Bt基因甜菜叶绿体转化载体构建及毒蛋白表达

Vector Construction with Bt Gene for Sugar Beet Chloroplast Transformation and Expression Toxin-protein

利用植物叶绿体基因组在进化中高度保守的特点,根据烟草、菠菜、水稻叶绿体基因组全序列资料设计合成引物,PCR扩增并克隆了甜菜叶绿体两个重要功能基因rbcL和atpB(GenBank登录号分别为DQ067450和DQ067451),并以其作为定点整合外源基因的同源重组片段,构建了Bt基因CryIAc甜菜叶绿体定点转化载体pSKARBt,酶切鉴定表明:所构建载体符合预期设计。对克隆菌菌体总蛋白进行了生物杀虫试验,结果表明:Bt基因CryIAc能够在叶绿体特异性启动子及终止子的调控下表达,并对二龄末甘蓝夜蛾有很强的毒杀作用。该载体构建对培育甜菜高抗虫品种具有重要应用价值。叶绿体转化及后续工作正在进行中。

Based on the high conservation of plant chloroplast genomes in evolution, the PCR primers were designed and synthesized according to the corresponding sequences in the chloroplast genomes of Nicotiana tobacum, Spinacia and Oryza sativa for amplifying and cloning two important chloroplast genes from sugar beet, rbcL gene and atpB gene （with accessory No. as DQ067450 and DQ067451 in GenBank respectively） as homologous recom-bination fragments to direct targeted foreign genes into the chloroplast genome. Constructed vector of Bt gene CrylAc for sugar beet chloroplast transformation. The constructed vector contain homologous recombination fragments, Bt gene CrylAc, aadA gene, strong chloroplast promoter Prrn and TpsbA. The results of restriction enzyme analysis were in accord with the desired. Bioassays using crude expressed products from host strain of the vector showed that Bt CrylAc gene was expressed and its products were strongly toxic to larvae of Barathra brassicae. The chloroplast transformation vector will be useful value for sugar beet chloroplast genetic transformation and the insect resistant crop improvement. Transformation and consequent works are in progress.