Inyigisho zitandukanye zo kurengera ibimera zitanga ubuyobozi bw'ingenzi mu gusobanura imiterere y'imikorere y'ibimera byihariye, ariko ubuhanuzi bwabyo bw'ingenzi buracyari kugeragezwa. Aha, twakoresheje isesengura rya tandem mass spectrometry (MS/MS) ridafite uruhande rushingiyeho kugira ngo dusuzume uburyo metabolome y'ubwoko bw'itabi buterwa n'ibimera bitandukanye kuva ku bimera bitandukanye kugeza ku moko n'ibindi binyabuzima bifitanye isano rya hafi, kandi twakoze ubushakashatsi bwinshi ku miterere y'ibintu bifatika bishingiye kuri spectra ivanze mu makuru. Urwego rwo kugerageza ubuhanuzi bw'ingenzi bw'ubwirinzi bwiza (OD) n'inyigisho zigenda zigana ku ntego (MT). Igice cy'amakuru cya metabolome y'ibimera gihuye n'inyigisho ya OD, ariko kinyuranyije n'ubuhanuzi bw'ingenzi bwa MT ku miterere ya metabolome iterwa n'ibimera. Kuva kuri micro kugeza ku gipimo cy'ihindagurika rya macro, ikimenyetso cya jasmonate cyagaragaye nk'ikintu nyamukuru kigena OD, mu gihe ikimenyetso cya ethylene cyatanze uburyo bwo kunoza igisubizo cyihariye cy'ibimera cyagaragajwe n'urusobe rw'uturemangingo rwa MS/MS.
Metabolite zidasanzwe zifite imiterere itandukanye ni zo zigize uruhare runini mu guhuza ibimera n’ibidukikije, cyane cyane mu kurinda abanzi (1). Gutandukana gutangaje kwa metabolite yihariye iboneka mu bimera byatumye habaho ubushakashatsi bwimbitse mu myaka myinshi ku mikorere yabyo myinshi mu bidukikije, kandi byashyizeho urutonde rurerure rw’inyigisho zo kwirinda ibimera, ari zo iterambere ry’ibidukikije n’iterambere ry’ibidukikije ry’ibimera n’udukoko. Ubushakashatsi bushingiye ku buhanga butanga ubuyobozi bw’ingenzi (2). Ariko, izi nyigisho zo kwirinda ibimera ntizakurikije inzira isanzwe yo gutekereza ku buryo bw’ibitekerezo, aho ibyahanuwe by’ingenzi byari ku rwego rumwe rw’isesengura (3) kandi byageragejwe mu buryo bw’igerageza kugira ngo biteze imbere uruziga rukurikira rw’iterambere ry’ibitekerezo (4). Inzitizi za tekiniki zigabanya ikusanyirizo ry’amakuru ku byiciro byihariye bya metabolite kandi zikuraho isesengura ryuzuye rya metabolite zihariye, bityo bikabuza igereranya ry’ibice bitandukanye ari ingenzi mu iterambere ry’ibitekerezo (5). Kutagira amakuru yuzuye ya metabolite n’amafaranga ahuriweho yo kugereranya imikorere y’uburyo metabolite ikorwa hagati y’amatsinda atandukanye y’ibimera bibangamira iterambere rya siyansi ry’urwego.
Iterambere riheruka mu rwego rwa tandem mass spectrometry (MS/MS) metabolomics rishobora kugaragaza neza impinduka za metabolike muri no hagati y’ubwoko bw’ubwoko runaka, kandi rishobora guhuzwa n’uburyo bwo kubara kugira ngo hamenyekane isano iri hagati y’iyi mvange ikomeye. Ubumenyi bwa kera bwa chimie (5). Guhuza ikoranabuhanga rigezweho mu isesengura no kubara bitanga urufatiro rukenewe rwo kugerageza igihe kirekire ibyahanuwe byinshi byakozwe n’inyigisho z’ibidukikije n’iz’ubwihindurize bw’ubutandukanye bwa metabolike. Shannon (6) yatangije inyigisho y’amakuru ku nshuro ya mbere mu nyandiko ye y’ingenzi mu 1948, ashyiraho urufatiro rw’isesengura ry’amakuru mu mibare, ryakoreshejwe mu nzego nyinshi zitari ikoreshwa ryayo rya mbere. Muri genomics, inyigisho y’amakuru yakoreshejwe neza kugira ngo hamenyekane amakuru ahoraho (7). Mu bushakashatsi bwa transcriptomics, inyigisho y’amakuru isesengura impinduka muri rusange muri transcriptom (8). Mu bushakashatsi bwabanje, twakoresheje urufatiro rw’ibarurishamibare rw’inyigisho y’amakuru kuri metabolike kugira ngo dusobanure ubuhanga bwa metabolike ku rwego rw’ingingo mu bimera (9). Aha, duhuza imikorere ishingiye kuri MS/MS n'icyerekezo cy'ibarurishamibare cy'inyigisho y'amakuru, irangwa n'ubusumbane bw'imikorere y'ibinyabuzima mu mafaranga asanzwe, kugira ngo tugereranye ibyahanuwe by'ingenzi by'inyigisho y'ubwirinzi bw'ibimera ku miterere y'ibinyabuzima iterwa n'ibimera.
Imiterere y’inyigisho zo kurinda ibimera ubusanzwe ihuriweho kandi ishobora kugabanywamo ibyiciro bibiri: ibyo kugerageza gusobanura ikwirakwizwa rya metabolite zihariye ku bimera bishingiye ku mikorere y’ubwirinzi, nko kwirwanaho neza (OD) (10), kwimuka kw’intego (MT) (11)) n’ishusho (12), mu gihe ibindi bishakisha ibisobanuro bya tekiniki ku buryo impinduka mu kuboneka k’umutungo zigira ingaruka ku mikurire y’ibimera no kwibumbira muri metabolite zihariye, nka karuboni: igitekerezo cyo kuringaniza intungamubiri (13), igitekerezo cy’igipimo cy’iterambere (14), n’igitekerezo cyo kuringaniza gukura no gutandukanya (15). Ibyiciro bibiri by’inyigisho biri ku rwego rutandukanye rw’isesengura (4). Ariko, inyigisho ebyiri zijyanye n’imikorere yo kwirwanaho ku rwego rw’imikorere ziganza ibiganiro ku bijyanye n’ubwirinzi bw’ibimera n’ubudahangarwa: inyigisho ya OD, ivuga ko ibimera bishora imari mu bwirinzi bwabyo buhenze bwa shimi gusa iyo bikenewe, urugero, iyo binyowe Iyo inyamaswa y’ibyatsi iteye, bityo, hakurikijwe ubushobozi bwo kugaba igitero mu gihe kizaza, ikintu gifite imikorere yo kwirwanaho gihabwa (10); Igitekerezo cya MT kivuga ko nta murongo w’impinduka za metabolite mu cyerekezo, ariko metabolite ihinduka ku buryo butunguranye, bityo bigatuma habaho uburyo bwo kuzitira "intego y'imikorere" y'ibimera birya ibyatsi. Mu yandi magambo, izi nyigisho ebyiri zitanga ubuhanuzi butandukanye ku bijyanye no kuvugurura imikorere ya metabolite bibaho nyuma y'igitero cy'ibimera birya ibyatsi: isano iri hagati yo kwirundanya kwa metabolite mu cyerekezo kimwe hamwe n'imikorere yo kwirwanaho (OD) n'impinduka za metabolite zitayobowe (MT) (11).
Ibitekerezo bya OD na MT ntibireba gusa impinduka ziterwa na metabolome, ahubwo binareba ingaruka z’ibidukikije n’iz’ihindagurika ry’iyi metabolite, nk’ikiguzi cyo guhangana n’ingaruka n’inyungu z’izi mpinduka mu mikorere y’umubiri mu bidukikije byihariye (16). Nubwo ibi bitekerezo byombi byemera imikorere yo kwirwanaho ya metabolite yihariye, ishobora kuba ihenze cyangwa idahenze, ubuhanuzi bw’ingenzi butandukanya ibitekerezo bya OD na MT buri mu cyerekezo cy’impinduka mu mikorere y’umubiri ziterwa. Iteganyagihe rya OD ryabonye igerageza rikomeye kugeza ubu. Ibi bizamini birimo kwiga imikorere y’ubwirinzi butaziguye cyangwa butaziguye bw’ingingo zitandukanye z’ibintu byihariye mu bidukikije no mu bidukikije, ndetse n’impinduka mu cyiciro cy’iterambere ry’ibimera (17-19). Ariko, kugeza ubu, kubera kutagira imikorere n’imiterere y’ibarurishamibare byo gusesengura ku isi hose ubwoko bw’ibinyabuzima, itandukaniro rikomeye hagati y’ibi bitekerezo bibiri (ni ukuvuga icyerekezo cy’impinduka mu mikorere y’umubiri) riracyagomba kugeragezwa. Hano, dutanga isesengura nk’iryo.
Kimwe mu bintu by'ingenzi biranga metabolite zihariye ku bimera ni ubudasa bwazo bukabije mu miterere yazo ku nzego zose kuva ku bimera bimwe, amoko kugeza ku bwoko bumwe (20). Impinduka nyinshi mu mibare mu metabolite zihariye zishobora kugaragara ku rwego rw'ubwinshi bw'ibinyabuzima, mu gihe itandukaniro rikomeye mu miterere rikunze kugaragara ku rwego rw'ubwoko (20). Kubwibyo, ubwinshi bw'ibimera mu mikorere ni cyo kintu cy'ingenzi mu budasa bw'imikorere, bigaragaza ubushobozi bwo guhuza n'uduce dutandukanye, cyane cyane utundi duce dufite ubushobozi butandukanye bwo kwibasirwa n'udukoko twihariye n'inyamaswa zisanzwe zirya ibimera (21). Kuva mu nyandiko ya Fraenkel (22) ivuga ku mpamvu zo kubaho kwa metabolite zihariye ku bimera, imikoranire n'udukoko dutandukanye byafashwe nk'ibitutu by'ingenzi byo guhitamo, kandi iyi mibanire ikekwa ko yahinduye imiterere y'ibimera mu gihe cy'ubwihindurize. Inzira ya metabolite (23). Itandukaniro hagati y'ubwoko mu budasa bw'ibimera byihariye rishobora no kugaragaza uburinganire bw'imiterere bujyanye no kwirinda ibimera bigizwe n'ibimera n'ingamba zo kurwanya ibimera, kuko ubwoko bubiri bukunze kuba bufite isano mbi hagati yabwo (24). Nubwo bishobora kuba ingirakamaro kugumana ubwirinzi bwiza igihe cyose, impinduka za metabolike ku gihe zijyanye no kwirwanaho zitanga inyungu zigaragara mu kwemerera ibimera gutanga umutungo w'agaciro ku zindi shoramari mu by'umubiri (19, 24), no kwirinda gukenera ubufatanye. Kwangirika kw'ingwate (25). Byongeye kandi, uku kuvugurura metabolike zihariye ziterwa n'udukoko twangiza ibimera bishobora gutuma habaho ikwirakwizwa ryangiza mu baturage (26), kandi bishobora kugaragaza impinduka zikomeye mu kimenyetso cya aside ya jasmonic (JA), gishobora kuguma mu baturage. Ibimenyetso bya JA byinshi n'ibiciriritse ni ubucuruzi hagati yo kwirwanaho ku bimera byangiza ibimera no guhangana n'ubwoko runaka (27). Byongeye kandi, inzira zihariye za metabolike zizatakaza kandi zihinduke vuba mu gihe cy'ubwihindurize, bigatuma metabolike ikwirakwizwa mu moko afitanye isano ya hafi (28). Izi polymorphisms zishobora gushyirwaho vuba bitewe n'impinduka mu miterere y'ibimera byangiza ibimera (29), bivuze ko ihindagurika ry'amatsinda y'ibimera byangiza ari ikintu cy'ingenzi gituma habaho itandukaniro mu mikorere ya metabolike.
