| Wheat(Triticum aestivum L.)is the most widely cultivated food crop in the world.Abiotic stress is the main factor restricting wheat production,which dramaticaly affects its growth and development and leads to remarkable yield reduction.In the scenario of climate change,the impact of abiotic stress on wheat production will be further intensified.Understanding the underlined mechanisms of abiotic stress resistance is the premise and basis for the development of high yield varieties with improved tolerances to abiotic stress.Plant adaptation to abiotic stress involves a variety of fine-tuning mechanisms,among of which protein ubiquitination is one of the effective and fast ways to cope with environmental changes.Accumulated documents shows that plant U-box E3 ubiquitin ligases(PUB)are invoved in the response to various stresses.Gene expression assays showed that TaPUB57-2A was induced by a variety of stresses.In order to decipher the relationship between TaPUB57-2A and stress resistance and reveal its molecular mechanism involved in stress response,TaPUB57-2A was transferred into wild type rice and Ospub57 mutant by cross,and a series of experiments were performed,including cis-acting element assay in promoter region,gene expression pattern analysis,in vitro ubiquitination assay,protein interaction analysis and transgenic rice phenotyping.The main results are as follows:(1)TaPUB57-2A contains 12 exons and 11 introns,and encods a 666 amino acid residue(AAR)polypeptide.It has three domains,an ICP0-binding domain of Ubiquitin-specific protease 7(IBD/USP7),a protein kinases catalytic domain(PKCD),and a U-box domain.Secondary structure prediction revealed that TaPUB57-2A has 32 ?-helixes and 17 ?-sheets.(2)Cis-acting element assays showed the presence of a variety of abiotic stress responsive elements in the upstream promoter region,including drought response element MBS,ABA response element ABRE,high temperature stress response element HSE,low temperature stress response element LTR and methyl jasmonate response element CGTCA / TGACG motif.(3)Gene expression assays showed that TaPUB57-2A expressed in root,stem,leave and ear.Its transcript was up-regulated under high salt,drought,ABA and low temperature conditions,and an early response was observed when exposed to exogenous ABA.The results were consistent with the presence of multiple types of cis-acting element in the promoter.(4)Two candidate interaction proteins,TaEXPB3 and TaXTH2 were identified by screening the yeast library constructed with wheat cDNA.The interactions between TaPUB57-2A and TaEXPB3 and TaXTH2 were confirmed by Y2 H,split luciferase and pull-down assays.(5)In vitro ubiquitination analysis showed that TaPUB57-2A had E3 ubiquitin ligase activity,and TaEXPB3 was its downstream substrate.(6)Overexpression of TaPUB57-2A in rice resulted in improved agronomic traits,including increased main panicle length,higher spike number and 1,000-grain weight,more panicle branches and enhanced grain length.However,the plant height was significantly reduced.(7)Under high salinity conditions,overexpression of TaPUB57-2A led to significantly lowered cell membrance stability,chlorophyll content,chlorophyll fluorescence,SOD activity and survival rate relative to the WT control.However,the CAT activity of transgenics was higher than the WT control.No difference was observed in proline contents.TaPUB57-2A transgenic rice were highly sensitive to high salinity,suggesting that SOD might play a major role in response to salt stress. |
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