Genetic Transformation And Resistant Analysis Of Phytophthora Capsici Resistance Related Gene Cannadph And Canml

Pepper is a multi-year or annual vegetable crops of Solanaceae Capsicum genus, and is cultivated widely in China. Pepper blight?has occurred in many parts of China, and has become one of the important constraints to pepper production. CanNADPH gene and CanML gene of Pepper are cloned by team of pepper of College of Horticulture Northwest A & F University with pepper varieties ,which have high resistance to Phytophthora capsici ,AA3 and AA11 as materials,which were infected by Phytophthora capsici. In this study, plant expression vector pBI121-NADPH and pBI121-ML were constructed by using vector pBI121 and were introduced into pepper variety B12, which is susceptible to Phytophthora capsici, via Agrobacterium-mediated genetic transformation technologies. The transgenic plants, resistance to Phytophthora capsici were identified with in vitro leaf inoculation technique.The major findings are as follows ,1.Plant expression vector pBI121-NADPH was constructed. CanNADPH gene which?was loaded at both ends of the restriction sites, and pBI121vector were digested with BamHⅠand SacⅠ, CanNADPH gene fragment and vector fragment of pBI121 were recovered by?using gel extraction kit and connected with T4 DNA Ligase. The products were transformed into E. coli DH5α. Positive plasmid was extracted by using plasmid extraction kit and transformed into Agrobacterium tumefaciens EHA105 by means of rapid frozen thaw method successfully. PCR analysis and restriction enzyme digestion analysis showed that the expression vector pBI121-NADPH was constructed successfully.2.Plant expression vector pBI121-ML was constructed with the same strategy. PCR analysis and restriction enzyme digestion analysis showed that the expression vector pBI121- ML was constructed successfully.3.Transgenic plants carrying CanNADPH gene and CanML gene were obtained. The seeds of pepper susceptible Cv.B12 were cultured in 1/2MS medium after surface sterilized. The robust seedlings were cut as explants after about two weeks. Explants were infected by Agrobacterium bacteria fluid after pre-cultured, and were cultured on the resistance bud differentiation, elongation and rooting medium with kanamycin .The PCR and RT-PCR molecular detection showed that the five introduced CanNADPH gene transgenic lines and four introduced CanML gene transgenic lines were obtained.4.Introduced CanNADPH gene transgenic pepper,s resistance to Phytophthora capsici was identified with in vitro leaf technique. Results showed that the incidence of transgenic peppers were reduced about 22.2% than those of the non-transgenic peppers, the disease index of transgenic peppers were also reduced 46.5%...