苏云金芽孢杆菌(Bt)晶体毒蛋白基因在烟草叶绿体中的表达

Expression of Bacillus thuringiensis (Bt) Crystal Toxin Gene in the Chloroplast of Tobacco

将全长３．５ｋｂ的Ｂｔ基因３'端缺失，得到长为２．１ｋｂ、１．８ｋｂ的基因。分别将这３个长度 （１．８ｋｂ２．１ｋｂ、３．５ｋｂ）的基因置于水稻叶绿体ｐｓｂＡ基因的启动于和终止子调控之下，并与选择 标记基因ａａｄＡ（编码氨基糖苷－３'－腺苷酸转移酶，具壮观霉素抗性）表达盒相连５以烟草叶绿 体基因ｔｒｎＨ－ｐｓｂＡ－ｔｒｎＫ为同源片段，构建成叶绿体转化载体ｐＢＴ３、ｐＢＴ８和ｐＢＴ２２。用基因枪 把Ｂｔ基因导入烟草叶绿体中，以壮观霉素筛选，获得转化再生植株。经Ｓｏｕｔｈｅｒｎ、Ｗｅｓｔｅｒｎ检测 分析证明Ｂｔ基因已整合进入烟草叶绿体基因组中并得到表达。且子代呈现壮观霉素抗性，即 外源基因得到稳定的遗传。利用转基因植株叶片对棉铃虫进行杀虫实验，有些转化植株表现 出较强的抗虫性。总体上来说，转Ｂｔ全长基因的烟草植株，其杀虫效果最好，其余两种差异不 大。首次报道将Ｂｔ基因成功转入高等植物叶绿体并获得表达。

The 3.5kb wild-type Bt Cry I A(c) gene and its 3' truncated forms (2.1 kb, l.8kb) were placed under the control of plastid expression signals consisting of the strong light-induced psbA promoter and its 3' untranslated region with the aadA cassette (Prrn, aadA and psbA3' ) as a selectable marker. The resulting vectors PBT3, PBTS and PBT22 also contain flanking tobacco plastid DNA homology regions to direct insertion of the Bt transgene into the tobacco plastid genome between psbA and trnK by homologous recombination. Transformed plastid genomes were selectively amplified by growing the cells on spectinomycin medium. Several independently transformed lines were obtained at last The results of Southern and Western blot demonstrated that these three kinds of Bt genes had been introduced into tobacco plants, and their filial generations are resistant to spectinomycin. Insecticidal activity assay with transgenic tobacco leaves indicate that some plants have strong toxicity to cotton bollworm. This is the first report in China that Bt gene has been introduced and successfully expressed in the chloroplast of higher plants.