Isolation And Expression Analysis Of MhWRKY15 In Malus Hupehensis (Pamp) Rehd.

Transcription factors were proteins that binding with specific DNA sequences of target gene, which could regulate gene expression by enhancing or inhibiting the efficiency of gene transcription. Regulation of the gene expression in eukaryotic was mainly at the transcriptional level. WRKY transcription factor was an important inducible regulatory factor which participated in a variety of important physiological activities, such as plant defense responses, growth regulation and metabolism regulation.Malus hupehensis (Pamp) Rehd. var pinyiensis Jiang (Chinese name is Ping Yi Tian Cha, PYTC) was the unique resource in our country, which was used widely as a common rootstock of apple and ornamental crabapple. In this thesis, the full-length cDNA of MhWRKY15 gene was isolated based on the materials of PYTC roots, the character and function of the gene sequence and the protein that it encoded was analyzed by bioinformatics. At the same time, the expression pattern of MhWRKY15 during growth process and under various sresses was studied. Otherwise, transgenic arabidopsis thaliana transformed with RNAi vector of MhWRKY15 gene was obtained by floral dip transformation.The main results were as follows:1. The full longth cDNA of MhWRKY15 was obtained. According to the homologous sequences from other plants, degenerate primers were designed to amplify specific DNA fragment using cDNA prepared from PYTC roots. MhWRKY15 was 1091bp in length, containing an open reading frame of 810bp and encoding 269 amino acids, and it was belonged to group II of WRKY transcription factors and was one number of WRKY15 family, and the accession number of GeneBank was GU576874.2. Bioinformatics analysis indicated that the protein encoded by MhWRKY15 was a soluble protein, with the molecular formula was C₁₂₆₃H₁₉₄₁N₃₆₅O₄₂₅S₁₂, the relative molecular weight was 29423.2Da and isoelectric point was 5.30. There was one conserved WRKY domain in the protein sequence, and the sequence identity to M. domestica and G. max was over 96%. The protein was located in nucleus, and there were many phosphorylation sites, N glycosylation sites and O glycosylation sites, but no transmembrane domain and signal peptide. The predicted secondary structure demonstrated that random coil was the most important structural conformation.3. The pCAMBIA1390-MhWRKY15-GFP vector was constructed to study subcellular localization of MhWRKY, and the result showed that MhWRKY15 was localized in nucleus.4. The RNAi vector of pFGC5941-RNAi-MhWRKY15 was also constructed in this study and transformed Arabidopsis thaliana col-0 using the floral dip method via agrobacterium mediated. Harvest seeds were put on MS medium containing glufosinate in order to select the resistant seedlings. Genomic DNA of resistant seedlings was extracted for PCR detection. In the end, 11 transgenic seedlings were identified, which lay the foundation for the further study of the function of MhWRKY15.5. According to the analysis of semi RT-PCR,MhWRKY15 gene expression could be induced by high salinity stress, drought stress, low temperature and mechanical injury, and had tissue specificity, the expression level in leaf was higher than root and stem.