Aha, twakemuye ibibazo bikurikira by'umwihariko. (I) Ni gute udukoko two mu bwoko bw'ibimera duhindura imiterere ya metabolome y'ibimera? (Ii) Ni ibihe bice by'ingenzi by'amakuru ajyanye n'imiterere ya metabolome bishobora gupimwa kugira ngo hamenyekane ibyahanuwe mu nyigisho y'ubwirinzi bw'igihe kirekire? (Iii) Niba ari ngombwa kuvugurura metabolome y'ibimera mu buryo bwihariye ku gitera, niba ari byo, ni uruhe ruhare imisemburo y'ibimera igira mu guhindura uburyo bwihariye bwo gusubiza metabolome, kandi ni izihe metabolome zigira uruhare mu kwihutisha imiterere y'ubwoko bw'ibinyabuzima? (Iv) Kubera ko ibyahanuwe n'inyigisho nyinshi zo kurinda bishobora kwaguka mu nzego zose z'ibinyabuzima, twabajije uburyo igisubizo cya metabolome giterwa gihuye kuva ku kugereranya imbere kugeza ku kugereranya ubwoko? Kugira ngo tubigereho, twize ku buryo bunonosoye metabolome y'amababi ya nikotine y'itabi, ikaba ari ikimera cy'icyitegererezo cy'ibidukikije gifite metabolisme yihariye, kandi gikora neza mu kurwanya inyo z'ibimera bibiri bisanzwe, Lepidoptera Datura (Ms) (Irakaze cyane, ikunze kuryoha). Kuri Solanaceae na Spodoptera littoralis (Sl), inzoka zo mu mababi y'ipamba ni ubwoko bwa "genus", hamwe n'ibimera bya Solanaceae n'ibindi binyabuzima byo mu yandi moko n'imiryango. Twasesenguye MS/MS metabolomics spectrum tunakuramo ibisobanuro by'ibarurishamibare by'amakuru kugira ngo tugereranye inyigisho za OD na MT. Kora amakarita yihariye kugira ngo hagaragazwe imiterere y'ibinyabutabire by'ingenzi. Isesengura ryagejejwe ku baturage kavukire ba N. nasi n'ubwoko bw'itabi bufitanye isano rya hafi kugira ngo dusesengure neza itandukaniro riri hagati y'ibimenyetso by'imisemburo y'ibimera n'ibyinjizwa bya OD.
Kugira ngo dufate ikarita rusange yerekeye ubuziranenge n'imiterere ya metabolome y'amababi y'itabi rikomoka ku bimera, twakoresheje uburyo bwo gusesengura no kubara bwakozwe mbere kugira ngo dukusanye neza kandi dukureho amakuru ya MS/MS spectra yigenga mu buryo bwo hejuru iva mu bimera (9). Ubu buryo butandujwe (bwitwa MS/MS) bushobora gukora spectra y'ibimera idasubirwaho, hanyuma igakoreshwa mu isesengura ryose ry'urwego rw'ibimera ryasobanuwe hano. Ibi bimera bitasubiwemo ni amoko atandukanye, agizwe n'amagana kugeza ku bihumbi bya metabolome (hafi 500-1000-s/MS/MS hano). Aha, dusuzuma ubuziranenge bw'ibimera mu buryo bw'amakuru, kandi dupima ubusumbane n'ubunyamwuga bwa metabolome dushingiye kuri Shannon entropy y'ikwirakwizwa ry'impinduka za metabolome. Dukoresheje formula yakoreshejwe mbere (8), twabaze urutonde rw'ibipimo bishobora gukoreshwa mu gupima ubusumbane bwa metabolome (ikimenyetso cya Hj), kwibanda ku miterere ya metabolome (ikimenyetso cya δj) n'imiterere ya metabolome imwe (ikimenyetso cya Si). Byongeye kandi, twakoresheje Relative Distance Plasticity Index (RDPI) kugira ngo dupime metabolome inducibility y'inyamaswa zirya inyama (Ishusho ya 1A) (30). Muri uru rwego rw'ibarurishamibare, dufata MS/MS spectrum nk'ishami ry'amakuru y'ibanze, kandi tugatunganya ubwinshi bwa MS/MS mu ikarita yo gukwirakwiza inshuro, hanyuma tugakoresha Shannon entropy kugira ngo tumenye ubusumbane bwa metabolome buva kuri yo. Isesengura rya metabolome ripimirwa n'ikigereranyo cy'umwihariko wa MS/MS imwe. Kubwibyo, ukwiyongera k'ubwinshi bwa zimwe mu nzego za MS/MS nyuma yo gukwirakwiza herbivore bihinduka inducibility ya spectral, RDPI na specialization, ni ukuvuga ukwiyongera k'ikimenyetso cya δj, kuko metabolites nyinshi zihariye zikorwa kandi hagakorwa igipimo cya Si kinini. Kugabanuka k'ikimenyetso cya Hj diversity index bigaragaza ko umubare wa MS/MS wakozwe ugabanuka, cyangwa ikwirakwizwa ry'ubwinshi bw'imiterere rihinduka mu cyerekezo kitari kimwe, mu gihe bigabanya ubusumbane bwabyo muri rusange. Binyuze mu kubara igipimo cya Si, birashoboka kugaragaza MS/MS iterwa n'inyamaswa zimwe na zimwe zirya inyama, ahubwo, MS/MS idasubiza iyo induction, ari cyo kimenyetso cy'ingenzi cyo gutandukanya MT na OD.
(A) Ibisobanuro by'ibarurishamibare bikoreshwa ku bimera (H1 kugeza kuri Hx) MS/MS-inducibility (RDPI), ubudasa (Hj index), umwihariko (δj index) n'imiterere ya metabolite (Si index). Ukwiyongera k'urwego rw'umwihariko (δj) bigaragaza ko, mu busanzwe, hazabaho metabolite nyinshi zikomoka ku bimera, mu gihe kugabanuka k'ubudasa (Hj) bigaragaza kugabanuka k'umusaruro wa metabolite cyangwa ikwirakwizwa ritari ryitezwe rya metabolite mu ikarita ikwirakwizwa. Agaciro ka Si gasuzuma niba metabolite ari yihariye ku kibazo runaka (aha, ibimera) cyangwa iguma ku rwego rumwe. (B) Ishusho y'ibitekerezo by'iteganyagihe ry'inyigisho y'ubwirinzi hakoreshejwe umurongo w'inyigisho y'amakuru. Inyigisho ya OD ihanura ko igitero cy'ibimera kizongera metabolite y'ubwirinzi, bityo kikongera δj. Muri icyo gihe, Hj iragabanuka kuko imiterere itunganywa ku kugabanuka k'amakuru ashingiye ku mikorere ya metabolite. Inyigisho ya MT ihanura ko igitero cy'ibimera kizatuma habaho impinduka zitari mu cyerekezo cya metabolite, bityo Hj ikaba ikimenyetso cy'ubwiyongere bw'amakuru ashingiye ku mikorere ya metabolite kandi bigatera ikwirakwizwa ritunguranye rya Si. Twanatanze igitekerezo cy’uburyo buvanze, MT nziza cyane, aho bimwe mu bintu bifite agaciro ko kwirwanaho byiyongera cyane (agaciro ka Si gakomeye), mu gihe ibindi bigaragaza ibisubizo bitunguranye (agaciro ka Si gake).
Dukoresheje ibisobanuro by’inyigisho z’amakuru, dusobanura inyigisho ya OD kugira ngo duhanure ko impinduka zidasanzwe za metabolite ziterwa n’ibimera mu buryo butatewe n’ibimera mu buryo budasanzwe zizatuma (i) kwiyongera k’imiterere yihariye ya metabolite (Si index) bitera imiterere yihariye ya metabolite (δj index) Kwiyongera kwa) amatsinda amwe yihariye ya metabolite afite agaciro ko hejuru k’ubwirinzi, na (ii) kugabanuka k’ubwinshi bwa metabolite (Hj index) bitewe n’impinduka mu gukwirakwiza ubwinshi bwa metabolite kugera ku gukwirakwiza kw’umubiri wa leptin. Ku rwego rwa metabolite imwe, hateganijwe ko ikwirakwizwa rya Si riteganyijwe, aho metabolite izongera agaciro ka Si hakurikijwe agaciro kayo k’ubwirinzi (Ishusho ya 1B). Muri uru rwego, dusobanura inyigisho ya MT kugira ngo duhanure ko gushyuha bizatuma (i) impinduka zitari mu cyerekezo mu metabolite zigabanuka bigatuma igipimo cya δj kigabanuka, na (ii) kwiyongera k’igipimo cya Hj bitewe n’ubwinshi bw’ibitagenda neza mu metabolite. Cyangwa guhindagurika, bishobora gupimwa na Shannon entropy mu buryo bw’ubwinshi rusange. Ku bijyanye n’imiterere ya metabolite, inyigisho ya MT izahanura ikwirakwizwa rya Si mu buryo butunguranye. Dukurikije ko metabolite zimwe na zimwe ziba mu bihe byihariye mu bihe byihariye, n'izindi zitaba mu bihe byihariye, kandi agaciro kazo k'ubwirinzi gaterwa n'ibidukikije, twanatanze icyitegererezo cy'ubwirinzi buvanze, aho δj na Hj bikwirakwizwamo kabiri kuri Si Inyongera mu mpande zose, amatsinda amwe ya metabolite, afite agaciro ko hejuru k'ubwirinzi, ni yo yonyine azazamura Si cyane cyane, mu gihe andi azaba afite ikwirakwizwa ritunguranye (Ishusho ya 1B).
Kugira ngo tugerageze uburyo bushya bwo guhanura inyigisho z’ubwirinzi ku murongo w’isesengura ry’inyigisho z’amakuru, twakusanyije inzobere (Ms) cyangwa generalist (Sl) z’ibimera ku mababi ya Nepenthes pallens (Ishusho ya 2A). Dukoresheje isesengura rya MS/MS, twakuyemo 599 MS/MS spectra idasubirwaho (dosiye y’amakuru S1) mu binyabutabire bya methanol byakusanyijwe nyuma yo kurya ibimera. Dukoresheje indexes za RDPI, Hj, na δj kugira ngo turebe uburyo amakuru asubirwamo muri dosiye za MS/MS agaragaza imiterere ishimishije (Ishusho ya 2B). Muri rusange, nk’uko byasobanuwe n’isesengura ry’amakuru, uko ibimera bikomeza kurya amababi, urwego rwo kuvugurura imikorere y’ibimera rugenda rwiyongera uko igihe kigenda: amasaha 72 nyuma y’uko ibimera birya, RDPI iriyongera cyane. Ugereranyije n’uburyo bwo kugenzura butarangiritse, Hj yagabanutse cyane, byatewe n’urwego rwo kwibanda ku miterere y’imikorere y’ibimera, rwapimwe n’imbonerahamwe ya δj. Iyi nzira igaragara ihuye n'ibyahanuwe muri iyo nyigisho ya OD, ariko ntihuye n'ibyahanuwe by'ingenzi muri iyo nyigisho ya MT, yemera ko impinduka zitunguranye (zitari iz'icyerekezo) mu rugero rwa metabolite zikoreshwa nk'uburyo bwo kwikingira (Ishusho ya 1B). Nubwo ingano y'ibikomoka ku gusohora ibimera mu kanwa (OS) n'imyitwarire yo kugaburira izi nyamaswa zombi bitandukanye, kugaburira kwazo mu buryo butaziguye byagize impinduka nk'izo mu cyerekezo cya Hj na δj mu gihe cy'amasaha 24 n'amasaha 72 yo gusarura. Itandukaniro ryonyine ryabaye mu masaha 72 ya RDPI. Ugereranyije n'ibyatewe no kugaburira Ms, metabolism rusange iterwa no kugaburira Sl yari hejuru.
(A) Igishushanyo mbonera cy’igerageza: ingurube isanzwe (S1) cyangwa impuguke (Ms) zirya ibyatsi zigaburirwa amababi y’ibimera byakuwemo umunyu, mu gihe ku bimera byifashishijwe mu buryo bwa "simulation", "OS" ya Ms (W + OSM) ikoreshwa mu guhangana n’ipfundo ry’ibibabi bisanzwe. S1 (W + OSSl) cyangwa "larvae" (W + W). "Control" (C) ni ikibabi kitangiritse. (B) "Inducibility" (RDPI) ugereranije n’imbonerahamwe y’igenzura), "diversity" (Hj index)" n’"specialization" (δj index) yashyizwe ku ikarita yihariye ya metabolite (599 MS / MS; dosiye y’amakuru S1). Inyenyeri zigaragaza itandukaniro rikomeye hagati yo kugaburira ibyatsi mu buryo butaziguye n’itsinda ry’igenzura ("Student's t-test" hamwe na "t-test", *P <0.05 na ***P <0.001). ns, ntabwo ari ngombwa. (C) Igipimo cy'igihe cy'ingenzi (agasanduku k'ubururu, aside amine, aside organic n'isukari; dosiye y'amakuru S2) na spectrum yihariye ya metabolite (agasanduku gatukura 443 MS/MS; dosiye y'amakuru S1) nyuma yo kuvurwa hifashishijwe imiti y'ibimera. Umugozi w'amabara ugaragaza igihe cy'icyizere cya 95%. Inyenyeri igaragaza itandukaniro rikomeye hagati y'ubuvuzi n'igenzura [isesengura rya quadratic ry'impinduka (ANOVA), rigakurikirwa n'itandukaniro rikomeye rya Tukey (HSD) ku igereranya ry'ibice byinshi nyuma ya hoc, *P<0.05, **P<0.01 na *** P<0.001]. (D) Kwibanda ku bice bya scatter na specifics zidasanzwe za metabolite (ibipimo bisubirwamo hamwe n'ubuvuzi butandukanye).
