri ahansi ezirikuruga omu kucwanyagura omu nshonga), okukyebera kuruga ahari m/z 50 kuhika ahari 1500 aha nkora y‘okugarukamu 5 Hz. Koresa nitrogen nk‘omuyaga gw‘okuteerana ahabw‘okushwijuma MS/MS, kandi okore ebipimo by‘okwepimira aha nkora ina ezi zitari zimwe na zimwe ezirikureetwaho okuteerana ezirikutaana: 20, 30, 40, na 50 eV. Omu nkora yoona ey‘okupima, quadrupole eine edirisa mpango y‘okwetaanisa aha buremeezi, kuruga ahari m/z 50 kuhika ahari 1500. Omubiri gw‘omu maisho m/z n‘okugyezesa kw‘obugazi bw‘okwetaanisaho kwaba kuteebwa ahari 200, orurengo rw‘oburemeezi nirukora kyonka ahabw‘okukoresa puroguraamu y‘okukoresa ekikwato na 0 Da. Sikaana ebicweka bya mass nk’omu muringo gwa mass gumwe. Koresa omubazi gwa sodium (miriyoni 50 z‘isopropanol, miriyoni 200 z‘omubazi gwa formic acid na miriyoni 1 z‘omubazi gw‘amaizi gwa NaOH 1M) ahabw‘okupima obuhango. Okukozesa enkora ya Bruker ey‘okugyezesa ey‘omutindo gw‘ahaiguru, fairo y‘amakuru neegyezesibwa bwanyima y‘okugyendera aha nkora ya spectrum eya purayimare omu bwire obuhairwe. Koresa omuringo gw’okushohoza puroguraamu y’okushwijuma amakuru v4.0 (Brook Dalton, Bremen, Bugirimaani) kuhindura fairo z’amakuru agataine nshonga omu muringo gwa NetCDF. Ekicweka ky‘amakuru kya MS/MS kibiikire omu nkora y‘okuhandiika ebiri omu nkora y‘eby‘amagara erikwetwa MetaboLights (www.ebi.ac.uk) n‘enamba y‘okutaahamu. 1471
Okuteerana kwa MS/MS nikubaasa kuhikirizibwa kurabira omu kucondooza aha nkoragana ahagati y‘obumanyiso bw‘omutindo gwa MS1 na MS/MS ahabw‘amaani g‘okuteerana g‘ahansi n‘ahaiguru n‘ebiragiro ebisya ebiteirwe omu nkora. Ekihandiiko kya R nikikoresibwa kumanyisa okushwijuma kw‘enkoragana y‘okugabwa kw‘ekirikubanza aha kihandiiko, kandi ekihandiiko kya C# (https://github.com/MPI-DL/indiscriminant-MS-MS-assembly-pipeline) nikikoresibwa kuta omu nkora ebiragiro.
Okubaasa kukyendeeza enshobi z‘ebishuba ezirungi ezirikureetwaho okuteera akaari k‘enyima n‘enkoragana y‘ebishuba erikureetwaho okushanga ebintu ebimwe ebya m/z omu sampo nkye zonka, nitukoresa omurimo gwa “enshonga eijwiire” ogwa R package XCMS (ahabw‘okutereeza okuteera akaari k‘enyima) Gushemerire kukoresibwa omu mwanya gwa “NA peuak) (entected peuak) . Omurimo gw‘okujwisa enshonga gwaba nigukoresibwa, hakiriho emihendo mingi y‘amaani “0′′ omu nkora y‘okuhandiika ebirikuza kukwata aha kubara kw‘enkoragana. Reero, nitugyeragyeranisa ebyarugamu omu kucondooza ebyatungirwe obu omuringo gw‘okujwisa enshonga gurikukoresibwa n‘obu omurimo gw‘okujwisa enshonga gutarikukoresibwa, kandi tubarira omuhendo gw‘enyima gw‘okuteera akaari ogwatebeekanisiibwe a espla the emihendo y‘amaani n‘omuhendo gw‘enyima ogwabariirwe Kandi tukateekateeka aha bintu byonka ebiine amaani agarikurenga emirundi eshatu aha muheendo gw‘enyima kandi twabitwara nk‘enshonda z‘enyima ez‘amazima. Aha kubara kwa PCC, obumanyiso bwa m/z bwonka obw‘eky‘okureeberaho ky‘okubanza (MS1) n‘ebicweka by‘amakuru ebiine haakiri enshonga munaana z‘amazima nibyo birikuteekateekwaho.
