Uloga transkripcionog faktora DREB2A i malih RNK miR398a/b i miR408 u odgovoru graška (Pisum sativum L.) na dehidrataciju

Abstract

Jedan od glavnih faktora abiotičkog stresa kod biljaka je dehidratacija. U ovom radu ispitivan je uticaj dehidratacije na ekspresiju gena za transkripcioni faktor DREB2A i malih RNK miR398 i miR408 graška, u cilju sagledavanja značaja ovih molekula u odgovoru graška na dehidrataciju i rehidrataciju. Parcijalna cDNK (PsDREB2A) koja kodira za DREB2A transkripcioni faktor graška je dobijena primenom metode PCR sa prajmerima dizajniranim na osnovu sekvenci DREB2A gena iz model leguminoze Medicago truncatula. Dobijena sekvenca (495 bp) deponovana je u GeneBank (HM229349). PoreĎenje nukletidnih sekvenci pokazalo je da je dobijena sekvenca najsličnija sa sekvencom za DREB2A iz M. truncatula, kao i sa EREBP (ethylene responsive element binding protein) iz iste vrste. Analiza izvedene aminokiselinske sekvence pokazala je da PsDREB2A ima strukturu karakterističnu za DREB2A protein - prisustvo konzervisanog AP2 domena (17-55 ak), kao i da ima najveću homologiju sa DREB2A i sa ERBP proteinom iz M. truncatula. Na osnovu kompjuterske predikcije posttranslacionih modifikacija proteina utvrĎeno je prisustvo jednog potencijalnog mesta za sumoilaciju proteina, i to na poziciji Lys157 (MKQE), kao i prisustvo nekoliko potencijalnih mesta za fosforilaciju, najverovatnije protein kinazom A. U cilju ispitivanja ekspresije gena za DREB2A transkripcioni factor graška vršene su analize iRNK korena i nadzemnog dela graška u normalnom fiziološkom stanju, kao i u uslovima dehidratacije u trajanju 7 i 10 dana, i rehidratacije. Primenjena je metoda Real-time PCR uz upotrebu proba specifičnih za PsDREB2A. Pokazano je da se profil ekspresije razlikuje u korenu i nadzemnom delu biljaka. Kod korena maksimalni nivo ekspresije PsDREB2A utvrĎen je nakon 10 dana tretmana, dok je kod nadzemnog dela biljaka maksimum dostignut nakon 7 dana dehidratacije...

Adverse environmental stresses, such as drought, low temperature and soil salinity have a strong influence on agricultural production and sustainability. A major limitation to yield and quality in many crop species is water availability throughout or at critical times during the growing season. We investigated the effect of dehydration on expression of DREB2A transcription factor in pea, as well as on the expression of conserved miRNAs-miR398a/b and miR408. Partial cDNA (PsDREB2A), which encodes a DREB2A transcription factor in pea, was obtained by PCR with primers designed according to DREB2A gene from the model legume Medicago truncatula. The resulting sequence (495 bp) has been deposited in Gene Bank (HM229349). Comparison of nucleotide sequences showed that the obtained sequence is most similar to the sequence of DREB2A from M. truncatula, and with EREBP (ethylene responsive element binding protein) from the same species. Analysis of deduced amino acid sequence showed that PsDREB2A has a structure characteristic of the DREB2A protein - presence of AP2 domains (17-55 a.a.), and has the highest homology with DREB2A and ERBP protein from M. truncatula. Computer prediction of post-translational modification of protein revealed the presence of a potential site for protein sumoylation, at the position of Lys157 (MKQE), and the presence of several potential sites for phosphorylation. To study the expression of PsDREB2A we performed the analysis of mRNA level in pea root and shoot in a normal physiological state, as well as during dehydration for 7 and 10 days, and rehydration. Real-time PCR using a probe specific for PsDREB2A has been applied. It has been shown that the expression profile was different in roots and aerial parts of the plants. In the roots the maximum level of expression PsDREB2A was determined after 10 days of treatment, while in the above-ground parts of plants the maximum was rached after 7 days of dehydration...