Identification Of Drought Resistance Of TaNRX-D1 Gene And Screening Of Its Interaction Protein In Wheat(Triticum Aestivum L.)

Wheat（Triticum aestivum L.）is one of the three major food crops in the world,and drought has had a serious impact on its yield and quality.The preliminary research results of our group have shown that the wheat nucleoredoxin gene（TaNRX-D1）is related to drought resistance.Validating its drought resistance and clarify its intracellular location and interaction protein can lay a foundation for studying its mechanism of drought resistance.In this study,the TaNRX-D1 gene was overexpressed in Arabidopsis by genetic transformation technology to verify its drought resistance;Subcellular localization technique was used to determine the subcellular location of TaNRX-D1 in wheat protoplasts;Yeast two-hybrid（Y2H）technology was used to obtain candidate interaction proteins of TaNRX-D1,and one-on-one verification by Y2H technology and bimolecular fluorescence complementary technology（BiFC）technology was used to further verify the authenticity of the interaction relationship.In order to enrich the types of interaction proteins,the bioinformatics was used to predict the candidate interaction proteins related to drought resistance of TaNRX-D1,and then the interaction relationship was verified by Y2H and BiFC technology.And yeast eukaryotic expression technology was used to ananlyze the drought resistance of the interaction proteins.The main results are as follows:1.Overexpression of TaNRX-D1 gene enhanced tolerance of Arabidopsis to drought stress.The plant overexpression vector was constructed and transformated to Arabidopsis thaliana,and relevant morphological and physiological indicators of drought resistance were measured and analyzed in transgenic Arabidopsis thaliana T₃ homozygous strain in seedling stage（growth 10 d）,seedling stage（growth 4 weeks）and adult stage（growth 8 weeks）,and the results showed that:（1）There was no significant difference in root length of wild-type Arabidopsis thaliana（WT）,Arabidopsis thaliana with empty vector（CK）and 5 transgenic lines on normal growth medium（P>0.05）.Although the root length of all Arabidopsis thaliana was significantly shortened under mannitol simulated drought stress,the root length of the five transgenic lines was significantly（P<0.05）or extremely significant（P<0.01）increased.（2）There was no significant difference in the MDA content between WT,CK and5 transgenic lines under normal water supply conditions（P>0.05）.After PEG6000simulated drought stress treatment,the MDA content of the five transgenic lines was significantly lower than that of WT and CK（P<0.01）.（3）There was no significant difference in the Pro content in leaves of WT,CK and 5 transgenic lines under normal water supply conditions（P>0.05）.After PEG6000 simulated drought stress treatment,the Pro content of the five transgenic lines was significantly higher than that of WT and CK（P<0.01）.（4）The results of dehydration rate of in vitro leaves at seedling stage showed that the five transgenic lines showed lower water loss rate at five time points,and there was extremely significant difference in different lines,different time and interaction between line and time were（P<0.01）.（5）The results of DAB staining in seedling leaves showed that under normal water supply conditions,no significant difference could be seen between WT,CK and 5 transgenic lines.After 3 days of PEG6000 simulated stress treatment,all the leaves were found to have different degrees of color enhancement.,and the five transgenic lines were significantly lighter than WT and CK.（6）The drought-rehydration experiment in the adult stage（rehydration 5 d after drought 25 d）showed that the survival rate of the five transgenic lines was significantly higher than that of WT and CK（P<0.01）.2.TaNRX-D1 was localized in the nucleus,cytoplasm and cell membrane.By expressing TaNRX-D1-e GFP fusion protein in wheat protoplasts,it was that Green fluorescence was observed mainly in the nucleus,cytoplasm and cell membrane,indicating that TaNRX-D1 localizes in the nucleus,cytoplasm and cell membrane in wheat cells.3.TaVDAC1,TaPDI,TaTRX-h,TaPP2Ac,and TaNRX-D1 all interacted with TaNRX-D1.The interaction proteins of TaNRX-D1 andΔTaNRX-D1（the first two domains of TaNRX-D1）was screened by yeast two-hybrid library,and the same interaction protein type I voltage-dependent anion channel protein（VDAC1）was obtained.According to BiFC,TaNRX-D1 and TaVDAC1 can interact in tobacco,and the interaction sites were mainly in the nucleus and cell membrane.In order to obtain more drought-related interaction proteins,more than 60 proteins that may interact with TaNRX-D1 were obtained through software prediction and previous studies,and five possible drought-related proteins that named ferritin（TaFd）,glutaredoxin（TaGRX）,disulfide isomerase（TaPDI）,protein phosphatase 2A catalytic subunit（TaPP2Ac）and h-type thioredoxin（TaTRX-h）were selected as candidate interaction proteins of TaNRX-D1.;and the five candidate interaction protein genes were cloned from the drought-tolerant wheat variety Jinmai 47,and the relevant expression vector were constructed.And three proteins TaPDI,TaTRX-h and TaPP2Ac were verified that can interact with TaNRX-D1 by Y2H and BiFC,and the interaction sites mainly in the nuclear and cell membrane.And TaNRX-D1 can interact with itself to form a dimer which was verified by Y2H and BiFC.4.Overexpression of TaTRX-h,TaPDI,TaPP2Ac and TaVDAC1 alleviated the inhibition of yeast growth by mannitol simulated drought stress.The yeast overexpression vector was constructed and the toxicity analysis showed that TaPP2Ac had a slight inhibitory effect on yeast growth.TaTRX-h,TaPDI and TaVDAC1 had no toxic effects on yeast growth.TaTRX-h,TaPDI,TaPP2Ac and TaVDAC1 were overexpressed in yeast,and the growth state of the yeast under simulated drought stress under mannitol was observed.Compared to the yeast strains grown under normal conditions,the four proteins had different degrees alleviation to inhibition of drought stress on yeast growth.And TaVDAC1 had the strongest effect,TaTRX-h and TaPP2Ac had weaker effects,and TaPDI had intermediate effect,indicating that TaPDI,TaPP2Ac,TaVDAC1 and TaTRX-h all have positive regulation effects on drought stress.