Cloning And Expression Analysis Of Powdery Mildew Resistance-related Gene Mlo And γVPE In Chinese Wild Vitis Pseudoreticulata

Powdery mildew(Erysiphales) is a pathogen caused by biotrophic fungi, which is one of the most common plant pathogenic bacteria. The grapevine powdery mildew(Erysiphe necator Schw. [syn. Uncinula necator(Schw.) Burr.]) is a fungi pathogen, which is seriously harm to the production of grapevine. Many years of research showed that the Chinese wild Vitis pseudoreticulata accession.Baihe-35-1 was highly resistant to the powdery mildew. In this study, we have identified 19 MLO proteins from the grape genome, and six VpMlo genes were cloned from Chinese wild Vitis pseudoreticulata accession.Baihe-35-1. We constructed six plant over expression vectors to transformed into Arabidopsis thaliana mutants, and obtained the T3 generation of transgenic Arabidopsis plants. The expression level of six Mlo genes after powdery mildew induced in different periods were analyzed. Meanwhile, VpγVPE gene was cloned from Chinese wild Vitis pseudoreticulata accession.Baihe-35-1, and the expression level of VpγVPE gene after powdery mildew induced in different periods were analyzed. The main results were as followings:1、VpMlo3,VpMlo6,VpMlo7,VpMlo8,VpMlo11 and VpMlo13 genes were cloned from the full-length cDNA library of Chinese wild Vitis pseudoreticulata accession.Baihe-35-1. The length of cDNA was 1596 bp, 1703 bp, 1803 bp, 1703 bp, 1827 bp and 1843 bp, respectively. The length of ORF was 1527 bp, 1638 bp, 1620 bp, 762 bp, 1752 bp and 1764 bp, respectively. The number of coding amino acids is 508, 545, 539, 253, 583 and 587, respectively. The gene structure analysis revealed that the VpMlo8 gene had a stop codon, which was not translated into the MLO protein.2、We have identified 19 MLO proteins from the grape genome by biological informatics analysis. The phylogenetic analysis showed that VpMLO3, VpMLO6, VpMLO7, VpMLO8, VpMLO11 and VpMLO13 with SlMLO1, AtMLO2, At MLO6, AtMLO12 were lacted in the same clade. Except for VpMLO8, the number of the transmembrane in other VpMLOs is 6 to 8. The alignment of the amino acid sequence and conservative analysis of transmembrane domain of MLO proteins showed that, except for VpMLO8, the structure of other VpMLOs includes an CaMBD and 2 conserved domain, and conservative of these domains were strong.3、We have constructed six plant over expression vector pCAMBIA1301/VpMlo. These vectors were transformed into Arabidopsis thaliana mutants by Agrobacterium mediated method. All the six target genes haove been transformed into Arabidopsis thaliana mutants by the PCR detection of T1 generation of transgenic Arabidopsis thaliana plants, and we have obtained the T3 generation seed.4、The real-time quantitative PCR was performed to analyze the expression level of six VpMlo genes after powdery mildew induced in different periods, and six Mlo genes exhibited different expression patterns. The expression level of VpMlo3, VpMlo8 and VpMlo11 gene was respond slightly within 8 h. The expression pattern of Vp Mlo6 and VpMlo7 gene was similar, which increased significantly within 8 h and reached the highest level at 8 h during the process of induction. The expression level of VpMlo13 gene was respond significantly at 8 h and 168 h.5、VpγVPE gene was cloned from the full-length cDNA library of Chinese wild Vitis pseudoreticulata accession.Baihe-35-1. The length of cDNA was 1724 bp, the length of ORF was 1482 bp, coding 493 amino acids. The alignment of the amino acid sequence revealed that a substrate pocket one of three key amino acids Ser 395 was mutated to Ala, which in Vitis pseudoreticulata accession.Baihe-35-1, Vitis vinifera cv.Thompson Seedless, and Vitis vinifera cv. Pinot Noir.6、The real-time quantitative PCR was performed to analyze the expression level of VpγVPE gene after powdery mildew induced in different periods. The expression level of VpγVPE gene was affected by powdery mildew induction.