Kugira ngo dusuzume niba impinduka ziterwa n’ibimera ku rwego rwa metabolome zigaragarira mu mpinduka mu rwego rwa metabolite ku giti cyazo, twabanje kwibanda ku bimera byizweho mbere mu mababi ya Nepenthes pallens bifite ubushobozi bwo kurwanya ibimera. Amide za phenolic ni hydroxycinnamamide-polyamine conjugates zikusanya mu gihe cy’uburyo udukoko twangiza ibimera kandi bizwiho kugabanya imikorere y’udukoko (32). Twashakishije ibintu byabanjirije MS/MS bihuye maze dushushanya imiyoboro yabyo ya kinetic (Ishusho S1). Ntibitangaje ko ibikomoka kuri phenol bitagira uruhare rutaziguye mu kwirinda ibimera, nka aside chlorogenic (CGA) na rutin, bigabanuka nyuma y’ibimera. Mu buryo bunyuranye, ibimera bishobora gutuma phenol amide zikomera cyane. Gukomeza kugaburira ibimera bibiri byatumye habaho spectrum imwe ya phenolamides, kandi iyi shusho yari igaragara cyane ku ikorwa rya de novo rya phenolamides. Ibyo bizagaragara mu gihe cyo gusuzuma inzira ya 17-hydroxygeranyl nonanediol diterpene glycosides (17-HGL-DTGs), itanga umubare munini wa diterpenes zisanzwe zifite imikorere myiza yo kurwanya ibimera (33), muri zo Ms Feeding with Sl yateje imiterere isa (Ishusho S1)).
Ingaruka mbi zishobora kubaho mu igerageza ryo kugaburira inyamaswa zirya ibyatsi ni itandukaniro mu gihe cyo kurya amababi n'igihe cyo kugaburira inyamaswa zirya ibyatsi, bigatuma bigorana gukuraho ingaruka zihariye z'inyamaswa zirya ibyatsi ziterwa n'ibikomere n'inyamaswa zirya ibyatsi. Kugira ngo dukemure neza imiterere y'ubwoko bw'inyamaswa zirya ibyatsi ku bijyanye n'uburyo amababi abyangiza, twakoresheje uburyo bwo kugaburira inyamaswa za Ms na Sl duhita dushyira OS nshya yakusanyijwe (OSM na OSS1) ku gice gisanzwe cya W cy'amababi ahoraho. Ubu buryo bwitwa W + OS treatment, kandi bugena uburyo bwo kubyaza umusaruro hakoreshejwe igihe nyacyo igisubizo cyatanzwe n'inyamaswa zirya ibyatsi kitabangamiye ingaruka z'itandukaniro mu muvuduko cyangwa ingano y'ibisigazwa by'uturemangingo (Ishusho ya 2A) (34). Dukoresheje uburyo bwo gusesengura no kubara bwa MS/MS, twakuyemo 443 MS/MS spectra (dosiye y'amakuru S1), yahuriranye na spectra yakusanyijwe mbere mu igerageza ryo kugaburira ubwatsi. Isesengura ry’amakuru kuri iyi seti y’amakuru ya MS/MS ryagaragaje ko gusubiramo gahunda ya metabolome zihariye ku mababi hakoreshejwe kwigana ibyatsi byagaragaje ko OS itera imbaraga (Ishusho ya 2C). By’umwihariko, ugereranije n’ubuvuzi bwa OSS1, OSM yatumye habaho kongera ubumenyi bwa metabolome mu masaha 4. Ni ngombwa kumenya ko ugereranije n’amakuru y’igerageza ry’ibimera byakorewe ku buryo butaziguye, uburyo metabolic kinetics bwagaragaye mu mwanya w’ibice bibiri hakoreshejwe Hj na δj nk’ibihuza n’icyerekezo cya metabolome specialization mu gusubiza uburyo plantation yakozwe ku byatsi yakozwe uko igihe kigenda gihita. Kongera guhoraho (Ishusho ya 2D). Muri icyo gihe, twapimye ingano ya aside amine, aside organic n’isukari (dosiye y’amakuru S2) kugira ngo turebe niba uku kwiyongera kwa metabolome mu buhanga bwatewe no kuvugurura imiterere ya metabolisme ya karubone hagati mu gusubiza plantation yakozwe ku byatsi byakozwe (Ishusho ya S2). Kugira ngo dusobanure neza iyi miterere, twakomeje gukurikirana uburyo metabolic kinetics yavuzwe mbere ya phenolamide na 17-HGL-DTG. Uburyo bwo gutera inyama mu buryo bwihariye bwa OS buhinduka uburyo bwo guhinduranya ibintu mu buryo butandukanye muri metabolism ya phenolamide (Ishusho S3). Amide za phenolic zirimo coumarin na caffeoyl moieties ziterwa cyane na OSS1, mu gihe OSM zitera uburyo bwihariye bwo guteranya ferulyl conjugates. Ku nzira ya 17-HGL-DTG, uburyo butandukanye bwo guteranya OS hakoreshejwe malonylation na dimalonylation products byabonetse (Ishusho S3).
Hanyuma, twize uburyo bwo gukora transcriptome ishingiye kuri OS dukoresheje amakuru ya microarray yakoreshejwe mu gihe runaka, agaragaza ikoreshwa rya OSM mu kuvura amababi y'ibibabi by'ibimera bya rosette mu bimera. Uburyo bwo gukora sampling kinetics buhura cyane n'uburyo bwo gukora metabolome bukoreshwa muri ubu bushakashatsi bwa metabolome (35). Ugereranije no guhindura imiterere ya metabolome aho metabolic plasticity yiyongera cyane uko igihe kigenda gihita, tubona ko transcription bursts mu mababi iterwa na Ms, aho transcriptome inducibility (RDPI) na specialization (δj) biri kuri 1 Hari ubwiyongere bugaragara mu masaha, kandi ubudasa (Hj) muri iki gihe, igaragara rya BMP1 ryagabanutse cyane, hagakurikiraho kuruhuka kwa transcriptome specialization (Ishusho ya S4). Imiryango ya gene ya metabolism (nka P450, glycosyltransferase, na BAHD acyltransferase) igira uruhare mu gikorwa cyo guteranya metabolites zidasanzwe ziturutse mu bice by'imiterere bikomoka kuri metabolism y'ibanze, dukurikije icyitegererezo cyavuzwe haruguru cyo gukora high-specialization. Nk'inyigo, inzira ya phenylalanine yarasesenguwe. Isesengura ryemeje ko jeni z’ingenzi mu mikorere ya phenolamide ziterwa cyane na OS mu bimera birya inyama ugereranije n’ibimera bitakururwa, kandi zihuye neza mu mikorere yazo. MYB8 transcription factor na genes structural PAL1, PAL2, C4H na 4CL mu gice cyo hejuru cy’iyi nzira byagaragaje ko transcription itangizwa hakiri kare. Acyltransferases igira uruhare mu guteranya kwa nyuma kwa phenolamide, nka AT1, DH29, na CV86, igaragaza imiterere y’izamuka ry’ingufu (Ishusho S4). Ibyavumbuwe haruguru bigaragaza ko gutangira hakiri kare transcriptome specialization no kongera nyuma metabolomics specialization ari uburyo buhuriweho, bushobora guterwa na synchronous regulatory system itangira response ikomeye yo kwirwanaho.
Ivugurura ry’imiterere y’imisemburo y’ibimera rikora nk’urwego rugenzura ruhuza amakuru y’ibimera kugira ngo hakorwe gahunda y’imiterere y’ibimera. Nyuma yo kwigana ibyatsi, twapimye imiterere y’ibyiciro by’ingenzi by’imisemburo y’ibimera maze tubona uburyo bwo guhuza ibimera [Pearson correlation coefficient (PCC)> 0.4] (Ishusho ya 3A). Nkuko byari byitezwe, imisemburo y’ibimera ifitanye isano na biosynthesis ihuzwa mu rusobe rw’imisemburo y’ibimera. Byongeye kandi, imiterere y’ibimera (Si index) ishyirwa kuri uru rusobe kugira ngo hagaragazwe imisemburo y’ibimera iterwa n’uburyo butandukanye bwo kuvura. Ahantu habiri h’ingenzi hagaragazwa uburyo bwo gusubiza ibimera: kimwe kiri mu itsinda rya JA, aho JA (ifite imikorere yabyo mu buryo bw’ibinyabuzima JA-Ile) n’izindi derivatives za JA bigaragaza amanota menshi ya Si; ikindi ni ethylene (ET). Gibberellin yagaragaje ukwiyongera guciriritse k’imiterere y’ibimera, mu gihe izindi misemburo z’ibimera, nka cytokinin, auxin, na aside abscisic, zari zifite imiterere mike y’ibimera. Ugereranije no gukoresha W + W gusa, kwiyongera kw'agaciro gakomeye k'ibikomoka kuri JA binyuze muri porogaramu ya OS (W + OS) bishobora guhinduka ikimenyetso gikomeye cya JA. Mu buryo butunguranye, OSM na OSS1 bifite ibikomoka kuri encitor bitandukanye bizwiho gutera JA na JA-Ile kwegeranya kimwe. Bitandukanye na OSS1, OSM iterwa cyane na OSM, mu gihe OSS1 idatanga umusaruro mwinshi ku bikomere byo hasi (Ishusho ya 3B).
(A) Isesengura ry’urusobe rw’amakuru rishingiye ku kubara PCC ku buryo bwo gukusanya imisemburo y’ibimera biterwa n’ibyatsi. Aka gapfundo kagaragaza imisemburo imwe y’ibimera, kandi ingano y’aka gapfundo igaragaza igipimo cya Si cyihariye kuri imisemburo y’ibimera hagati y’uburyo ivurwa. (B) Kwiyongera kwa JA, JA-Ile na ET mu mababi biterwa n’uburyo butandukanye bwo kuvura bugaragazwa n’amabara atandukanye: apricot, W + OSM; ubururu, W + OSMl; umukara, W + W; imvi, C (igenzura). Inyenyeri zigaragaza itandukaniro rikomeye hagati yo kuvura no kugenzura (ANOVA y’uburyo bubiri ikurikirwa na Tukey HSD post hoc multiple comparison, *** P <0.001). Isesengura ry’amakuru kuri (C)697 MS/MS (dosiye y’amakuru S1) muri JA biosynthesis na ircoimplied perception spectrum (irAOC na irCOI1) na (D)585 MS/MS (dosiye y’amakuru S1) muri ETR1 hamwe n’ikimenyetso cya ET kibi. Uburyo bubiri bwo kuvura ibimera bwateje imirongo y’ibimera n’ibimera bigenzurwa n’imodoka (EV). Inyenyeri zigaragaza itandukaniro rinini hagati yo kuvura W+OS no kugenzura bitangiritse (ANOVA y'impande ebyiri ikurikirwa na Tukey HSD post hoc multiple comparison, *P<0.05, **P<0.01 na ***P<0.001). (E) Imbonerahamwe zitatanye zerekana uburyo butandukanye bwo guhangana n'ubuhanga. Amabara agaragaza ubwoko butandukanye bwahinduwe mu buryo bw'uturemangingo; ibimenyetso bigaragaza uburyo butandukanye bwo kuvura: mpandeshatu, W + OSS1; urukiramende, W + OSM; uruziga C
Hanyuma, dukoresha ubwoko bwa Nepenthes bwahinduwe mu buryo bwa genetiki (irCOI1 na sETR1) mu ntambwe z'ingenzi za JA na ET biosynthesis (irAOC na irACO) no kumva (irCOI1 na sETR1) kugira ngo dusesengure metabolism y'izi hormone ebyiri z'ibimera ku bimera byangiza ibinyabuzima. Uruhare rw'ivugurura ry'ibimera. Dukurikije igerageza ryabanje, twemeje ko herbivore-OS yashyizwe mu bimera bya empty carrier (EV) (Ishusho ya 3, C kugeza kuri D) ndetse no kugabanuka muri rusange k'igipimo cya Hj giterwa na OSM, mu gihe igipimo cya δj cyiyongereye. Igisubizo kiragaragara cyane kuruta igisubizo cyatewe na OSS1. Imbonerahamwe y'imirongo ibiri ikoresha Hj na δj nk'ibihuza igaragaza igabanuka ry'imikorere yihariye (Ishusho ya 3E). Icyerekezo kigaragara cyane ni uko mu bwoko budafite ikimenyetso cya JA, itandukaniro rya metabolome n'impinduka ziterwa n'ibimera byangiza ibinyabuzima hafi ya byose (Ishusho ya 3C). Ibinyuranye n'ibyo, uburyo ET igaragara bucecetse mu bimera bya sETR1, nubwo ingaruka rusange ku mpinduka mu mikorere y'ibimera birya ibyatsi ziri hasi cyane ugereranyije n'uburyo JA ikora, bigabanya itandukaniro mu bipimo bya Hj na δj hagati y'ibishyushya bya OSM na OSS1 (Ishusho ya 3D n'Ishusho ya S5). Ibi bigaragaza ko uretse imikorere y'ingenzi yo guhindura ibimenyetso bya JA, guhindura ibimenyetso bya ET binakora nk'uburyo bwo gutunganya neza uburyo ibinyabuzima birya ibyatsi byitabira imikorere yabyo. Bijyanye n'iyi mikorere yo gutunganya neza, nta mpinduka yabayeho mu buryo bwose bwo guhindura imikorere ya sETR1. Ku rundi ruhande, ugereranije n'ibimera bya sETR1, ibimera bya irACO byateje impinduka nk'izo muri rusange zatewe n'ibimera birya ibyatsi, ariko byagaragaje amanota atandukanye cyane ya Hj na δj hagati y'ibibazo bya OSM na OSS1 (Ishusho ya S5).