Obuhango bw‘omutindo gw‘okubanza omu sampo yoona bwaba nibukwatagana munonga n‘okukyendeeza kw‘amaani g‘omutindo nigwo gumwe ogurikuteganisibwa amaani g‘okuteerana makye nari maingi, kandi omutindo gutahandiikirweho nk‘enshonga y‘isotopu na CAMERA, nikibaasa kwongyera kushoboororwa. Bwanyima, omu kubara ebirikubaasika byona eby‘okubanza-ebirikurugamu bibiri omu 3 s (edirisa y‘obwire bw‘okurinda oburikuteberezibwa ahabw‘okurinda aha mutwe), okweshwijuma kw‘enkoragana nikukorwa. Omuhendo gwa m/z gwaba guri ahansi y‘omuhendo gw‘okubanza kandi okucwanyagura kwa MS/MS kurikubaho omu mwanya gumwe gw‘eky‘okureeberaho omu nkora y‘okucondooza nk‘eky‘okubanza eki kirikurugamu, niho gwonka ogurikutwarwa nk‘ekicweka.
Okurugirira aha biragiro ebi bibiri ebyanguhi, nitwihaho ebicweka ebimanyirwe ebiine emihendo ya m/z erikukira m/z y‘omubazi ogumanyirwe, kandi turikwegamira aha mwanya gw‘eky‘okureeberaho ahu omubazi gurikureebeka n‘ekicweka ekimanyirwe. Kandi nikibaasika kutoorana emitwarize y‘omutindo erikuruga omu bicweka bingi eby‘omu ntandikwa ebirikuruga omu muringo gwa MS1 nk‘ebirikubanza eby‘okuhakanisa, obwo birikureetaho ebirungo bya MS/MS ebitarikwetengyesa. Okubaasa kukyendeeza aha kugarukagaruka kw’ebihandiiko ebi, okushushana kwa NDP kw’ebicweka by’omubiri byarenga 0.6, kandi biri omu “pcgroup” ya chromatogram ehandiikirwe CAMERA, nitwija kubigaita. Eky‘aha muheru, nitugaita ebyarugamu byona bina ebya CE ebiine akakwate n‘ekirikubanza n‘ebicweka omu nkora y‘aha muheru ey‘okuteerana (deconvoluted composite spectrum) turikutoorana enshonga y‘amaani erikukirayo omu nshonga zoona ezirikwesimbaho ​​eziine omuhendo gwa m/z gumwe aha maani g‘okuteerana gatari gamwe na gamwe. Amatembezo g‘okukora agarikukuratira nigarugiirira aha nteekateeka ya composite spectrum kandi nigatwara omu nkora embeera za CE zitari zimwe na zimwe ezirikwetengyesa kutunguura okubaasa kw‘okucwanyagura, ahabw‘okuba ebicweka ebimwe nibibaasa kumanyirwa ahansi y‘amaani g‘okuteerana agamanyirwe gonka.
RDPI (30) ekakoresibwa kubara obutabaasa kureetaho enkora y‘okuhindura eby‘okurya. Obutashushana bw‘eby‘okurya (Hj index) niburuga omu bwingi bw‘ebintu ebirikubanza MS/MS barikukoresa Shannon entropy y‘okugabwa kw‘emirundi ya MS/MS barikukoresa enkora egi erikushoboororwa Martínez na bagyenzi be. (8). Hj = −∑i = 1mPijlog2(Pij) ahu Pij erikuhikaana n‘emirundi y‘okugyeragyeranisa ey‘omubaro gw‘eby‘okureeberaho bya j (j = 1, 2,..., m) (i = 1, 2, ..., m) t).
Metabolic specificity (Si index) neeshoboororwa nk‘okumanyisa kw‘okworeka kwa MS/MS erikuheebwa omu nkoragana n‘emirundi eri ahagati y‘eby‘okureeberaho ebirikuteekateekwaho. MS/MS obumanyiso nibubarwa nka Si = 1t (∑j = 1tPijPilog2PijPi)
Koresa enkora egi kupima omubaro gw‘eby‘okurya ebirimu ebiriisa bya metabolome δj index ya buri j sampo, n‘omubaro gw‘eby‘okurya bya MS/MS δj = ∑i = 1mPijSi .
