Cloning Of STCYP51and STCYP77A2Genes From Wild Eggplant And Analysis On Its Disease Resistance To Verticillium Dahliae

Cytochrome P450is a class of heme oxygenase enzymes, which exists in bacteria, plants and animals. The enzyme carries out the electron transfer by reversible in living cells and is involved in a variety of important biological metabolic processes. In eukaryotes, as a membrane-binding protein, it is mainly distributed in the endoplasmic reticulum, microsomal and mitochondrial inner membrane. Plant cytochrome P450has a wide range of catalytic activity and its function concerns the biosynthetic pathway and biological detoxification pathway. Previous studies have shown that the CYP450family is involved in the defense of Solanum torvum against Verticillium dahliae.In this study, StCYP51and StCYP77A2cDNAs were cloned from S. torvum by homologous cloning method. After that, the transgenic tobacco plants of those genes were generated by genetic transformation, and further their resistance to V. dahliae was analyzed. The results obtained are as follows:The cloning and analysis of StCYP51gene. The open reading frame of the gene was1464bp long with a start codon ATG and a stop codon TAA. The sequence encoded a protein of487amino acid residues with protein molecular weight of55.33kDa and theoretical isoelectric point at8.35. In order to analyze the function of StCYP51, over-expression vector35S-StCYP51was constructed using the vector PCB2008E. Six StCYPS1transformed tobacco plants were generated by Agrobacterium-mediated transformation. One independent transgenic plant with single copy was confirmed by Southern blot analysis. Disease resistance analysis showed that both infected plant incidence and disease index of StCYP51transgenic plant were lower than the control.The cloning and analysis of StCYP77A2gene. The ORF of the gene was1536bp long with a start codon ATG and a stop codon TAA. The cDNA sequence encoded a protein of487amino acid residues with protein molecular weight of58.08kDa and theoretical isoelectric point at9.17. In order to analyze the function of StCYP77A2, over-expression vector35S-StCYP77A2was constructed using the vector PCB2008E. Eight StCYP77A2transformed tobacco plants were generated by Agrobacterium-mediated transformation. Two independent transgenic plants with double copies were confirmed by Southern blot analysis. Disease resistance analysis showed that both infected plant incidence and disease index of StCYP77A2transgenic plants were lower than the control.Threse results show that both StCYP51and StCYP77A2have inhibitory effect on V. dahliae proliferation and might be involved in the defense of S. torvum against V. dahliae.