Studying On RNAi Mediated To Resistance WMV,ZYMV,CMV In Transgenic Melon Materials

RNAi caused by target gene expression silencing though into small segments of double-stranded RNA, that can be applied to restrain or weaken the muskmelon virus physiological and biochemical genes to achieve disease resistance effect. This research use RNAi technology developed transgenic melon seedling, that is basis to learn the distribution, physiological mechanism of the antiviral and muskmelon resistance. This research that through the cp gene sequence analysis about WMV, CMV and ZYMV to find a conservative sequence fragment as interference by DNAMAN5.0?CLUSTAL X and MEGA5.O software, than to build the RNAi plant expression vector; Using the Agrobacterium-mediated transformation to obtain transgenic muskmelon plants.The main research results as follows:(1)The cp gene sequence alignment of the WMV, ZYMV and CMV showed that nucleotide sequence homology more than 86.25%,93.37% and 83.13%, they have higher nucleotide sequence homology; Studies have shown that WMV have 2 subgroups by Genetic and phylogenetic analyses,1 subgroup in Nanjiang and 2 subgroups in Beijiang; Studies have shown that CMV have 4 subgroups by Genetic and phylogenetic analyses, three isolates in genetic and phylogenetic relationship were near with isolates in Beijing, Shandong, Shanxi and Taiwan; ZYMV have 3 subgroups by Genetic and phylogenetic analyses, The part of the isolates in genetic and phylogenetic relationship were near with three areas both in China and abroad; There are base mutations on the cp gene in xinjiang.(2)The multiple RT-PCR system was establishment for WMV, ZYMV, and CMV:The best volume of template cDNA was 1.8 ?l; When the total volume was 2?l that best ratio was WMV:CMV:ZYMV=9:6:5; The optimum concentration of dNTPs was 0.4 mmol/L; The optimum nuit energy of Taq DNA polymerase was 0.75U; The optimum concentration of Mg2+ was 1.5 mmol/L; The optimum annealing temperature was 50?.(3)Tissue culture technique and regeneration system for 'XHXC' and '86-1' were established. This study shows that concentration of 5% NaClO to deal with 20min was the best; The optimum concentration callus induction of 6-BA were 1.2 mg/L and 1.0 mg/L correspond to '86-1' and 'XHXC'; Bud differentiation frequency was higher in MS10; MS19 was the optimum medium for '86-1' to dventitious buds elongate, MS20 was the optimum medium for 'XHXC' to dventitious buds elongate; he best medium of rooting of '86-1' and 'XHXC' were MS24.(4)The successful access to 2 transgenic muskmelon plants about pCAMBIA2300 WMV-SA of '86-1',2 transgenic muskmelon plants about pCAMBIA2300CMV-SA,4 transgenic muskmelon plants about pCAMBIA2300CMV-SA; The successful access to 'XHXC' transgenic muskmelon seedling were two pCAMBIA2300WMV-SA, three pCAMBIA2300CMV-SA and two pCAMBIA2300 ZYMV-SA.