Abstract:

Bacillus thuringiensis could produce insecticide crystal proteins (ICPs), which had strong toxicity to sensitive insects, but no toxicity to higher animals and human. A full length of 2.1 kb cry1I gene fragment was obtained by PCR amplification using primers designed according to cry1I-type gene sequences with the plasmid DNA of Bt LB52 strain as template. It could express 79.9 kDa protein in the Escherichia coli by the expression vector pEB. By AlginX analysis, its protein was composed of 712 amino acids. The molecular weight was 79.9 kDa, and the isoelectric point was 6.54, a weak acid protein. By blasting on NCBI, the cry1Ib6s amino acid sequences had 98% similarity with Cry1Ib3, with 12 amino acids different. The gene has been registered in GenBank with accession number ADK38579 and was named as cry1Ib6 by International Nomenclature Committee. Cry1Ib6 protein products had high toxicity to Plutella xylostella, with Lethal concentration (LC50) 1.196 μg/mL, which provided a new gene for  insect-resistant transgenic plants.

Key words: Bacillus thuringiensis, cry1Ib6 gene, cloning, expression, biology activity

摘要：

苏云金芽胞杆菌(Bacillus thuringiensis)能产生杀虫晶体蛋白(Insecticida Crystal Proteins, ICPs),对敏感昆虫有强烈毒性,而对高等动物和人无毒性。ICPs由cry或cyt基因编码,根据cry1I型基因设计引物,以Bt LB52菌株的质粒DNA为模板,扩增出了全长为2.1 kb的cry1I基因,其能通过表达载体pEB在大肠杆菌中高效表达为79.9 kDa的蛋白。经过AlginX软件分析该蛋白由712个氨基酸组成,分子量为79.9 kDa,等电点为6.54,为弱酸性蛋白质,NCBI Blast比对该蛋白的氨基酸序列与Cry1Ib3的相似性最高为98%,有12个氨基酸的差异。该基因已在GenBank中注册,登录号为ADK38579,并被国际基因命名委员会正式命名为cry1Ib6。它的表达产物对小菜蛾具有较高的毒力,LC50为1.196 μg/mL,为抗虫转基因植物研究提供了新的基因。

关键词: 苏云金芽胞杆菌;cry1Ib6基因;克隆;表达;生物活性