Kugira ngo tumenye metabolite zihariye zifite uruhare runini mu gusubiza inyamaswa zirya inyama no kunoza umusaruro wazo binyuze mu bimenyetso bya ET, twakoresheje uburyo bwa MS/MS bwari bwarateguwe mbere. Ubu buryo bushingiye ku buryo bwo gukusanya ibintu bibiri kugira ngo twongere dusuzume umuryango wa metabolite uhereye ku bice bya MS/MS [normalized dot product (NDP)] n'amanota asa hakurikijwe igihombo cya neutral (NL). Amakuru ya MS/MS yubatswe binyuze mu isesengura ry'imirongo ya ET transgenic yakozwe 585 MS/MS (dosiye y'amakuru S1), yakemuwe no kuyashyira mu byiciro birindwi by'ingenzi bya MS/MS (M) (Ishusho ya 4A). Zimwe muri izi modules zirimo metabolite zihariye zari zisanzwe zizwi: urugero, M1, M2, M3, M4 na M7 zikungahaye ku bintu bitandukanye bya phenol derivatives (M1), flavonoid glycosides (M2), acyl sugar (M3 na M4), na 17-HGL-DTG (M7). Byongeye kandi, amakuru yihariye ya metabolite (Si index) ya metabolite imwe muri buri module arabarirwa, kandi ikwirakwizwa ryayo rya Si rishobora kugaragara mu buryo bwihuse. Muri make, MS/MS spectra igaragaza imiterere y’ibimera byinshi n’imiterere ya genotype bigaragazwa n’agaciro ka Si kari hejuru, kandi imibare ya kurtosis igaragaza ikwirakwizwa ry’ubwoya ku mfuruka y’iburyo. Imwe muri iyo miterere ya colloid yoroheje yabonetse muri M1, aho phenol amide yagaragaje igice kinini cya Si (Ishusho ya 4B). Ingano ya 17-HGL-DTG yavuzwe mbere yakuwemo ibimera muri M7 yagaragaje amanota ya Si ari hagati, yerekana urwego rwo hagati rw’itandukaniro hagati y’ubwoko bubiri bwa OS. Mu buryo bunyuranye, metabolite nyinshi zikozwe mu buryo bwihariye, nka rutin, CGA, na acyl sugar, ziri mu manota make ya Si. Kugira ngo harebwe neza imiterere n’ikwirakwizwa rya Si hagati ya metabolite zihariye, hakozwe urusobe rwa molekile kuri buri module (Ishusho ya 4B). Igitekerezo cy'ingenzi cy'inyigisho ya OD (yavuzwe muri Figure 1B) ni uko kuvugurura imiterere ya metabolite zihariye nyuma y'ibimera bigomba gutuma habaho impinduka mu buryo bumwe mu metabolite zifite agaciro kanini ko kwirwanaho, cyane cyane binyuze mu kongera umwihariko wazo (bitandukanye no gukwirakwira mu buryo butunguranye) Uburyo) Metabolite yo kwirwanaho yahanuwe n'inyigisho ya MT. Inyinshi mu bikomoka kuri phenol byakusanyijwe muri M1 bifitanye isano n'imikorere y'udukoko (32). Mu kugereranya agaciro ka Si muri metabolite ya M1 hagati y'amababi yatewe n'amababi agize ibimera bigenzura EV mu masaha 24, twabonye ko imiterere ya metabolite nyinshi nyuma y'udukoko two mu bimera ifite icyerekezo gikomeye cyo kwiyongera (Figure 4C). Ukwiyongera kwihariye kw'agaciro ka Si kwabonetse gusa muri phenolides zo kwirwanaho, ariko nta kwiyongera kw'agaciro ka Si kwabonetse mu zindi fenoli na metabolite zitazwi zibaho muri iyi module. Iyi ni moderi yihariye, ifitanye isano n'inyigisho ya OD. Ibyerekeye impinduka mu mikorere ya metabolite ziterwa n'ibimera byo mu bimera birahuye. Kugira ngo tugerageze niba ubu buryo bwihariye bwa phenolamide spectrum bwatewe na OS-specific ET, twashushanyije metabolite Si index maze dutera itandukaniro hagati ya OSM na OSS1 muri genotypes za EV na sETR1 (Ishusho ya 4D). Muri sETR1, itandukaniro rya phenamide hagati ya OSM na OSS1 ryagabanutse cyane. Uburyo bwo gukusanya kabiri bwakoreshejwe kandi ku makuru ya MS/MS yakusanyijwe mu moko adafite JA ihagije kugira ngo hamenyekane modules z'ingenzi za MS/MS zijyanye na JA-regulated metabolic specialization (Ishusho ya S6).
(A) Ibisubizo bya 585 MS/MS bishingiye ku gice cyasangiwe (NDP similarity) n'igihombo cyasangiwe (NL similarity) bituma module (M) ihura n'umuryango w'ibice bizwi, cyangwa bitewe n'imiterere ya Metabolite itazwi cyangwa idatunganyijwe neza. Iruhande rwa buri module, hagaragara ikwirakwizwa rya metabolite (MS/MS) yihariye (Si). (B) Urusobe rw'amamolekile ya modular: Nodes zigaragaza MS/MS n'impande, amanota ya NDP (umutuku) na NL (ubururu) ya MS/MS (igabanywa,> 0.6). Igipimo cya metabolite specificiality index (Si) cyashyizwe mu ibara hashingiwe kuri module (ibumoso) kandi gishushanyijwe ku rusobe rw'amamolekile (iburyo). (C) Module M1 y'ikimera cya EV mu buryo buhuriweho (control) na induced state (simulated herbivore) mu masaha 24: imbonerahamwe y'urusobe rw'amamolekile (Si value ni node, defensive phenolamide igaragara mu ibara ry'ubururu). (D) Ishusho y'urusobe rw'amakuru ya M1 y'umurongo wa spectrum sETR1 ufite ikibazo cyo kumva EV na ET: imvange ya phenolic igaragazwa n'agace k'uruziga rw'icyatsi kibisi, n'itandukaniro rikomeye (agaciro ka P) hagati yo kuvurwa na W + OSM na W + OSS1 nk'ingano ya Node. CP, N-caffeoyl-tyrosine; CS, N-caffeoyl-spermidine; FP, N-ferulic aside ester-uric aside; FS, N-ferulyl-spermidine; CoP, N', N “-Coumarolyl-tyrosine; DCS, N', N”-dicaffeoyl-spermidine; CFS, N', N”-caffeoyl, feruloyl-spermidine; Lycium barbarum muri wolfberry Son; Nick. O-AS, O-acyl isukari.
Twakomeje kwagura isesengura kuva kuri genotype imwe ya Nepenthes yagabanutse kugeza ku miterere karemano, aho impinduka zikomeye mu rugero rwa JA rukomoka ku bimera n'urwego rwihariye rwa metabolite zavuzwe mbere mu miterere karemano (26). Koresha iyi mibare kugira ngo urebe imiterere ya germplasm 43. Izi germplasm zigizwe n'ubwoko 123 bw'ibimera bivuye muri N. pallens. Ibi bimera byakuwe mu mbuto zakusanyijwe mu duce dutandukanye tw'ibinyabuzima muri Utah, Nevada, Arizona, na Kaliforuniya (Ishusho S7), twabaruye imiterere ya metabolome (aha hitwa urwego rw'abaturage) β diversity) n'umwihariko uterwa na OSM. Dukurikije ubushakashatsi bwabanje, twabonye impinduka nyinshi za metabolite ku miterere ya Hj na δj, bigaragaza ko germplasm zifite itandukaniro rikomeye mu buryo imikorere yazo ya metabolite ku bimera birya ibyatsi (Ishusho S7). Iyi gahunda yibutsa ibyabanje ku birebana n'imiterere ya JA iterwa n'ibimera birya ibyatsi, kandi yakomeje kugira agaciro kanini cyane mu mubare umwe (26, 36). Mu gukoresha JA na JA-Ile kugira ngo tugerageze isano rusange hagati ya Hj na δj, twasanze hari isano ikomeye hagati ya JA n'ikimenyetso cy'ubusumbane bwa metabolome β n'umwihariko (Ishusho S7). Ibi bigaragaza ko ubusumbane buterwa n'ibimera mu gutera JA bugaragara ku rwego rw'abaturage bushobora guterwa na polymorphisms z'ingenzi za metabolisme ziterwa no gutoranya udukoko twangiza inyamaswa.
Ubushakashatsi bwakozwe mbere bwagaragaje ko ubwoko bw'itabi butandukanye cyane mu bwoko bwaryo no mu buryo rishingira ku bwishingizi bw'umubiri buterwa n'uburyo rikoreshwa mu kurwanya indwara. Bikekwa ko izi mpinduka mu gutanga ibimenyetso byo kurwanya indwara n'ubushobozi bwo kwirinda zigengwa n'umuvuduko w'udukoko, ubuzima bw'ibimera, n'ikiguzi cyo gukora ubwirinzi mu gace runaka aho ubwoko runaka bukurira. Twize ku buryo metabolome y'amababi ihinduka bitewe n'ibimera by'ubwoko butandatu bwa Nicotiana bukomoka muri Amerika ya Ruguru na Amerika y'Epfo. Ubwo bwoko bufitanye isano rya hafi na Nepenthes yo muri Amerika ya Ruguru, ari yo Nicolas Bociflo. La, N. nicotinis, Nicotiana n. ibyatsi biremereye, Nicotiana tabacum, itabi rigororotse, itabi (Nicotiana spegazzinii) n'itabi ry'amababi y'itabi (Nicotiana obtusifolia) (Ishusho ya 5A) (37). Ubwoko butandatu muri ubu bwoko, harimo n'ubwoko buzwi neza bwa N. please, ni ibimera by'umwaka bya petunia clade, naho obtusifolia N. ni ibimera bihoraho bya sister clade Trigonophyllae (38). Nyuma yaho, W + W, W + OSM na W + OSS1 byakozwe kuri ubu bwoko burindwi kugira ngo higwe uburyo bwo kuvugurura imikorere y’uturemangingo mu kugaburira udukoko.
(A) Igiti cya phylogenetic cya bootstrap gishingiye ku bushobozi ntarengwa [bwo gukora glutamine ya kirimbuzi (38)] n'aho ubwoko bw'amoko arindwi ya Nicotiana ahuriye (amabara atandukanye) (37). (B) Urutonde rw'ubwoko bwihariye bw'ubwoko ... Agasanduku kerekana (D) ubwinshi bw'ibinyabuzima na (E) kwibanda kuri buri bwoko nyuma yo kwigana ibyatsi na methyl JA (MeJA). Akanyenyeri kerekana itandukaniro rinini hagati ya W + OS na W + W cyangwa lanolin hamwe na W (Lan + W) cyangwa Lan hamwe na MeJA (Lan + MeJa) na Lan control (isesengura ry’uburyo bubiri bw’ubwihindurize, rikurikirwa na Tukey's HSD post hoc multiple comparison, *P <0.05, **P <0.01 na ***P <0.001).
