The Effect Of Overexpression Of Elicitins From Phytophthora Capsici On Disease Resistance In Tobacco

Phytophthora capsici is one of the important diseases on Hot pepper, which is no effective method of prevention and cure disease, cultivation and promotion application resistant varieties is the most fundamental solution pepper diseases, and effective technical measures for cultivating modern genetic engineering disease-resistance species provides a powerful method and has shown that broad application prospects in the resistance gene engineering, development has application value genes and expounds its mechanism is the very important research subject. Elicitins is derived from Phytophthora and Pythium two plants of the genus pathogenic bacteria of the curriculum stimulate the son eggs, it can activate the plant defense responses in plant resistance and solanaceae genetic engineering in a very important potential use value. At the same time, Elicitins and solanaceae plant interaction of plant resistance system for the defense signaling pathway offers a great model. In this paper , two capsici elicitins gene construct full-length cDNA as a template over expression vector, and using Agrobacterium-mediated genetic transformation system in tobacco leaves, the elicitins gene into the tobacco, then on the T1 transgenic plants for further functional analysis, further study is designed to elicitins in theSolanaceae plant disease resistance genes and its use value in the mechanism of action may lay the foundation.The main results are as followings:(1) Using Gateway technology to build ultra-containingelicitins gene expression vector. Full-length cDNA of two elicitins gene as template, specific primers were amplified by two-step PCR fragment with attB joints, and then with purpose vehicle pDONR ? 207 to get started BP reaction cloning vector, and then cloned by LR reaction to obtain ultra-expression vector pk7wg2 on purpose expression vector. Through the agrobacterium-mediated technique to the tobacco in total, turned 60 explant, each received 45 and 68 regeneration plant, of which there were 40 and 65 positive for cloning. The results, the charge for flower seed preservation. Set aside.(2) GM seeds will be harves and control tobacco seeds with K326 resistance training of the T1generation. Select the two lines of each gene experiments, grow to 4-5 in the tobacco leaves were inoculated on pepper Phytophthora capsici Leonian and tobacco Alternaria alternata, and then observe the phenotypic changes of the GM plant and wild-type . The results show that, elicitin 7 and elicitin 24 overexpression in tobacco have disease resistance after inoculated peppers Phytophthora capsici Leonian to control type, and relatively obvious; elicitin 7 overexpression in tobacco inoculated with resistance to Alternaria alternate than elicitin 24 resistance is weak, but the relative resistance of wild-type. And then the highly resistant strains elicitin24-3 were screened out.(3) Construction of two elicitins prokaryotic expression vector, and fusion with the inducing agent induces the expression of the protein. Will receive the artificial fusion protein were injected with pepper P120 leaves and tobacco K326 features , while the BSA protein as control further elicitin protein on dicotyledonous plants. The results showed that after injection of the fusion protein, can induce production of tobacco and pepper HR activity. This shows that the original expression does not make pET32a: elictins loss of biological activity of the fusion protein and induce production of tobacco and pepper HR activity.