Abstract：Thioredoxin (TRX) is a kind of functionally diverse proteins with disulfide active sites, and plays an important role in many aspects including plant stress response. Nucleotoreoxin (NRX), a novel member of the TRX superfamily, was found to be associated with drought resistance in wheat (Triticum aestivum), but the mechanism of drought resistance is still unclear. Exploring the interaction protein of TaNRX1 will be helpful to further reveal the drought-resistant mechanism of TaNRX1. Based on previous studies of other researchers, the protein phosphatase 2A catalytic subunit (PP2Ac) was selected as candidate interaction protein. And according to bioinformatics analysis in STRING website (https://string-db.org/), other 2 proteins, protein disulfide isomerase (PDI) and TRX-h were also selected as TaNRX1-D candidate interaction proteins in this study. Then, the ORFs of TaPDI-A, TaPDI-B, TaPDI-D, TaTRX-h-A, TaPP2Ac-B, and TaPP2Ac-D were acquired by homologous cloning from drought-resistant cultivar 'Jinmai 47'. Furthermore, the yeast two-hybrid (Y2H) and bimolecular fluorescence complementation (BiFC) were used to verify the interaction proteins. The correctly sequenced recombinant plasmids pGADT7-TaTRX-h, pGADT7-TaPDI, and pGADT7-TaPP2Ac were co-transformed into yeast (Saccharomyces cerevisiae) strain Y2H Gold with pGBKT7-TaNRX1, and then transferred to SD/-Trp-Leu-His-Ade/X-α-gal screening medium. Then on the medium, colonies were found to grow normally and turn blue, which indicated that all the 3 proteins could interact with TaNRX1-D. In order to eliminate false positives, plasmids pCAMBIA1302-CeYFP-TaPDI, pCAMBIA1302-CeYFP-TaPP2Ac, pCAMBIA1302-CeYFP-TaTRX-h and pCAMBIA1302-NeYFP-TaNRX1 were paired, respectively. Then the 3 paired plasmids were co-transformed into Agrobacterium tumefaciens GV3101 and injected into Nicotiana benthamiana leaves. Yellow fluorescent signal was found in the fluorescence confocal microscope, and the yellow signal was mainly located in the nucleus and cell membrane. To verify whether the 3 interacted proteins would respond to drought stress, mannitol was used to simulate drought stress. The recombinant plasmid pYES2-TaPDI, pYES2-TaPP2Ac and pYES2-TaTRX-h and the empty vector pYES2 were transfected into yeast strain BY4741, respectively and cultured in SD/-Ura medium containing 100 mmol/L mannitol, and the growth of the yeasts was observed. The results showed that the growth of yeasts which heterologous expressing TaPDI, TaPP2Ac and TaTRX-h was significantly better than that of empty vector colonies after drought stress, suggesting that TaPDI, TaPP2Ac and TaTRX-h all have positive regulation to mannitol stress. This study validated interaction of 3 candidate proteins with TaNRX1, and simulated drought stress analysis on yeast was conducted, which provide a reference for further elucidating the molecular mechanism of TaNRX1 drought resistance.
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