海岛棉GbTCP5基因沉默和过量表达载体的构建及拟南芥转化

Construction of GbTCP5 Gene Silencing and Over-expression Vectors and Transformation of Arabidopsis

海岛棉具有优良的纤维品质,TCP蛋白参与细胞生长和增殖调控,本研究构建海岛棉GbTCP5基因沉默和过量表达植物表达载体。以GbTCP5基因pGEM-T Easy-GbTCP5质粒为模板进行PCR扩增,并将该基因构建到植物表达载体pCAMBIA3301中,利用冻融法转入农杆菌GV3101菌株,通过农杆菌浸染法转化拟南芥植株,初步证明已获得转化海岛棉GbTCP5基因的拟南芥。利用重叠PCR方法替换拟南芥AtMIR319的成熟链和互补链序列,构建含有amiRTCP5前体的植物表达载体amiRTCP5-pCAMBIA3301,为深入研究海岛棉GbTCP5基因的生物学功能奠定基础,为研究棉花纤维发育机制提供理论依据。

Gossypium barbadense has the best cotton fiber and TCP protein is involved in the regula- tion of cell growth and proliferation. GbTCP5 gene silencing and over-expression vector were con- structed in this study. TheGbTCP5 gene pGEM-T Easy-GbTCP5 plasmid were used as a template for PCR amplification. The gene was constructed into the plant expression vector pCAMBIA3301, trans- formed into Agrobacterium tumefaciens strain GV3101 by freeze-thaw method, and transformed into Arabidopsis plants by Agrobacterium tumefaciens method. It was initially proved that GbTCP5 gene of Arabidopsis thaliana was obtained. To construct a plant expression vector amiRTCP5-pCAM- BIA3301 containing theamiRTCP5 precursor by overlapping PCR method for replacing mature Arabi- dopsis thaliana At-MIR319 ,so as tolay the foundation for further study on the biological function of GbTCP5 gene and providing a theoretical basis for studying development mechanism of cotton fiber.