Ebishushani bya MS/MS nibishushanisibwa omu bubiri bubiri, kandi okushushana nikubarwa kurugiirira aha bubonero bubiri. Eky‘okubanza, orikukoresa NDP ey‘omutindo (erikumanywa nk‘omuringo gw‘okuteerana kwa cosine), kozesa ekibaro eki ahaifo kutunga obubonero bw‘okushushana kw‘ekicweka ahagati y‘erangi NDP = (∑iS1 & S2WS1, iWS2, i) 2∑iWS1, i2∑iWS2, i2 ahu S1 na S2 nk‘oku i Okuhikaana, ahabw‘erangi nk‘oku, 1.1. i nikijwekyera oburemeezi oburikurugiirira aha nshonga y‘amaani ngu entaaniso y‘enshonga ya i-th eya buriijo ahagati y‘ebishushani bibiri eri ahansi ya 0.01 Da. Oburemeezi nibubarwa omu muringo ogu: W = [amaani g‘ahaiguru] m [omutindo] n, m = 0.5, n = 2, nk‘oku MassBank erikuhabura.
Enkora ya kabiri ey‘okuha obubonero ekateebwa omu nkora, eyabaire erimu okushwijuma NL erikugabwa ahagati ya MS/MS. Okubaasa kuhikiiriza eki, tukakoresa enkalala 52 za ​​NL ezirikukira kushangwa omu bwire bw‘omuringo gw‘okucwanyagura MS omu nkora y‘okucwanyagura, na NL erikukirayo (data fairo S1) eyaabaire ehandiikirwe enyima ahabw‘omubaro gwa MS/MS gw‘ebicweka by‘omubiri eby‘akabiri eby‘ebika bya Nepenthes ebiine amaani makye ( 9, 26). Taho vekita ya binary eya 1 na 0 ahari buri MS/MS, erikuhikaana n‘ebiriho hati n‘ebitariho bya NL ezimwe nk‘oku biri. Okurugirira aha kushushana kw’oburaingwa bwa Euclidean, obubonero bw’okushushana kwa NL nibubarwa ahari buri pair ya vectors za NL eza binary.
Okukora okuteerana kw‘ebicweka bibiri, tukakoresa ekipapura kya R DiffCoEx, ekirikurugiirira aha kwongyera aha kucondooza kw‘okumanyisa ebiri omu nkora y‘obuzaarwa (WGCNA). Twakoresa obubonero bwa NDP na NL obw‘okuha obubonero bwa MS/MS spectra, tukakoresa DiffCoEx kubara obubonero bw‘okugyeragyeranisa enkoragana. Okuteerana kw‘ebicweka bibiri nikukorwa orikutaho “cutreeDynamic” paramita aha nkora = “eky‘omutaano”, cutHeight = 0.9999, deepSplit = T, na minClusterSize = 10. Koodi y‘entandikwa ya R eya DiffCoEx ekaruga omu fairo y‘okwongyeraho 1 eyakozirwe Tesson na bagyenzi be. (57); Ekicweka kya puroguraamu ya R WGCNA erikwetengyesa nikibaasa kushangwa omu mukutu gwa intaneeti.
Okubaasa kukora okucondooza kw‘omutimbagano gwa molekyu za MS/MS, tukabara okutaahagana kw‘erangi z‘omubiri kurugiirira aha miringo y‘okushushana kwa NDP na NL, kandi twakoresa puroguraamu ya Cytoscape kureeba omu maisho topology y‘omutimbagano turikukoresa enkora y‘ebihangwa omu nkora y‘okutunguura enkora ya CyFilescape yFiles.
Koresa R version 3.0.1 kukora okucondooza kw‘embariirira aha byafaayo. Obukuru bw‘embariirira bukakyeberwa barikukoresa okucondooza kw‘emiringo ebiri (ANOVA), kukuratirwaho okukyebera kwa Tukey okwa bwanyima y‘okukyebera entaaniso y‘obwesigwa (HSD). Okubaasa kushwijuma entaaniso eri ahagati y‘okujanjaba eby‘okurya by‘ebishaka n‘okukyebera, okugabwa kw‘emikira ebiri kw‘ebicweka bibiri by‘eby‘okureeberaho ebiine entaaniso emwe kukakyeberwa barikukoresa ekigyezo kya Student‘s t.
Okumanya ebihandiiko by’okwongyeraho eby’ekihandiiko eki, reeba aha http://advances.sciencemag.org/cgi/ebirimu/omu bwijwire/6/24/eaaz0381/DC1.
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Enteekateeka y‘amakuru neeha esente z‘ensi yoona ez‘okugyeragyeranisa metabolomes z‘omutaano n‘okuteebereza enteekateeka z‘okwerinda kw‘okugyezibwa.
Enteekateeka y‘amakuru neeha esente z‘ensi yoona ez‘okugyeragyeranisa metabolomes z‘omutaano n‘okuteebereza enteekateeka z‘okwerinda kw‘okugyezibwa.
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