| Cotton fiber is the most important textile material, plays an important role in ournational economy. So, it is important to elucidate fiber development process by molecularbiology.To characterize the precise function ofβ-mannosidase in cotton fiber, in this study,the sense and antisense coding sequence of theβ-mannosidase gene were fused withfiber-specific E6 promoter to build the constructs of sense gene and antisense RNA gene,and with which to transform into cotton via Agrobacterium-mediated transformation, and tomake them over expression, or suppression the expression of the endogenesis gene in thecotton genome in the purpose of studying the real function of this gene in fiberdevelopment.By DNA recombination technology, the sense and antisense coding sequence of theβ-mannosidase gene droved by fiber-specific E6 promoter were constructed using the binaryvector pBI121 as backbone vector. PCR with the special primer and sequencing of the PCRproduct and digestions with SmaI and NotI proved the two vectors were constructedsuccessfully and can be used for transformation. By Agrobacterium mediatedtransformation, the above-mentioned twoβ-marmosidase constructs were successfullytransferred to the cotton cultivars, Simian3 and W0, Around 500 T0 regeneration plantsfrom 53 different calli lines were obtained.PCR amplification and Southern blot analysis of the transgenic cotton plants wereused to confirm the integration of the transgenes. The primers were designed according tothe sequences of NPTⅡand E6-β-mannosidase gene and applied to PCR analyses of T0transgenic plants. For the sense vector, the percentage of the plants with positive signals inNPTⅡprimer reaction is 97.92%, that of the plants in E6-β-mannosidase primer reactionis 87.23%; and for the antisense vector, the percentage of the plants with positive signals inNPTⅡprimer reaction is 95.19%, that of the plants in E6-β-marmosidase primer reactionis 64.65%. The plants showing both positive signals of NPTⅡand E6-β-mannosidaseprimers were regarded as positive transgenic plants primarily and were regenerated by grafting. In this research, 230 positive plants were grafted and 196 of them were survived. 8To plants which were regarded as positive transgenic plants primarily by PCR reaction werefurther analyzed with Southern blotting. Using NPTⅡgene as the probe, the results showedthat the NPTⅡgene had been inserted to the cotton genome, using E6 promoter as theprobe, the results showed that the E6 promoter had been integrated into the cotton genomein some T0 plants. PCR analysis of T1 and T2 transgenic plants using the E6-β-mannosidase gene special primers showed that the segregation ratio of ManA2 gene inseven transgenic lines were inherited in a classical Mendelian manner (3:1). But Southernblot showed that there was more than one copy of ManA2 gene in some transgenic lines,which perhaps was because of mutiple-copy integration in one site of the cottonchromosome. |
