Study On The Function Of Drought-resistant Gene VyP5CR In Chinese Wild Vitis Yeshanensis

Grape?Vitis vinifera L.?is the third largest fruit tree in the world and has a high economic value.Drought is one of the main stress factors,which seriously affects the growth and development of plants.The northwestern region is the most important grape-producing area in China.It is an arid and semi-arid area with low annual rainfall and long-term dry soil and water shortage,which seriously restricts the development of the grape industry.Therefore,cultivating drought-resistant grape varieties is one of the fundamental ways to solve production problems.Vitis yeshanensis,a wild grape species native to North China,has strong drought resistance and is a very important drought resistant gene resource.Research and utilize of its key drought resistant genes is of great significance for revealing its drought resistance mechanism and further molecular breeding.This study is based on the transcriptome sequencing analysis of the drought-resistant V.yeshanensis accession'Yanshan-1'and the sensitive-drought North American V.riparia Michx.accession'Hean???'.One of the significantly differentially expressed genes,pyrroline-5-carboxylate reductase gene?P5CR?,has been undergone preliminary functional studies.The specific results obtained are as follows:1.The VyP5CR gene was isolated from the acc.'Yanshan-1'.The gene has a full length of 1198 bp and an ORF?open reading frame?of 831 bp,encoding 276 amino acids.Bioinformatics analysis found that VyP5CR is located on chromosome 8.The homology analysis showed that the sequence homology between VyP5CR and Vv P5CR protein was the highest,which reached 95.89%.Through tobacco epidermal subcellular localization,it was found that VyP5CR exists in the cell membrane and cytoplasm,mainly in the cell membrane.2.The potted plants of'Yanshan-1'and'Hean???'were subjected to drought stress,and grape leaves at different stress time points were collected for real-time quantitative PCR?q RT-PCR?to analyze the expression pattern of P5CR gene.The results showed that with the increase of drought stress time,the P5CR gene of V.yeshanensis decreased first and then increased,and reached the highest peak at 16 d,which is about 5.7 times the highest expression level of V.riparia.In V.riparia,it rose first and then fell,and reached its highest level on 8 d.To detect the expression level of VyP5CR gene in different tissues of grape genotypes,the roots,stems and leaves of'Yanshan-1'were collected for real-time quantitative PCR analysis.The results showed that VyP5CR was expressed in the roots,stems and leaves of'Yanshan-1',with the highest expression level in leaves,which was about 5 times that in roots and stems.3.The V.yeshanensis'Yanshan-1'was treated with different stresses?ABA and salt?,and then the grape leaves at different stress time points were collected for q RT-PCR to analyze the expression changes of VyP5CR.The results showed that during ABA treatment,the expression of VyP5CR first increased,then decreased and then increased,reached the maximum value at 12 h,and then decreased at 24 h.After Na Cl treatment,the expression of VyP5CR gradually accumulated and reached a peak at 8 h.Then it drops and rises again at 24 h.In summary,in addition to being induced by drought,the VyP5CR gene can also be induced by salt and ABA.4.The V.vinifera cultivar'Tompson Seedless'was treated with different stresses?low temperature,salt,SA and ABA?,and grape leaves at different stress time points were collected for q RT-PCR to analyze the expression changes of Vv P5CR.The results showed that a small expression of Vv P5CR was observed within 2 h after 4?treatment,and then gradually decreased.After Na Cl treatment,the expression level of Vv P5CR gradually increased,reaching a peak at 8 h,which was about 8.5 times of the initial level.During ABA treatment,the expression of Vv P5CR first decreased,then increased and then decreased.The expression reached the maximum value at 2 h,and then the expression decreased slowly.After SA treatment,the expression level of Vv P5CR did not change much in the early stage.The expression level was the highest at 4 h,about 6 times the initial value,and then decreased.Therefore,the Vv P5CR gene can be induced by low temperature,salt,SA and ABA.5.Through the Agrobacterium-mediated method,the VyP5CR gene over-expression vector was transformed into wild-type Arabidopsis thaliana,and a homozygous T3generation Arabidopsis plant transformed with the VyP5CR gene was obtained.After Kan resistance screening and PCR,q RT-PCR and Western blot detection,three transgenic Arabidopsis lines were obtained.The four-week-old wild-type?WT?and transgenic Arabidopsis thaliana were subjected to drought treatment.The results showed that compared with WT,the lines over-expressing the VyP5CR gene have significantly improved the drought tolerance and have smaller stomatal apertures.Under mannitol stress,transgenic lines could grow better and have a longer taproot.Also,they have lower MDA,H₂O₂and O₂^-content,higher proline content and SOD,POD activity in terms of physiological and biochemical indexes,which indicates that over-expression of VyP5CR gene can improve the drought resistance of Arabidopsis.In addition,under drought stress,the over-expression of VyP5CR in the transgenic lines induced the expression of drought-related genes KIN1,NCED3,DREB2A,RD29A,COR15A and COR47,and the expression of these genes was significantly up-regulated.Therefore,this study shows that the VyP5CR gene has the function of drought resistance.6.Screening the yeast library with V.yeshanensis VyP5CR as bait,the two candidate interacting proteins Vy COP9 and Vy RPS20 were obtained by yeast two-hybrid.Through bimolecular fluorescence complementation experiments,it was further confirmed that P5CR interacted with Vy COP9 and Vy RPS20 respectively,and the interaction occurred in the cell membrane and cytoplasm.