Cloning And Function Analysis Mlo Gene Related To Powdery Mildew Resistance In Grapevine

There are many problems in production which seriously influence the healthy development of grape industry in china. The cultivated varieties are almost from the Europe,which have high quality but poor stress-resistance. Previous study from our lab showed that the Chinese wild Vitis pseudoreticulata accession.Baihe-35-1 is highly resistant to powdery mildew. Therefore, further study and isolation of powdery mildew resistance genes have important value in the research of grape disease resistance mechanism and the use of disease-resistant genetically modified European grape varieties. The research group has cloned VpMlo3,VpMlo6,VpMlo7,VpMlo8,VpMlo11 and VpMlo13 from the Chinese wild Vitis pseudoreticulata accession.Baihe-35-1. On the basis of this result, VvCMlo3, VvCMlo6,VvCMlo7, VvCMlo11, VvCMlo13 were cloned from Vitis vinifera cv.Carinena. The responsive pattern of Mlo gene to pathogen had been analyzed. The subcellular localization of VpMlo gene had also been investigated and further verified whether the VpMlo proteins interact with CaM protein. At last, the ability of transgenic Arabidopsis thaliana plants to resist powdery mildew was identified. The main results are described as followings:1. VvCMlo3, VvCMlo6, VvCMlo7, VvCMlo11, VvCMlo13 genes were cloned from Vitis vinifera cv. Carinena. The length of cDNA was 1600 bp, 1701 bp, 1690 bp, 1823 bp, 1841 bp,respectively. The length of ORF was 1329 bp, 1638 bp, 1620 bp, 1752 bp, 1764 bp,respectively. The number of coding amino acids is 442, 545, 539, 583, 587, respectively.2. Transmembrane domain structure of Mlo proteins were analyzed by SMART software. The numbers of transmembrane in VvCMlo3, VvCMlo6 were one less than VpMlo3, VpMlo6, respectively. VpMlo8 gene appeared terminator in advance, so it only contained 2 transmembrane domains. The structure of other 3 MLO proteins included 7transmembrane domains, a CaMBD domain and 2 conserved domains. The conservative of these domains were strong.3. The real-time quantitative PCR was performed to analyze the expression level of six Mlo genes between disease-resistant grape and susceptible grape after powdery mildew induced in different periods. Six Mlo genes exhibited different expression patterns. The expression patterns of Mlo6 and Mlo7 gene were similar, which increased significantlywithin 8 h and the expression level of treat group was 10 fold than control group during the process of induction. VpMlo11 gene was respond to powdery mildew at 8 h and 72 h in Vitis vinifera cv. Carinena but not respond in Vitis pseudoreticulata accession.Baihe-35-1.Overall, the expression level of Mlo11 and Mlo13 genes were lower than that of Mlo6 and Mlo7 genes.4. We had built five subcellular localization vectors pBI221-GFP/VpMlo. These vectors were transformed into Arabidopsis thaliana protoplasts by PEG mediated. Green fluorescence signal was observed under confocal laser microscopy. The experiment results showed that VpMlo genes localized in cell membrane.5. CaM gene was cloned from Vitis pseudoreticulata accession.Baihe-35-1. The length of ORF was 450 bp, coding 149 amino acids. We had built five bait vectors pGBKT7/VpMlo and a pGADT7/CaM vector and verified whether the VpMlo proteins interact with CaM protein by yeast two-hybrid. The experiment results showed that VpMlo7 protein interact with CaM protein.6. Transgenic Arabidopsis thaliana plants were inoculated with powdery mildew. After that we observed the spore germination and growth situation. The results showed that the transgenic Arabidopsis thaliana plants restored sensitivity to powdery mildew.