Study On The Function Of Powdery Mildew Resistance And Interacting Proteins Screening Of VqLecRK1 In Vitis Quinquangularis

Grape is a very old fruit tree,its fruit nutritional value and economic value are high,known as one of the ‘four fruits’.In the process of grape planting and growth,grape powdery mildew is often affected by external conditions,and powdery mildew is a very serious factor.Grape powdery mildew occurs under a wide range of conditions,and it is more harmful to the grape under facility cultivation.Therefore,it is of great significance to explore the powdery mildew resistance gene and study the powdery mildew resistance function of grape for the creation of new powdery mildew resistance germplasm materials.In the early stage of this study,transcriptomic sequencing technology was used to discover a gene up-regulated by powdery mildew infection named VqLecRK1,and the function of powdery mildew resistance was analyzed through overexpression transformation model plant arabidopsis.On this basis,VqLecRK1 was cloned from Vitis quinquangularis‘Shang-24’ with high resistance to powdery mildew.The function of VqLecRK1 gene against powdery mildew was studied by transient transformation of grape leaves and genetic transformation of ‘Seedless white’ problastoid mass,and the interaction protein of VqLecRK1 was screened to lay a foundation for revealing the molecular mechanism of powdery mildew resistance.The main results are as follows:1.Cloning and sequence analysis of VqLecRK1 from Vitis quinquangularis.VqLecRK1 gene was cloned from powdery mildew-resistant grape ‘Shang-24’ with a total length of2412 bp encoding 803 amino acids,which was 98.01% similar to the amino acid sequence of Vitis vinifera ‘Pinot noir’,and contained a ‘B-lectin’ conserved domain,belonging to G-type lectin receptor kinase.2.The function of VqLecRK1 gene that resists powdery mildew was further determined by transient transformation test.The expression vector VqLecRK1-GFP was constructed to instantaneously transform susceptible grape ‘Red globe’ and ‘Seedless white’,and interference vector RNAi-VqLecRK1 was constructed to instantaneously transform resistant grape ‘Shang-24’ and ‘Baihe-35-1’.By observing the phenotype and Trypan blue staining of leaves,it was found that compared with the control group.There were fewer powdery mildew fungi in the leaves with transient gene expression,while there were more powdery mildew fungi in the leaves with instantaneous silence VqLecRK1 gene and their mycelia were more dense.It was preliminarily verified that VqLecRK1 gene could enhance powdery mildew resistance in grape.3.Screening of VqLecRK1 interacting proteins.The interaction protein VqPGDH of VqLecRK1 was screened by yeast two-hybrid technology,and the interaction between the two proteins was verified by yeast response verification,split-Luc test and bimolecular fluorescence complementation test.According to the results of subcellular localization test,VqPGDH was located in chloroplast,cell membrane and cytoplasm.Quantitative analysis of the leaves of ‘Shang-24’ was conducted to confirm that VqPGDH was up-regulated by powdery mildew.In addition,quantitative analysis results showed that VqPGDH was positively induced by VqLecRK1 in the transient transformation test.4.Genetic transformation of grape mediated by Agrobacterium.Through Agrobacterium mediated genetic transformation of ‘Seedless white’ problastoid mass,the transgenic lines were identified by agarose gel electrophoresis,semi-quantitative and quantitative test and western-blot test respectively.Finally,three overexpressed transgenic grape lines were obtained.