The Function Study Of Grapevine VqBAK1 Gene Resistant To Powdery Mildew And Identification Of VvG-LecRK Gene Family

Grape berries have a high economic value in China,and the land area allotted for grape cultivation in the country is ranked among the highest in the world.The European grapevine(Vitis vinifera L)plays a leading role in grape production because of its good quality and high yield.Yet the European grape suffers from poor disease resistance,particularly against powdery mildew,anthracnose,gray mold,downy mildew,and other fungal diseases,which can severely impact both the quality and yield of grapes.As one centre of origin of grapes in the world,China has a large number of wild grape germplasm resources.Chinese wild grape Vitis quinquangularis Shang-24 is highly resistant grape germplasm and an important material for studying the resistance of grapes to powdery mildew.Receptor-like kinases(RLKs)are structurally similar protein superfamily that are involved in defense against pathogens in plant.The group previous research conducted transcriptome sequencing and gene expression analysis of Chinese wild grapes resistant to powdery mildew and susceptible to powdery mildew,and identified some differentially expressed powdery mildew-resistant-related receptor-like kinases,including some SERK(somatic embryogenesis receptor kinase)and G type Lec RK(lectin receptor-like kinase).On this basis,this study identified SERK family genes and G-Lec RK family genes in grape,and the structure,evolution,expression,the possible biological regulation processes of SERK genes and G-Lec RK genes were analyzed.After the above-mentioned bioinformatics analysis,expression analysis of different treatments,and combined with recent research in plants,this study speculates that BAK1 have the function of resistant to powdery mildew.In this study,the Vq BAK1 gene was cloned from the grapevine Shang-24,and its interaction protein was screened and the function of regulating grape powdery mildew resistance was studied.The main research results obtained are as follows:1.Analysis of grapevine’s somatic embryogenesis receptor kinase(SERK)gene family.According to the previous research in the laboratory,we downloaded 5 Vv SERK protein sequences from NCBI through the gene sequence accession number.The 5 Vv SERK protein sequences were compared with the entire grape genome protein sequence,and found that the Vv SERK1 gene is located on the 18 chromosome of grape,Vv SERK2 is located on chromosome 7,Vv SERK3,Vv SERK4 and Vv SERK5 are located on chromosome 12.At the same time,the structure of SERK proteins was analyzed.Subcellular localization experiments confirmed that the Vq SERK family is indeed localized to the plasma membrane,but Vq SERK3 also localized to the cytoplasm.The expression of Vq SERK1 was up-regulated in SA and ETH treatment;Vq SERK3 was up-regulated in SA,JA and ABA treatment;Vq SERK4 was up-regulated in ABA treatment;Vq SERK5 was up-regulated in ETH treatment.2.Functional analysis of Vq BAK1 gene resistance to powdery mildew in Chinese wild grapevine(Vitis quinquangularis).SERK3 is known to be involved in the brassinolide(BR)signaling pathway,in which it functions as non-ligand binding coreceptor,so it is also called BAK1(BRI1-Associated Receptor Kinase1).According to the co-localization,Vq BAK1 has the localization of Golgi complex.Plasma membrane,TGN/EE and endomembrane system.Vq BAK1 was overexpressed in Arabidopsis and grape Thompson Seedless(V.vinifera L),the transgenic lines were stained with trypan blue after inoculated with Erysiphe necator.The infection of the fungus was inhibited,and the spore germination rate and spore production of Erysiphe necator were reduced.The expression levels of disease resistance regulation-related genes PR1 and ARFA also changed to varying degrees.The expression level of PR1 gene was up-regulated in overexcession lines and down-regulated in RNAi-leaves;ARFA was down-regulated in RNAi-leaves but overexcession had little effect on it;At 24 h and 72 h after the inoculation of Erysiphe necator,PR1 and ARFA were up-regulated in both the overexcession lines and RNAi-leaves,but the expression levels were higher in the overexcession lines than wild type(WT),in RNAi-leaves,the expression level was lower than that of WT.The above results indicated that the resistance of Chinese wild grapes to powdery mildew was related to defense-related genes.3.The function study of Vq RLK5(receptor-like kinase 5)interacting with Vq BAK1 in Chinese wild grapevine(Vitis quinquangularis)resistant to powdery mildew.The interaction protein Vq RLK5 was screened from the yeast two-hybrid c DNA library induced by powdery mildew using Vq BAK1 deletion fragment as the bait protein.The interaction relationship between Vq BAK1 and Vq RLK5 was verified by using Bi FC and spilt-luc.Vq RLK5 was located on the cell membrane.It was verified that the overexpression of Vq RLK5 could improve the resistance to powdery mildew in grape.4.Identification and expression analysis of grapevine G-Lec RK family genes.According to the published grape genome sequencing information,a total of 94 Vv G-Lec RK family genes were identified,mainly distributed on 12 and 19 chromosome.A phylogenetic tree of Vv G-Lec RK family genes was constructed and they were divided into I-X groups.This study found that six segmental duplication events have occurred,and no tandem duplication gene was found.At 0-120 h after PM treatment,the expression of most of thecandidate genes was up-regulated at most time points compared with the control,and Vv G-Lec RK-V5 was up-regulated nearly 9-fold at 48 h,which was the highest among all Vv G-Lec RK-V4 and its homologous genes.In addition,Vv G-Lec RK-V8 was negatively regulated in response to powdery mildew.Vv G-Lec RK-V4 and its homologous genes responded differently to SA,Me JA,ABA,and ETH,and most genes responded positively to these four hormones.