Dukoresheje uburyo bwa "dual cluster", twabonye module 9 za 939 MS/MS (dosiye y'amakuru S1). Imiterere ya MS/MS yongeye guhindurwa hakoreshejwe uburyo butandukanye bwo kuvura iratandukanye cyane hagati ya module zitandukanye hagati y'ubwoko (Ishusho S8). Kureba Hj (ivugwa hano nka γ-diversity) na δj bigaragaza ko ubwoko butandukanye buhurira mu matsinda atandukanye cyane mu mwanya wa metabolism, aho igabanywa ry'ubwoko rikunze kugaragara cyane kuruta gushyuha. Uretse N. linear na N. obliquus, zigaragaza ingaruka nyinshi z'uburyo bwo kuvura (Ishusho ya 5B). Mu buryo bunyuranye, ubwoko nka N. purpurea na N. obtusifolia bufite uburyo metabolic response idasobanutse neza ku buvuzi, ariko metabolome iratandukanye cyane. Ikwirakwizwa ry'ubwoko bw'uburyo metabolism response yatewe ryatumye habaho isano iri hagati y'umwihariko n'uburyo gamma diversity (PCC = -0.46, P = 4.9×10-8). Impinduka ziterwa na OS mu rugero rwa JA zifitanye isano ryiza n’ubwiharike bwa metabolome, kandi zifitanye isano mbi n’ubwiyongere bwa gamma mu mikorere y’umubiri bugaragazwa na buri bwoko (Ishusho ya 5B n’Ishusho ya S9). Ni ngombwa kumenya ko ubwoko bwitwa ubwoko bwa "signal response" mu Ishusho ya 5C, nka Nepenthes nematodes, Nepenthes nepenthes, Nepenthes acute, na Nepenthes zagabanutse, byateje ibimenyetso bikomeye mu minota 30. Icyorezo cya vuba aha cya JA na JA-Ile byihariye muri OS, mu gihe izindi bagiteri zitwa "signal response", nka Nepenthes mills, Nepenthes powdery na N. obtusifolia zigaragaza gusa JA-Ile Edge induction nta buryo bwihariye bwa OS (Ishusho ya 5C). Ku rwego rwa metabolisme, nk'uko byavuzwe haruguru, kuri Nepenthes zagabanutse, ibintu bisubiza ibimenyetso byagaragaje umwihariko wa OS kandi byongera cyane δj, mu gihe bigabanya Hj. Iyi ngaruka ya OS yihariye ntiyabonetse mu bwoko bwashyizwe mu bwoko bwa signal non-reactive species (Ishusho ya 5, D na E). Metabolite zihariye za OS zikunze gusangirwa hagati y’ubwoko butanga ibimenyetso, kandi izi tsinda ry’ibimenyetso rihuriweho n’ubwoko bufite ibimenyetso bidakomeye, mu gihe ubwoko bufite ibimenyetso bidakomeye bugaragaza isano nke (Ishusho S8). Ibi bisubizo bigaragaza ko imikorere ya OS ya JAs n’imiterere ya OS ya metabolome iri hasi bihujwe ku rwego rw’ubwoko.
Hanyuma, twakoresheje paste ya lanolin irimo methyl JA (MeJA) kugira ngo tuvure ibimera kugira ngo turebe niba ubwo buryo bwo guhuza bubangamiwe no kuboneka kwa JA ikoreshwa na JA yo hanze, izaba iri muri cytoplasm y'ibimera. Guhinduka vuba kw'ibimera ni JA. Twasanze hari impinduka imwe y'ubwoko butitabira ibimenyetso bujya ku bwoko butitabira ibimenyetso buterwa no gukomeza gutanga JA (Ishusho ya 5, D na E). Muri make, uburyo bwo kuvura MeJA bwahinduye cyane metabolome za linear nematodes, N. obliquus, N. aquaticus, N. pallens, na N. mikimotoi, bituma δj yiyongera cyane kandi Hj. N. purpurea igabanuka gusa, ariko Hj. N. obtusifolia, yagaragaye ko yiyongera cyane muri δj, nayo ikira nabi mu kuvura MeJA mu bijyanye no kuvugurura metabolome. Ibi bisubizo bigaragaza ko umusaruro wa JA cyangwa uburyo bwo kuvura ibimenyetso bigabanuka mu buryo bw'umubiri mu bwoko butitabira ibimenyetso. Kugira ngo tugerageze iki gitekerezo, twize ubwoko bune (N. pallens, N. mills, N. pink na N. microphylla) buterwa na W + W, W + OSM na W + OSS1 Transcriptome (39). Dukurikije imiterere yo kuvugurura metabolome, ubwoko butandukanye neza mu mwanya wa transcriptome, muri bwo N. attenuated yagaragaje RDPI iterwa na OS nyinshi cyane, mu gihe N. gracilis yari ifite hasi cyane (Ishusho ya 6A). Ariko, byagaragaye ko ubwinshi bwa transcriptome buterwa na N. oblonga bwari hasi cyane mu bwoko bune, bitandukanye n'ubwinshi bwa metabonomike bwa N. oblonga bwagaragajwe mbere mu bwoko burindwi. Ubushakashatsi bwakozwe mbere bwagaragaje ko itsinda ry'uturemangingo dufitanye isano n'ibimenyetso byo kwirwanaho hakiri kare, harimo n'ibimenyetso bya JA, bisobanura umwihariko w'uburyo bwo kwirwanaho hakiri kare buterwa n'ibikomoka ku bimera mu bwoko bwa Nicotiana (39). Kugereranya inzira zo kwitabazwa kwa JA hagati y'ubu bwoko bune byagaragaje imiterere ishimishije (Ishusho ya 6B). Inyinshi mu ngirabuzimafatizo muri ubu buryo, nka AOC, OPR3, ACX na COI1, zagaragaje urwego rwo hejuru rw’ubwiyongere muri ubwo bwoko bune. Ariko, gene y’ingenzi, JAR4, ihindura JA mu buryo bwayo bw’ibinyabuzima bw’inyandiko zakusanyijwe za JA-Ile, kandi urwego rwayo rwo kwandika ruri hasi cyane, cyane cyane muri N. mills, Nepenthes pieris na N. microphylla. Byongeye kandi, inyandiko y’indi ngirabuzimafatizo ya AOS ni yo yonyine yabonetse muri N. bifidum. Izi mpinduka mu miterere ya gene zishobora kuba nyirabayazana w’imiterere ikabije iterwa n’umusaruro muke wa JA mu bwoko bwa signal anergic hamwe n’ubwiyongere bwa N. gracilis.
(A) Isesengura ry’amakuru ku ivugurura ry’ibisubizo bya mbere by’ubwoko bune bw’itabi bufitanye isano rya hafi byafashwe mu minota 30 nyuma yo kwinjizwa mu byatsi. RDPI ibarwa hagereranywa amababi yatewe na OS y’ibyatsi n’uburyo bwo kugenzura ibikomere. Amabara agaragaza ubwoko butandukanye, kandi ibimenyetso bigaragaza uburyo butandukanye bwo kuvura. (B) Isesengura ry’imvugo ya gene mu nzira zo gutanga ibimenyetso bya JA mu bwoko bune. Inzira ya JA yoroshye igaragara iruhande rw’agasanduku. Amabara atandukanye agaragaza uburyo butandukanye bwo gutunganya. Inyenyeri igaragaza ko hari itandukaniro rinini hagati yo kuvura W + OS n’uburyo bwo kugenzura W + W (ku ikizamini cya t cy’Umunyeshuri ku itandukaniro ry’ibice bibiri, *P <0.05, **P <0.01 na ***P <0.001). OPDA, aside 12-oxophytodienoic; OPC-8: aside 0,3-oxo-2(2′(Z)-pentenyl)-cyclopentane-1-octanoic.
Mu gice cya nyuma, twize uburyo udukoko two mu bwoko butandukanye bw’udukoko dushobora kuvugurura metabolome y’ibimera bitandukanye bishobora kurwanya ibimera birya ibyatsi. Ubushakashatsi bwabanje bwibanze ku bwoko bwa Nicotiana. Ubudahangarwa bwabyo kuri Ms n’ibihumyo bitandukanye cyane (40). Aha, twize isano iri hagati y’ubu buryo n’uburyo imikorere yabyo ihinduka. Dukoresheje ubwoko bune bw’itabi bwavuzwe haruguru, kandi tugerageza isano iri hagati y’ubudahangarwa n’umwihariko wa metabolome iterwa n’ibimera birya ibyatsi ndetse n’ubudahangarwa bw’ibimera kuri Ms na Sl, twasanze ubudahangarwa, ubudahangarwa n’umwihariko kuri Sl All rusange bifitanye isano nziza, mu gihe isano iri hagati yo kudahangarwa n’abagore b’inzobere n’umwihariko ari nke, kandi isano iri hagati y’ubudahangarwa n’ubwoko ntabwo igaragara (Ishusho S10). Ku bijyanye no kudahangarwa na S1, N. chinensis na N. gracilis zombi zagabanutse, zagaragaye mbere ko zigaragaza urugero rwa JA signal transduction na metabolome plasticity, zari zifite ibisubizo bitandukanye cyane ku buryo bwo kudahangarwa n’ibimera, kandi zagaragaje ubudahangarwa bungana. Igitsina.
Mu myaka mirongo itandatu ishize, inyigisho yo kurengera ibimera yatanze urufatiro rw’inyigisho, aho abashakashatsi bahanuye umubare munini w’impinduka n’imikorere y’ibimera byihariye. Inyinshi muri izi nyigisho ntizikurikiza inzira isanzwe y’ibitekerezo bikomeye (41). Batanga ibitekerezo by’ingenzi (3) ku rwego rumwe rw’isesengura. Iyo igerageza ry’ibyahanuwe by’ingenzi ryemerera inyigisho runaka gusesengurwa, bizatuma urwego rukomeza gutera imbere. Fata inkunga, ariko uhakane izindi (42). Ahubwo, inyigisho nshya itanga ibitekerezo ku nzego zitandukanye z’isesengura kandi yongeraho urwego rushya rw’ibitekerezo bisobanuye (42). Ariko, inyigisho ebyiri zatanzwe ku rwego rw’imikorere, MT na OD, zishobora gusobanurwa byoroshye nk’ubuhanuzi bw’ingenzi bw’impinduka zihariye za metabolike ziterwa n’ibimera: inyigisho ya OD yemera ko impinduka mu "mwanya" wihariye wa metabolike zigana icyerekezo gikomeye. Inyigisho ya MT yemera ko izi mpinduka zitari mu cyerekezo kandi zigashyirwa mu mwanya wa metabolike, kandi zikunze kugira metabolike ifite agaciro kanini k’ubwirinzi. Isuzuma ryabanje ry’ibyahanuwe bya OD na MT ryageragejwe hakoreshejwe itsinda rito ry’ibintu bya mbere byitwa "ubwirinzi". Ibi bizamini bigamije gusuzuma ingano n'inzira byahinduye imiterere ya metabolome mu gihe cyo kuvura ibimera, kandi ntibituma ibizamini mu buryo buhamye bw'ibarurishamibare bisaba guhanura ibintu by'ingenzi bishobora gufatwa muri rusange. Gupima impinduka mu mikorere ya metabolome y'ibimera. Aha, twakoresheje ikoranabuhanga rishya mu mikorere ya metabolome dushingiye kuri MS y'amakuru kandi dukora isesengura rya MS ryo gukuraho ihindagurika mu buryo rusange bw'amakuru kugira ngo tugerageze itandukaniro riri hagati y'ibyo bibiri byatanzwe ku rwego rw'isi rwa metabolome. Igitekerezo cy'ingenzi cy'iyi nyigisho. Inyigisho y'amakuru yakoreshejwe mu nzego nyinshi, cyane cyane mu rwego rw'ubushakashatsi ku binyabuzima n'imirire (43). Ariko, uko tubizi, iyi ni yo porogaramu ya mbere ikoreshwa mu gusobanura umwanya w'amakuru ku bimera no gukemura ibibazo by'ibidukikije bijyanye n'impinduka z'igihe gito mu gusubiza ibimenyetso by'ibidukikije. By'umwihariko, ubushobozi bw'ubu buryo buri mu bushobozi bwabwo bwo kugereranya imiterere iri hagati y'ubwoko bw'ibimera no gusuzuma uburyo ibimera byahindutse bivuye mu bwoko butandukanye bikagera ku mikorere ya macroevolutionary mu byiciro bitandukanye by'ubwihindurize. Metabolism.
