Analysis Of Cold Resistance Function Of VaDi19-3 Gene In Vitis Amurensis

Grape(Vitis vinifera L.)is one of the largest economic crops in the world and is widely used for fresh food,wine making,drying,juice and canning.The main grape cultivation areas in China are mostly arid or semi-arid areas,which are usually cold and dry in winter,so the freezing damage is a prominent problem in grape production.The way to solve this problem is to breed new grape varieties with strong cold resistance through hybridization technology,but this method has a long breeding cycle and slow effect.The development of molecular biology provides a new way for grape cold-resistant breeding.It is of great significance to study the key genes for cold-resistant Vitis amurensis,which is the most cold-resistant species in Vitis,and to cultivate new cold-resistant grape varieties through molecular breeding.On the basis of obtaining Vitis amurensis VaDREB gene and screening out an interaction protein VaDi19-3 of VaDREB in our previous study,we cloned VaDi19-3 gene and analyzed its cold resistance function,in order to lay a foundation for further revealing its cold resistance molecular mechanism.The main results obtained are as follows:1.The VaDi19-3 gene from V.amurensis acc.‘Shuangyou’ was cloned.The CDS of this gene is 663 bp,encodes 220 amino acids,and contains two conserved domains: zf-Di19 and Di19＿C.Subcellular location shows that VaDi19-3 was located on the nucleus and cytomembrane.After treatment at a low temperature of 4℃,the the expression level of Di19-3 in V.amurensis ‘Shuangyou’ and V.vinifera ‘Red Globe’ plants was analyzed by qRT-PCR.The results showed that with the extension of low temperature treatment,the expression of Di19-3 continued to increase,and the expression levelt in ’Shuangyou’ was significantly higher than that in ‘Red Globe’.The expression of Di19-3 was found in the roots,stems,leaves and tendrils of ‘Shuangyou’ and ‘Red Globe’,but the expression levels were different.The highest expression level of Di19-3 in the stems of ‘Shuangyou’ and in the roots of ‘Red Globe’,respectively.2.The overexpression vector of VaDi19-3 was ransformed into Arabidopsis thaliana through the Agrobacterium-mediated method,and the homozygous T3 transgenic Arabidopsis plants were obtained.Three transgenic lines OE-1,OE-2 and OE-5 were obtained by PCR,qRT-PCR and Western blot detection.Four-week-old wild-type and transgenic Arabidopsis plants were treated with low temperature(-6℃).The results showed that after freezing treatment,the survival rate of transgenic Arabidopsis plants was higher than that of wild-type,the REL and MDA contents in transgenic plant were lower than in WT,and the contents of proline,soluble sugar and the activities of SOD,POD and CAT in the former were higher than in the latter.After treatment at a low temperature of 0℃,the expression levels of the cold resistance-related genes were analyzed in WT and transgenic plants by qRT-PCR.The results showed that the expression levels of CBF1,CBF2,CBF3,COR15 A,P5CS2,COR47,RD29 A and KIN1 genes in transgenic plants were significantly up-regulated,while the expression level of NCED3 did not change significantly.This indicates that VaDi19-3 regulates the expression of cold-responsive genes through the ABA-independent CBF pathway,thereby the cold tolerance of transgenic Arabidopsis is improved.3.The promoter fragments of Di19-3 in ‘Shuangyou’ and ‘Red Globe’ were cloned,respectively,and the cis-acting elements of them were analyzed.It was found that both P-VaDi19-3 and P-Vv Di19-3 contained the cis-acting elements AE-box,Box4,P-box,W box and TC-rich repeats etc.,but there were some slight differences in quantity.The results of GUS histochemical staining and GUS protein activity analysis showed that both P-VaDi19-3 and P-Vv Di19-3 could induced by low temperature,but the expression activity of P-VaDi19-3 was stronger than that of P-Vv Di19-3.4.VaDi19-3 has transcriptional activation activity in yeast.Using a fragment of VaDi19-3(VaDi19-3 d6)as bait plasmid,three candidate interaction proteins VaTLP,VaPR10 and VaP5 CS were screened by yeast two-hybrid.Bimolecular fluorescence complementarity test further confirmed that there was an interaction between VaDi19-3 and the three candidate proteins,and the interaction occurred in the nucleus and cell membrane.