Pseudomona's NahG And Pepper's LOX Gene Cloning And Its Research Of Transformation Of Tobacco

In nature ecosystem, plants interact with a wide range of microbial pathogens and herbivorous insects. During plants co-evolution with a variety of pathogenic microorganisms, it has evolved a series of complex and effective defense response mechanisms. Including plant cells under stress perception, signal transduction and changes expression of defense-related gene and so on. Among this Plant Response to Stress Signaling which received extensive attention. Now we known SA, JA and other plant hormones involved in plant stress response signal transduction, but its mechanism is not clear. In order to study the molecular mechanism of Solanaceae plant defense response, analysis JA,SA and other endogenous hormones in regulation of different downstream defense response and its possible mechanism. This study selected lipoxygenase (LOX) and salicylic acid and enzyme (nahG) as the target, isolated part or full of their length cDNA.I construction its expression vector and obtained the corresponding transgenic tobacco plants by Agrobacterium tumefaciems. The main result is as follows.1. Isolated LOX gene's positive cDNA clones from UV irradiation chili pepper (Capsicum annuum L.) cDNA library. Its sequence analysis showed that the positive clone length 285 bp, containing 95 amino acids. The base sequence and deduced amino acid sequence with LOX cDNA which in other plants has been isolated and identified were 92% and 96% homology, so we suggesting that the positive clone is the part cDNA of chili LOX gene.2.Obtain nahG gene from the nahG prokaryotic expression vector. Its length is 1305 bp, containing 435 amino acid open reading frame. The base sequence and deduced amino acid sequence with nahG gene which in other microbes are already isolated and identified the more than 99% of nahG gene homology, suggesting that the positive clone is full-lengthcDNA of nahG gene.3. I constructed RNAi vectors of LOX gene and overexpression vector of nahG gene.4. I transformation the two vectors into tobacco K326 by Agrobacterium tumefaciems. We obtain 73 strains T0 generation of transgenic tobacco regeneration plant, 54 strains were PCR positive plants. RT-PCR showed that LOX gene expression was inhibited in transgenic tobacco, nahG gene has transcription expression in transgenic tobacco.The transgenic Plants abtained above lay fundation for analysising the relationship between candidate gene and JA and SA signal pathway and providing the basis for analysising function of candidate gene.