Isesengura ry’ibice by’ingenzi (PCA) rihindura amakuru yashyizwe mu buryo butandukanye mu mwanya wo kugabanya ingano kugira ngo icyerekezo cy’ingenzi cy’amakuru gishobore gusobanurwa, bityo akenshi rikoreshwa nk'uburyo bwo gusesengura amakuru, nko gukuraho metabolome. Ariko, kugabanya ingano bizatakaza igice cy’amakuru ari mu makuru, kandi PCA ntishobora gutanga amakuru y’imibare yerekeye imiterere ijyanye cyane n’inyigisho z’ibidukikije, nko: uburyo inyamaswa zirya inyama zihindura imiterere y’ubusumbane mu nzego zihariye (urugero, ubukungu, ikwirakwizwa) N’ubwinshi) ibinyabuzima bikomoka kuri ibinyabuzima? Ni ibihe bintu bikomoka kuri ibinyabuzima bikomoka kuri inyamaswa bigena imiterere y’inyamaswa runaka? Ukurikije uburyo bwihariye, ubwinshi n’ubushobozi bwo kubyaza umusaruro, amakuru ari mu buryo bwihariye bw’ibinyabuzima bikomoka ku mababi arangirika, kandi byagaragaye ko kurya inyamaswa zikomoka kuri inyamaswa bishobora gutuma imikorere y’ibinyabuzima ihinduka. Mu buryo butunguranye, twabonye ko, nk'uko byasobanuwe mu bipimo by'amakuru byashyizwe mu bikorwa, imiterere y'imikorere y'umubiri ihinduka cyane nyuma y'ibitero by'inyamaswa ebyiri zirya inyama (Sl isanzwe irya nijoro) n'impuguke ya Solanaceae Madamu. Nubwo imyitwarire yazo yo kurya n'uburyo zishyira mu byiciro bitandukanye cyane. Itangiriro rya aside amine (FAC) muri OS (31). Mu gukoresha OS y'ibimera mu kuvura ibikomere bisanzwe, ubuvuzi bwakozwe mu buryo bwakozwe bwagaragaje icyerekezo nk'icyo. Ubu buryo busanzwe bwo kwigana uburyo ibimera byitwara ku bitero by'ibimera birya inyama bukuraho ibintu bitesha umutwe biterwa n'impinduka mu myitwarire yo kurya kw'inyamaswa zirya inyama, bigatera kwangirika gutandukanye mu bihe bitandukanye (34). FAC, izwiho kuba impamvu nyamukuru ya OSM, igabanya JAS n'izindi misemburo y'ibimera muri OSS1, mu gihe OSS1 igabanya inshuro amagana (31). Ariko, OSS1 yateje urugero nk'urwo rwo kwikusanya kwa JA ugereranije na OSM. Byagaragaye mbere ko igisubizo cya JA muri Nepenthes zangiritse kigira ingaruka zikomeye kuri OSM, aho FAC ishobora gukomeza gukora kwayo nubwo yavangwa na 1:1000 hamwe n'amazi (44). Kubwibyo, ugereranije na OSM, nubwo FAC muri OSS1 ari nke cyane, irahagije kugira ngo itere indwara ya JA ihagije. Ubushakashatsi bwabanje bwagaragaje ko poroteyine zisa na porin (45) na oligosaccharides (46) zishobora gukoreshwa nk'ibimenyetso bya molekile mu gutera imbaraga zo kwirinda ibimera muri OSS1. Ariko, ntibiramenyekana neza niba izi exitors muri OSS1 ari zo zitera JA yiyongera muri ubu bushakashatsi.
Nubwo hari ubushakashatsi buke bugaragaza itandukaniro ry’ibikumwe by’umubiri biterwa no gukoresha inyamaswa zitandukanye zirya ibyatsi cyangwa JA cyangwa SA (aside salicylic) (47), nta wigeze abangamira imiterere y’ubwoko bw’inyamaswa zirya ibyatsi mu rusobe rw’ibyatsi n’ingaruka zabyo ku buryo bwihariye. Ingaruka rusange z’imikorere y’umubiri zigenzurwa ku buryo burambuye. Iri sesengura ryemeje kandi ko isano y’urusobe rw’imisemburo imbere n’izindi misemburo z’ibimera zitari JA zigira uruhare mu buryo bwihariye mu mikorere y’umubiri iterwa n’inyamaswa zirya ibyatsi. By’umwihariko, twasanze ET iterwa na OSM ari nini cyane kuruta iyatewe na OSS1. Ubu buryo buhuye n’ibipimo byinshi bya FAC muri OSM, ari ikintu gikenewe kandi gihagije kugira ngo habeho ET ikwirakwira (48). Mu rwego rw’imikoranire hagati y’ibimera n’inyamaswa zirya ibyatsi, imikorere ya ET ku mikorere y’ibinyabuzima byihariye iracyari isanzwe kandi yibanda gusa ku itsinda rimwe ry’ibimera. Byongeye kandi, ubushakashatsi bwinshi bwakoresheje ikoreshwa rya ET cyangwa ibyayibanjirije cyangwa ibiyibuza bitandukanye mu kwiga uburyo ET ikoreshwa, muri byo iyi mikoreshereze y’ibinyabutabire byo hanze izatanga ingaruka mbi nyinshi zitari izihariye. Uko tubizi, ubu bushakashatsi bugaragaza isuzuma rya mbere rinini ry’uruhare rwa ET mu ikoreshwa rya ET mu gukora no kubona ibimera bya transgenic byangiritse kugira ngo bihuze imikorere ya metabolome y’ibimera. Uburyo ET ikoreshwa mu buryo bwihariye bushobora guhindura uburyo metabolome ikorwamo. Igikomeye cyane ni uburyo ET ikoresha biosynthesis (ACO) na perception (ETR1) byagaragaje uburyo ET ikoresha nikotine mu buryo bwihariye. Byagaragaye mbere ko ET ishobora guhindura uburyo nikotine ikoreshwa mu buryo bushya ikorwamo. Byagaragaye ko ET ishobora guhindura uburyo JA ikoresha nikotine ikorwamo ikoresheje putrescine N-methyltransferase (49). Ariko, mu buryo bw’ikoranabuhanga, ntabwo byumvikana neza uburyo ET ikoresha phenamide mu guhindura uburyo phenamide ikorwamo. Uretse imikorere yo gutanga ibimenyetso bya ET, imikorere y’imikorere y’umubiri ishobora no koherezwa kuri S-adenosyl-1-methionine kugira ngo igenzure ishoramari rya polyaminophenol amide. S-adenosyl-1-methionine ni ET kandi ni yo ikoreshwa mu buryo bwa polyamine. Uburyo ikimenyetso cya ET gikoresha mu kugena urwego rwa phenolamide bukeneye kwigwaho byinshi.
Mu gihe kirekire, bitewe n'umubare munini w'ibintu byihariye bitazwi, kwibanda cyane ku byiciro byihariye bya metabolism ntibyashoboye gusuzuma neza impinduka z'igihe mu mikorere y'ibinyabuzima nyuma y'imikoranire yabyo. Kuri ubu, hashingiwe ku isesengura ry'amakuru, ingaruka nyamukuru yo kubona MS/MS spectrum hashingiwe kuri metabolites zitabogamye ni uko ibimera birya cyangwa biganisha ku bimera bikomeza kugabanya ubwinshi bw'imikorere y'ibimera muri metabolite y'amababi mu gihe byongera urwego rwabyo rw'umwihariko. Uku kwiyongera kw'igihe gito mu mikorere y'ibimera biterwa n'ibimera bifitanye isano no kwiyongera kw'uburyo bw'imikorere y'ibimera. Ikintu gikora cyane kuri ubu buryo bunini bwa metabolite (bufite agaciro ka Si kari hejuru) ni metabolite yihariye ifite imikorere y'ibimera mbere. Ubu buryo buhuye n'ubuhanuzi bw'inyigisho ya OD, ariko ubuhanuzi bwa MT bujyanye n'uburyo bwo kuvugurura metabolite ntabwo buhuye. Ariko, aya makuru ahuye kandi n'ubuhanuzi bw'uburyo buvanze (MT nziza; Ishusho ya 1B), kuko izindi metabolite zitaramenyekana zifite imikorere y'ubwirinzi itazwi zishobora gukomeza gukurikira ikwirakwizwa rya Si ritunguranye.
Uburyo bugaragara bwagaragajwe n'ubu bushakashatsi ni uko kuva ku rwego rw'ubwihindurize buto (ibimera bimwe n'itabi) kugeza ku rwego runini rw'ubwihindurize (ubwoko bw'itabi bufitanye isano rya hafi), urwego rutandukanye rw'imiterere y'ubwihindurize ruri mu "burinzi bwiza cyane". Hari itandukaniro rikomeye mu bushobozi bw'inyamaswa zirya ibyatsi. Moore et al. (20) na Kessler na Kalske (1) bigenga batanze igitekerezo cyo guhindura urwego rutatu rw'imikorere y'ibinyabuzima bitandukanye na Whittaker (50) byabanje guhindurwamo impinduka zishingiye ku gihe kandi ziterwa n'ubusumbane bw'ibinyabutabire; aba banditsi ntibatanze ibisobanuro ku buryo bwo gukusanya amakuru ya metabolome mu buryo bunini kandi ntibugaragaza uburyo bwo kubara ubusumbane bw'imikorere y'ibinyabuzima uhereye kuri aya makuru. Muri ubu bushakashatsi, impinduka nto ku ishyirwa mu byiciro ry'imikorere ya Whittaker zizareba ubusumbane bwa α-metabolic nk'ubusumbane bwa MS/MS spectra mu kimera runaka, na β-metabolic diversity nk'ishingiro rya metabolisme y'itsinda ry'abantu Space, na γ-metabolic diversity bizaba inyongera y'isesengura ry'ubwoko busa.
Ikimenyetso cya JA ni ingenzi ku buryo butandukanye bwo gusubiza imikorere y’ibinyabuzima bikomoka ku bimera. Ariko, hari ibura ry’isuzuma rikomeye ry’umubare w’uruhare rw’igenzura ry’imiterere ya JA mu buryo bwihariye mu miterere y’ibinyabuzima bikomoka ku bimera, kandi niba ikimenyetso cya JA ari ahantu rusange ho gukwirakwiza imikorere y’ibinyabuzima bitewe n’ihungabana ku rugero rwo hejuru rw’ihindagurika ry’ibinyabuzima bikiri urujijo. Twabonye ko imiterere y’ibinyabuzima bikomoka ku bimera bya Nepenthes itera umwihariko wa metabolome kandi ihindagurika ry’umwihariko wa metabolome mu bwoko bw’ibinyabuzima bya Nicotiana no mu bwoko bwa Nicotiana bifitanye isano rya hafi rifitanye isano nziza n’ibimenyetso bya JA. Byongeye kandi, iyo ikimenyetso cya JA cyangiritse, imiterere y’imikorere y’ibinyabuzima iterwa n’ubwoko bumwe bw’ibinyabuzima bikomoka ku bimera izahagarikwa (Ishusho ya 3, C na E). Kubera ko impinduka mu miterere y’imikorere y’ibinyabuzima bya Nepenthes byagabanutse mu buryo busanzwe ahanini ari imibare, impinduka mu miterere y’ibinyabuzima bikomoka kuri β n’imiterere y’ibinyabuzima muri iri sesengura zishobora guterwa ahanini n’ibyiciro bikomeye by’ibimera bikungahaye kuri metabolome. Ibi byiciro by’ibimera byiganje mu gice cy’imiterere ya metabolome kandi biganisha ku isano nziza n’ibimenyetso bya JA.
