Construction Of TtAk And TtCHI Binary RNAi Vector Of Tetranychus Turkestani And Preliminary Study On Its Insect Resistance

Object: Cotton is one of the most important economic crops in China. Due to its unique desert oasis irrigation agriculture, Xinjiang Province has became an important cotton production base in China. Dataes showed that Xinjiang cootton accounted for about two fifths of the whole planting area and three fifths of the fiber production in China. Along with the expanding of Xinjiang drip irrigation area, the cotton niche and farmland eclolgical environment have graduelly changed in recent years. What’s more, the long-term dependence on chemical prevention not only enhanced the cotton leaf mites resistance, but also killed a large number of natural enemies of the leaf mites. Furthermore, there has been no leaf mites-resistant cotton varieties so far. As a result, cotton leaf mites broke out year after year and cotton yield and quality were seriously affected. So cultivating leaf mite-resistance varieties to reduce the harm of cotton leaf mite is an important problem need to be resolved for the whole xinjiang cotton production. RNAi technique has been successfully applied to improve plant disease resistance, insect resistance and so on. Double-stranded RNA(ds RNA) of insect gene was expressed in plants using transgenic technology. Ds RNA could effectively inhibit the expression of insects corresponding target gene when herbivorous insects ate transgenic plants,which would lead to defect of insect’s development and growth and even kill them. Those functional genes in insects which encode important proteins were appropriate targets for RNAi. A large number of studies shown that the Arginine Kinase(Arginine Kinase, AK) wsa a key enzyme in ability to metabolizein, and Chitinase(Chitinase, CHI)was an important enzyme to ensure the completion of exuviate and regenerate of cadre peritrophic and trachea in the process of insect development. These two key enzyme genes played an important role in the growth and development of insects. In the study these two key genes were cloned from Tetranychus turkestani, the expression of two key enzyme genes were inhibited in spider mites through the plant mediated RNAi technology, and done some related research, for the purpose of creating new germplasm of leaf mites-resistance.Methods:( 1) Bioinformatics software was amplified to analyze two genes, partial functional region fragments of AK and CHI were cloneed, which based on the c DNA template of Turkestani Tetranychus.(2)The RNAi-vectors containing double valence gene constructed by commbining DNA recombinant technology and Gateway technology, the model plant tobacco and cotton were transformed through agrobacterium-mediated..( 3) A real-time PCR technique was used for identifying the integration of AK-CHI gene expression in different transgenic tobacco lines.(4)Though the contagion mites experiment analysis the effect on inhibit the fragment of AK-CHI fragment to Turkestani Tetranychus.Results and conclusion: 1. Bioinformatics software was amplified to analyze and contrast the two target genes functional region sequence of AK and CHI in Tetranychus turkestani which have been reported.Partial functional region fragments of AK and CHI were amplified(453 bp and 610 bp respectively).Specific primers were designed which contained specific enzyme loci. RNAi-vectors, p B7GWIWG2(Ⅱ)-AK-CHI and p ANDAHK35-AK-CHI, were successfully constructed by commbining DNA recombinant technology and Gateway technology.2. Tobacco leaves were transformed through agrobacterium-mediated transformation method with the interference expression vector p B7GWIWG2(Ⅱ)-AK-CHI, and we got 50 tobacco plants. Genomic DNA of the basta-resistanct plants were extracted, and 20 positive transgenic tobacco plants were verified by PCR. Seven positive transgenic tobacco plants were selected for further study. Total RNA was extracted,and reverse-transcribed to c DNA using PCR method. A real-time PCR technique was used for identifying the integration of AK-CHI gene expression on m RNA level. Three high expression transgenic lines of AK-CHI were chosen for contagion mites experiment and the non-transgenic tobacco was set as control.The results showed that, compared with control plants, Simultaneous Silencing of Tt AK and Tt CHI significantly reduced the survival rate of the mites, the spawning was reduced about 23-30%, the entire growth period of mites was shorten about four days, but there was no effect on the mite egg hatchability.3.The hypocotyls of cotton cv. YZ-1 were genetic transformated through agrobacterium-mediated method, which containing the interference expression vector p ANDAHK35-AK-CHI. We got Kana-resistanct embryonic callus, which were used for PCR verification at genomic level. The results showed that some embryonic callus were positive and the positive rate was up to 60%.