矮牵牛PhDFR基因和抗除草剂Bar基因植物表达载体的构建及对烟草的遗传转化研究

Construction of Plant Expression Vector Carrying Petunia PhDFR Genes and Herbicide Resistance Bar Gene and Its Transformation into Tobacco

[目的]构建矮牵牛PhDFR基因的植物表达载体,并对烟草进行遗传转化。[方法]从不同花色的矮牵牛中克隆了2个PhDFR基因,通过目的基因序列克隆、多步载体酶切、连接、转化等技术手段,将其连入通用植物表达载体pCAMBIA2300及pCAMBIA3301。利用冻融法将重组质粒导入农杆菌GV3101继而转化烟草。[结果]构建了含卡那霉素抗性筛选标记基因NPTII和除草剂抗性筛选标记基因Bar的GFP融合蛋白植物表达载体pCAMBIA2300-PhDFR-GFP和pCAMBIA3301-PhDFR-GFP。转基因植株的GUS染色结果进一步验证了所构建表达载体的正确性和实用性。[结论]所构建的表达载体为进一步研究PhDFR基因在植物花色调控效应的功能及开展植物花色改良基因工程研究奠定了基础,为植物基因工程科研工作提供了优良的备选植物表达载体。

[ Objective ] The aim was to construct a plant expression vector carrying Petunias PhDFR gene and to genetically transform it into to- bacco. [ Method] Two PhDFR gene was cloned from Petunia varieties with different flower color in the previous study. Through multi technical methods including sequence cloning of target genes, double enzyme digestion, plasmid vector connection and transformation, target gene PhDFR were introduced into the plant expression vector pCAMBIA2300 and pCAMBIA3301. These vectors were genetically transform into Agrobacterium tumefactions strain GV3101 and then applied in genetic transformation of tobacco recipient plants. [ Result] The plant over-expression vectors carrying target genes PhDFR, including pCAMBIA2300-PhDFR-GFP which contained kanamycin resistance selection marker gene NFFII and GFP-fused gene, and pCAMBIA3301-PhDFR-GFP which contained Basra resistance marker gene Bar and GFP-fused gene, were successfully constructed. The correctness and practicability of expression vector construct were further verified by GUS staining analysis of transgenic plants further. [ Conclusion] The constructed plant over-expression vectors provide a foundation for further study on the function of PhDFR genes in regulating plant flower color and genetic engineering related to modification of plant flower color. This study also provided excellent alternative plant ex- pression vectors for plant genetic engineering work.