Kubera ko uburyo bw'ibinyabutabire bw'ubwoko bw'itabi bufitanye isano rya hafi naryo butandukanye cyane, metabolites zigaragazwa by'umwihariko mu buryo bw'imiterere, bityo bikaba ari ugusesengura cyane. Uburyo inyigisho y'amakuru itunganya imiterere y'imyakura yafashwe igaragaza ko induction y'ibimera yongera ubucuruzi hagati y'ubusumbane bwa gamma mu mikorere n'ubwihariko. Ikimenyetso cya JA kigira uruhare runini muri ubu busumbane. Ukwiyongera kwa metabolome specialization bihuye n'ubuhanuzi bw'ingenzi bwa OD kandi bifitanye isano nziza n'ikimenyetso cya JA, mu gihe ikimenyetso cya JA gifitanye isano mbi n'ubusumbane bwa gamma mu mikorere. Izi moderi zigaragaza ko ubushobozi bwa OD bw'ibimera bugenwa ahanini n'uburyo JA ikora, haba ku gipimo cy'ihindagurika ry'ibintu cyangwa ku gipimo kinini cy'ihindagurika. Igerageza ryo gukoresha JA hanze ribuza ubusembwa bwa JA bikomeza kugaragaza ko ubwoko bw'itabi bufitanye isano rya hafi bushobora gutandukanywa mu bwoko butanga ibimenyetso n'ubutatanga ibimenyetso, kimwe n'uburyo bwazo bwa JA na metabolome plasticity biterwa n'ibimera. Ubwoko butanga ibimenyetso ntibushobora gutanga ibisubizo bitewe n'uko budashobora gukora JA y'umwimerere bityo bukaba bufite imbogamizi ku miterere y'umubiri. Ibi bishobora guterwa n'impinduka mu turemangingo tumwe na tumwe tw’ingenzi mu nzira ya JA signaling pathway (AOS na JAR4 muri N. crescens). Ibi bisubizo bigaragaza ko izi ngero z’ubwihindurize bw’ubwoko butandukanye zishobora guterwa ahanini n’impinduka mu buryo imisemburo igaragaramo no mu buryo ikora.
Uretse imikoranire hagati y’ibimera n’inyamaswa zirya ibyatsi, ubushakashatsi ku budasa bw’imikorere y’ibinyabuzima bufitanye isano n’iterambere ry’ingenzi mu bushakashatsi ku bijyanye no guhuza ibidukikije n’impinduka mu miterere y’ibintu bigoye. Bitewe n’ubwiyongere bw’amakuru aboneka mu bikoresho bigezweho bya MS, isuzuma ry’ubudasa bw’imikorere y’ibinyabuzima ubu rishobora kurenga itandukaniro ry’ibintu ku giti cyabyo/icyiciro cyabyo no gukora isesengura mpuzamahanga kugira ngo hagaragare imiterere itunguranye. Mu isesengura rinini, ikigereranyo cy’ingenzi ni igitekerezo cyo gutekereza amakarita afite akamaro ashobora gukoreshwa mu gusesengura amakuru. Kubwibyo, umusaruro w’ingenzi w’uruvange rw’imikorere y’ibinyabuzima bya MS/MS n’inyigisho y’amakuru ni uko bitanga igipimo cyoroshye gishobora gukoreshwa mu kubaka amakarita yo kureba budasa bw’imikorere y’ibinyabuzima ku bipimo bitandukanye bya taxonomike. Ni cyo kintu cy’ibanze gisabwa muri ubu buryo. Inyigo y’ihindagurika rya micro/macro hamwe n’ibidukikije by’abaturage.
Ku rwego rw’ubwihindurize bw’ibinyabuzima, ishingiro ry’inyigisho y’ubwihindurize bw’ibinyabuzima n’udukoko ya Ehrlich na Raven (51) ni uguhanura ko itandukaniro ry’ubwihindurize bw’ibinyabuzima bitandukanye ari yo mpamvu y’ubwihindurize bw’ibimera. Ariko, mu myaka mirongo itanu ishize iki gitabo cy’ingenzi gisohotse, iki gitekerezo nticyageragejwe cyane (52). Ibi ahanini biterwa n’imiterere y’ibinyabuzima by’imiterere y’ibinyabuzima bisa mu miterere y’ibinyabuzima mu miterere y’ibinyabuzima biva kure. Ubuke bw’ibi bintu bushobora gukoreshwa mu gushimangira uburyo bwo gusesengura intego. Imikorere ya MS/MS ikorwa ubu hifashishijwe inyigisho y’amakuru ipima imiterere ya MS/MS y’ibintu bitazwi (bitabanje gutoranywa) kandi igahindura izi MS/MS mu itsinda rya MS/MS, bityo mu mikorere y’ibinyabuzima by’umwuga. Imiterere y’ubwihindurize bw’ibinyabuzima igereranywa mu buryo bwo gushyira mu byiciro. Ibipimo byoroheje by’ibarurishamibare. Iyi gahunda isa n’isesengura ry’ibinyabuzima, rishobora gukoresha uburyo bwo guhuza imiterere kugira ngo ripime igipimo cy’ubwihindurize bw’ibinyabuzima cyangwa ubwihindurize bw’imiterere hatabayeho guhanura mbere.
Ku rwego rwa biochemical, igitekerezo cyo gusuzuma cya Firn na Jones (53) kigaragaza ko ubusumbane bw'imikorere y'umubiri buguma ku rwego rutandukanye kugira ngo haboneke ibikoresho fatizo byo gukoresha ibikorwa bya biologique bya metabolites zitari zifitanye isano cyangwa zasimbuwe. Uburyo bwo gutanga amakuru butanga urufatiro aho izi mpinduka zishingiye kuri metabolites ziba mu gihe cyo kwibanda kuri metabolites zishobora gupimwa nk'igice cy'uburyo bwo gusuzuma imikorere y'umubiri: guhindura imikorere y'ibinyabuzima kuva ku buryo budasobanutse neza kugeza ku buryo budasobanutse neza Mebolites zibujijwe zo mu gace runaka.
Muri rusange, mu ntangiriro z'ibinyabuzima bya molekile, inyigisho z'ingenzi zo kurinda ibimera zarateguwe, kandi uburyo bushingiye ku bitekerezo bufatwa nk'uburyo bwonyine bwo gutera imbere mu bya siyansi. Ibi ahanini biterwa n'imbogamizi za tekiniki zo gupima metabolome yose. Nubwo uburyo bushingiye ku bitekerezo ari ingirakamaro cyane mu guhitamo ubundi buryo butera imbere, ubushobozi bwabwo bwo guteza imbere ubumenyi bwacu ku miyoboro ya biochemical ni buke cyane ugereranyije n'uburyo bwo kubara buboneka muri siyansi ikoresha amakuru menshi muri iki gihe. Kubwibyo, inyigisho zidashobora guhanura zirenze kure amakuru aboneka, bityo uburyo bwo gupima/gupima iterambere mu rwego rw'ubushakashatsi ntibushobora gukurwaho (4). Turateganya ko imikorere y'ikoranabuhanga rya metabolome yashyizwe aha ishobora kongera gushishikazwa n'ibibazo biherutse (uburyo) n'ibya nyuma (impamvu) by'ubusumbane mu mikorere ya metabolome, kandi igatanga umusanzu mu gihe gishya cy'ubumenyi bw'amakuru buyobowe n'inyigisho. Icyo gihe cyongeye gusuzuma inyigisho z'ingenzi zahumekeye ibisekuru byabanje.
Guha inyama z’ibimera ubwatsi bikorwa hatezwa igihu cya kabiri cyangwa ikibuno cya Sl ku kibabi kimwe cy’igihingwa gifite ibara ry’umweru cy’igihingwa kimwe cy’indabyo z’iroza, hamwe n’ibimera 10 byiyongera kuri buri gihingwa. Udukoko twafashwe dukoresheje udukingirizo, hanyuma igice gisigaye cy’amababi gikusanywa nyuma y’amasaha 24 na 72 nyuma yo kwandura hanyuma kigakonjeshwa vuba, hanyuma ibyavuye mu bimera bigakurwamo.
Kwigana uburyo bwo kuvura ibimera mu buryo buhuriweho cyane. Uburyo ni ugukoresha amapine y'imyenda kugira ngo utobore imirongo itatu y'amahwa ku mpande zombi z'amababi atatu yagutse y'ikimera mu gihe cy'ikura ry'indabyo z'igitambaro, hanyuma uhite ushyiramo 1:5 Diluted Ms. Cyangwa ukoreshe intoki zifite uturindantoki kugira ngo ushyire S1 OS mu gikomere cyo gutobora. Kuramo no gutunganya ikibabi nk'uko byasobanuwe haruguru. Koresha uburyo bwavuzwe mbere kugira ngo ukuremo metabolites z'ibanze n'imisemburo y'ibimera (54).
Ku bijyanye no gukoresha JA hanze, amababi atatu ya petiole ya buri kimwe mu bimera bitandatu by'indabyo z'iroza bya buri bwoko avurwa na 20μl ya lanolin paste irimo 150μg MeJA (Lan + MeJA), na 20μl ya lanolin hamwe n'ibikomere (Lan + W), cyangwa agakoreshwa 20μl ya lanolin nk'uburyo bwo kugenzura. Amababi yasaruwe amasaha 72 nyuma yo kuvurwa, ashyirwa muri azote y'amazi, maze abikwa kuri -80°C kugeza igihe akoreshejwe.
Imirongo ine ya JA na ET transgenic, ari yo irAOC (36), irCOI1 (55), irACO na sETR1 (48), yagaragaye mu itsinda ryacu ry’ubushakashatsi. IrAOC yagaragaje cyane igabanuka ry’urugero rwa JA na JA-Ile, mu gihe irCOI1 itari ifite ubushobozi bwo kwita kuri JA. Ugereranyije na EV, ubwiyongere bwa JA-Ile bwariyongereye. Mu buryo nk’ubwo, irACO izagabanya umusaruro wa ET, kandi ugereranije na EV, sETR1, idakomeye kuri ET, izongera umusaruro wa ET.
Imashini ipima urumuri rw'amajwi (Sensor Sense ET-300 real-time ET sensor) ikoreshwa mu gupima ET idakoreshejwe amashanyarazi. Ako kanya nyuma yo kuvurwa, kimwe cya kabiri cy'amababi cyaciwe maze gishyirwa mu gacupa k'ikirahure ka mililitiro 4 gafunze, maze umwanya w'umutwe wemererwa gukusanya mu masaha 5. Mu gihe cyo gupima, buri gacupa kasutswemo litiro 2 ku isaha z'umwuka mwiza mu gihe cy'iminota 8, kari kanyuze mu cyuma cyatanzwe na Sensor Sense kugira ngo gikuremo CO2 n'amazi.
Amakuru ya microarray yasohotse bwa mbere muri (35) abikwa muri Database Comprehensive Center for Biotechnology Information (NCBI) Gene Expression Comprehensive Database (nomero yo kwinjira GSE30287). Amakuru ajyanye n'amababi yatewe no kuvurwa kwa W + OSMs hamwe n'uburyo bwo kugenzura butangiritse yakuwe muri ubu bushakashatsi. Ingufu z'ibanze ni log2. Mbere yo gusesengura imibare, ibanze ryahinduwe rigashyirwa ku kigero cya 75% hakoreshejwe porogaramu ya R.
Amakuru y’umwimerere ya RNA sequencing (RNA-seq) y’ubwoko bwa Nicotiana yakuwe muri NCBI Short Reading Archives (SRA), inomero y’umushinga ni PRJNA301787, yatangajwe na Zhou et al. (39) kandi ikomeza nkuko byasobanuwe muri (56). Amakuru y’umwimerere yatunganyijwe na W + W, W + OSM na W + OSS1 ajyanye n’ubwoko bwa Nicotiana yatoranijwe kugira ngo asesengurwe muri ubu bushakashatsi, kandi atunganywa mu buryo bukurikira: Ubwa mbere, ibyanditswe bya RNA-seq byahinduwe mu buryo bwa FASTQ. HISAT2 ihindura FASTQ muri SAM, naho SAMtools ihindura dosiye za SAM muri dosiye za BAM zatondekanijwe. StringTie ikoreshwa mu kubara igaragara rya gene, kandi uburyo bwayo bwo kugaragaza ni uko hari ibice ku bice igihumbi by’ibanze kuri miliyoni y’ibice byanditse byakurikiranye.
Inkingi ya Acclaim chromatographic (150 mm x 2.1 mm; ingano y'uduce 2.2μm) ikoreshwa mu isesengura n'inkingi y'uburinzi ya mm 4 x 4 mm igizwe n'ibikoresho bimwe. Ubu buryo bukurikira bwo gupima ibice bibiri bukoreshwa muri sisitemu ya Dionex UltiMate 3000 Ultra High Performance Liquid Chromatography (UHPLC): iminota 0 kugeza kuri 0.5, isocratic 90% A [amazi ya deionized, 0.1% (v/v) acetonitrile na 0.05% aside formic], 10% B (Acetonitrile na 0.05% aside formic); iminota 0.5 kugeza kuri 23.5, icyiciro cyo gupima ibice ni 10% A na 90% B, uko bikurikirana; iminota 23.5 kugeza kuri 25, isocratic 10% A na 90% B. Igipimo cyo gupima ibice ni 400μl/min. Ku isesengura ryose rya MS, shyiramo inkingi ya eluent mu cyuma gikora isesengura rya quadrupole n'igihe cyo kuguruka (qTOF) gifite isoko ya electrospray ikora mu buryo bwa ionization bwiza (voltage ya capillary, 4500 V; capillary outlet 130 V; Ubushyuhe bwo kumisha 200°C; kumisha umwuka litiro 10/iminota).
