苏云金杆菌毒性蛋白基因5′端的PCR合成、克隆及序列测定
PCR Synthesizing、 Cloning and Sequencing of 5’ End of BT Toxin Gene
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摘要: 以苏云金杆菌戈尔斯德亚种(Bacillus thuringiensis var. kurstaki HD-1)的总DNA为模板,用多聚酶链反应(PCR)的方法,成功合成了该菌毒性蛋白基因5′端片段,并克隆到大肠杆菌Bluescript质粒载体上,获得了重组质粒pQC20。核酸限制酶谱分析表明,pQC20的重组片段属于Kronstad的毒性蛋白基因限制酶片段长度多型性分类系统中5.3 Kb类型基因。对该基因片段的全序列分析结果证明,在以PCR合成的1959个核苷酸中与国外已发表的5.3 Kb类型基因的相应片段仅有一个核苷酸差异。该基因片段具有表达有生物活性的毒性蛋白所必需的全部核苷酸序列。Abstract: By Polymerase Chain Reaction(PCR), the 5’ end of Bacillus thuringieusis var. kurstaki HD-1 toxin gene has been synthesized, using the total DNA of the bacterium as template. The recombinant plasmid pQC20 has been obtained, cloning the PCR fragment into the Bluescript plasmid vector in E. coli. According to the restriction map, the insert of pQC20 belongs to the 5.3 Kb type gene in the Kronstad’s classification system for restriction fragment of length polytype of toxin protein gene. The sequencing result showed that, the synthesized 1959 bp nucleotides are almost the same as the published sequence of 5.3 Kb type toxin gene with only one-nucleotide difference. This 5’ end fragment contains the complete sequence for encoding the activated toxin.
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Keywords:
- BT toxin /
- PCR /
- Cloning /
- DNA sequencing
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[11] Shin-ichi Kondo, Nobuya Tamura et al. 1987. Cloning and nucleotide sequencing of two insecticida,endotoxin genes from Bacillus thuringiensis var. kurstaki HD-I DNA. Agri. Biol. Chem. 51:955-963
[12] Vacck M, A Reynaerts et al. 1987. Transgenic plants protected from insect attack. Nature. 328 j 33-37
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