Bc MF3反义基因植物表达载体的构建及其对拟南芥的遗传转化

Construction of Plant Expression Vector Containing the Antisense BcMF3 and Its Genetic Transformation of Arabidopsis Thaliana

根据编码白菜（Brassica rapa L．ssp．chinensis，syn B．campestris ssp．chinensis）果胶甲酯酶的BcMF3基因cDNA序列内部第1343～1797个碱基之间序列设计引物，从白菜花蕾eDNA中PCR扩增出455bp的片段，把该片段作为反义基因连接至双元载体pBI121上，得到了植物表达栽体pBI-B3，并用“三亲杂交”法导入农杆菌LBA4404菌株中，PCR扩增和酶切结果表明所构建的植物表达载体pBFB3含有BcMF3反义基因片断，并已导入了根癌农杆菌（Agrobacterinrn tume fuciens）中；采用花序浸润法转化拟南芥（Arabidopsis thaliana）后，播种筛选浸润植株的种子获得了5株卡那霉素抗性植株，PCR扩增证明BcMF3反义片段成功整合到拟南芥基因组中；转基因拟南芥子代（T2）的抗性分离符合孟德尔分离比例，表明外源基因是单一位点插入。

A 455 bp antisense gene fragment was amplified from nucleotides 1343 to 1797 of the cDNA sequence of BcMF3 gene that encodes a Pectin Methylesterase in Chinese cabbage-pak-choi.The produced fragment was introduced to the binary vector pBI121 to produce pBI-B3,and pBI-B3 was mobilized into Agrobacteriurn tumefaciens strain LBA4404 by triparental mating.PCR amplification and restriction digestion showed that the anti-gene fragment of BcMF3 was introduced into pBI121 and the generated pBI-B3 was transferred into the Agrobacterium tumefaciens successfully.Transformation of the A.tumefaciens strain LBA4404 harboring pBI-B3 to Arabidopsis thaliana was per- formed by floral dip,and 5 transgenic plants were obtained by kanamycin selection and confirmed by PCR amplification.The inheritance of selective marker gene transferred was in the model of the simple Mendel's fashion in progenies of the self-crossed genera- tion,indicating that a single copy of the foreign gene had integrated into Arabidopsis thaliana genome.