Kora isesengura ry'ibice bya MS / MS (byitwa MS / MS) ridafite aho rihuriye cyangwa ritatandukaniye n'amakuru kugira ngo ubone amakuru y'imiterere yerekeye imiterere rusange y'imikorere y'umubiri. Igitekerezo cy'uburyo bwa MS / MS budatoranyije gishingiye ku kuba quadrupole ifite idirishya rinini cyane ryo gutandukanya uburemere [bityo, tekereza ibimenyetso byose bya mass-to-charge (m/z) nk'ibice]. Kubera iyo mpamvu, kubera ko igikoresho cya Impact II kitashoboye gukora CE tilt, hakozwe isesengura ryigenga hakoreshejwe agaciro k'ingufu za collision-induced dissociation collision energy (CE)). Muri make, banza usesengure icyitegererezo ukoresheje UHPLC-electrospray ionization/qTOF-MS ukoresheje uburyo bwa single mass spectrometry (imiterere yo gutandukana hasi iterwa no gutandukana kw'isoko), ushakisha kuva kuri m/z 50 kugeza 1500 ku nshuro yo gusubiramo ya 5 Hz. Koresha azote nk'umwuka wo gukubitana mu isesengura rya MS/MS, kandi ukore ibipimo byigenga kuri voltage enye zitandukanye zikurikira zo gukubitana: 20, 30, 40, na 50 eV. Mu gihe cyose cyo gupima, quadrupole ifite idirishya rinini cyane ryo gutandukanya uburemere, kuva kuri m/z 50 kugeza 1500. Iyo igerageza ry'umubiri w'imbere m/z n'ubugari bwo gutandukanya rishyizwe kuri 200, urwego rw'uburemere ruhita rukoreshwa na porogaramu ikora y'igikoresho na 0 Da. Skena ibice by'uburemere nk'uko biri mu buryo bw'uburemere bumwe. Koresha sodium formate (50 ml isopropanol, 200 μl formic aside na 1 ml 1M NaOH aqueous solution) kugira ngo upime uburemere. Ukoresheje algorithme ya Bruker yo gupima neza cyane, dosiye y'amakuru irapimwa nyuma yo gukoresha spectrum mpuzandengo mu gihe runaka. Koresha function yo kohereza hanze ya porogaramu ya Data Analysis v4.0 (Brook Dalton, Bremen, Ubudage) kugira ngo uhindure dosiye z'amakuru mpapuro mu buryo bwa NetCDF. Amakuru ya MS/MS yabitswe muri database ya metabolomics ifunguye MetaboLights (www.ebi.ac.uk) hamwe na nomero yo kwinjira. MTBLS1471.
Guteranya MS/MS bishobora gukorwa binyuze mu gusesengura isano riri hagati y’ibimenyetso by’ubwiza bwa MS1 na MS/MS ku ngufu nke n’izikomeye zo gukubitana hamwe n’amategeko mashya ashyirwa mu bikorwa. Inyandiko ya R ikoreshwa mu gusesengura isano iri hagati y’ikwirakwizwa ry’ibikubiye mu gice cya mbere cy’ibicuruzwa, naho inyandiko ya C# (https://github.com/MPI-DL/indiscriminant-MS-MS-assembly-pipeline) ikoreshwa mu gushyira mu bikorwa amategeko.
Kugira ngo tugabanye amakosa y’impimbano aterwa n’urusaku rw’inyuma n’isano ry’impimbano riterwa no kubona ibintu bimwe na bimwe bya m/z mu ngero nke gusa, dukoresha imikorere ya "filled peak" ya paki ya R XCMS (yo gukosora urusaku rw’inyuma). Igomba gukoreshwa mu gusimbuza imbaraga za "NA" (intsinzi itagaragara). Iyo imikorere ya "fill peak" ikoreshejwe, haracyari agaciro kenshi ka "0″ intensity" mu makuru azagira ingaruka ku mibare y’impimbano. Hanyuma, tugereranya ibisubizo byo gutunganya amakuru byabonetse iyo imikorere ya "wuzuye peak" ikoreshejwe n’iyo imikorere ya "wuzuye peak" idakoreshejwe, hanyuma tukabara agaciro k’urusaku rw’inyuma dushingiye ku gaciro k’impuzandengo gakosowe, hanyuma tugasimbuza izi ngero za "0 intensity" n’agaciro k’inyuma kabazwe. Twanasuzumye gusa ibintu bifite imbaraga zirenze inshuro eshatu agaciro k’inyuma kandi tubifata nk' "intsinzi nyazo." Ku mibare ya PCC, ni ibimenyetso bya m/z by’icyitegererezo (MS1) n’uduce tw’amakuru dufite nibura impera umunani nyazo.
Iyo ubukana bw'ubwiza bw'ibipimo mu rugero rwose bufitanye isano rikomeye n'ubukana bugabanuka bw'ubwiza bumwe buterwa n'ingufu nke cyangwa nyinshi zo gukubitana, kandi iyo miterere ntiyitiriwe uburebure bwa isotope na CAMERA, ishobora gusobanurwa kurushaho. Hanyuma, hakozwe isesengura ry'imibanire yose ishoboka y'ibipimo mu masegonda 3 (igihe cyo kubika giteganijwe cyo kubika igipimo), gusa iyo agaciro ka m/z kari hasi y'agaciro k'ibipimo kandi MS/MS igacikagurika ahantu hamwe mu rugero mu makuru yakusanyijwe n'ibipimo byakomotseho, niho bifatwa nk'igice.
Dushingiye kuri aya mategeko abiri yoroshye, dukuramo ibice byavuzwe bifite agaciro ka m/z karuta m/z k’umubanjirije wagaragajwe, kandi dushingiye ku mwanya w’icyitegererezo aho umubanziriza agaragara n’igice cyavuzwe. Birashoboka kandi guhitamo imiterere myiza yakozwe n’ibice byinshi byakozwe mu buryo bwa MS1 nk’ibibanza bya mbere, bityo bigatanga ibinyabutabire bya MS/MS bidakoreshwa. Kugira ngo tugabanye ubu buryo bwo gusibanganya amakuru, niba NDP isa na spectra irenga 0.6, kandi ikaba iri muri chromatogram "pcgroup" yavuzwe na CAMERA, tuzayihuza. Amaherezo, duhuza ibisubizo bine bya CE bifitanye isano n’umubanziriza n’ibice muri spectrum ya nyuma ya composite deconvoluted duhitamo peak y’ubukana bwinshi hagati y’ibice byose bya candidate bifite agaciro ka m/z kamwe ku ngufu zitandukanye zo gukubitana. Intambwe zikurikiraho zo gutunganya zishingiye ku gitekerezo cya spectrum ya composite kandi zigashingira ku miterere itandukanye ya CE isabwa kugira ngo hongerwe amahirwe yo gucikamo ibice, kuko bimwe mu bice bishobora kugaragara gusa munsi y’ingufu runaka zo gukubitana.
RDPI (30) yakoreshejwe mu kubara ubushobozi bwo gupima imiterere y’umubiri. Ubwinshi bw’imiterere y’umubiri (Hj index) bukomoka ku bwinshi bw’ibintu byabanje bya MS/MS hakoreshejwe Shannon entropy y’ikwirakwizwa ry’inshuro za MS/MS hakoreshejwe ikigereranyo gikurikira cyasobanuwe na Martínez et al. (8). Hj = −∑i = 1mPijlog2(Pij) aho Pij ihuye n’inshuro ya MS/MS i-th mu gipimo cya j-th (j = 1, 2,…, m) (i = 1, 2, …, m) t).
Ubwinshi bwa metabolike (Si index) busobanurwa nk'imvugo y'itsinda rya MS/MS runaka ugereranyije n'inshuro iri hagati y'ibipimo biri gusuzumwa. Ubwinshi bwa MS/MS bubarwa nka Si = 1t (∑j = 1tPijPilog2PijPi)
Koresha formula ikurikira kugira ngo upime igipimo cya δj cya metabolome-specific cya buri gipimo cya j, hamwe n'impuzandengo y'imiterere ya MS/MS δj = ∑i = 1mPijSi
Spektara za MS/MS zihujwe mu buryo bwa kabiri, kandi imiterere ihuye ishingiye ku manota abiri. Ubwa mbere, ukoresheje uburyo bwa NDP busanzwe (buzwi kandi nka cosine correlation method), koresha iki gipimo gikurikira kugira ngo umenye imiterere ihuye hagati ya spectra NDP = (∑iS1 & S2WS1, iWS2, i) 2∑iWS1, i2∑iWS2, i2 aho S1 na S2. Mu buryo nk'ubwo, kuri spectra ya 1 na spectra ya 2, hamwe na WS1, i na WS2, i ihagarariye uburemere bushingiye ku bunini bw'impeshyi aho itandukaniro ry'impeshyi isanzwe ya i-th hagati ya spectra ebyiri riri munsi ya 0.01 Da. Uburemere bubarwa ku buryo bukurikira: W = [ubunini bw'impeshyi] m [ubwiza] n, m = 0.5, n = 2, nk'uko MassBank yabitangaje.
Uburyo bwa kabiri bwo gutanga amanota bwashyizweho, bwari bukubiyemo gusesengura NL ihuriweho hagati ya MS/MS. Muri urwo rwego, twakoresheje urutonde rwa NL 52 rukunze kugaragara mu gihe cyo gucikamo ibice bya MS, hamwe na NL yihariye (dosiye y'amakuru S1) yabanje gusobanurwa ku urwego rwa MS/MS rw'ibinyabutabire bya kabiri by'ubwoko bwa Nepenthes bwacitse intege (9, 26). Kora vekteri ya binary ya 1 na 0 kuri buri MS/MS, ijyanye n'ubukonje n'ubutabaho bwa NL zimwe na zimwe. Hashingiwe ku isano ya Euclidean, amanota ya NL asa abarwa kuri buri mpande ebyiri za vekteri ya NL.
Kugira ngo dukore dual clustering, twakoresheje paki ya R DiffCoEx, ishingiye ku kongera Weighted Gene Co-expression Analysis (WGCNA). Dukoresheje NDP na NL scoring matrices za MS/MS spectra, twakoresheje DiffCoEx kugira ngo tubare comparative correlation matrix. Binary clustering ikorwa hashyirwaho "cutreeDynamic" parameter kuri method = "hybrid", cutHeight = 0.9999, deepSplit = T, na minClusterSize = 10. Kode ya R source ya DiffCoEx yakuwe kuri dosiye y'inyongera ya 1 na Tesson et al. (57); Paki ya porogaramu ya R WGCNA ikenewe iboneka muri https://horvath.genetics.ucla.edu/html/CoexpressionNetwork/Rpackages/WGCNA.
Kugira ngo dukore isesengura rya MS/MS molekile network, twabaze uburyo bwo guhuza spectral dushingiye ku bwoko bwa NDP na NL, kandi twakoresheje porogaramu ya Cytoscape kugira ngo turebe imiterere y’urusobe rw’amakuru dukoresheje imiterere ya organic muri porogaramu yo kongera imiterere ya CyFilescape yFiles.
Koresha verisiyo ya R 3.0.1 kugira ngo ukore isesengura ry'imibare ku makuru. Akamaro k'imibare kapimwe hakoreshejwe uburyo bubiri bwo gusesengura itandukaniro (ANOVA), hagakurikiraho ikizamini cya Tukey cy'itandukaniro rikomeye (HSD) nyuma ya hoc. Kugira ngo hasesengurwe itandukaniro riri hagati yo kuvura ibimera n'uburyo bwo kugenzura, ikwirakwizwa ry'amatsinda abiri y'ibipimo bifite itandukaniro rimwe ryasesenguwe hakoreshejwe ikizamini cya t cy'umunyeshuri.
Ku bindi bikoresho by'inyongera kuri iyi nkuru, reba http://advances.sciencemag.org/cgi/content/full/6/24/eaaz0381/DC1
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Inyigisho y'amakuru itanga uburyo rusange bwo kugereranya metabolome zihariye no guhanura inyigisho z'ubwirinzi bw'ibizamini.
Inyigisho y'amakuru itanga uburyo rusange bwo kugereranya metabolome zihariye no guhanura inyigisho z'ubwirinzi bw'ibizamini.
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