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PCR-aided synthesis and stable expression in E.coli of the cryIA(c)Bt gene

文献类型： 外文期刊

作者： Peng, RH 1 ; Xiong, AS 2 ; Li, X 2 ; Fan, HQ 2 ; Huang, XM 2 ; Yao, QH 2 ;

作者机构： 1.Shanghai Acad Agr Sci, Shanghai Key Lab Agr Genet & Breeding, Shanghai 201106, Peoples R China

2.Shanghai Acad Agr Sci, Shanghai Key Lab Agr Genet & Breeding, Shanghai 201106, Peoples R China; Shanghai Acad Agr Sci, Agrobiotechnol Res Ctr, Shanghai 201106, Peoples R China

关键词： chemical synthesis;cryIA(c)Bt gene;gene stable expression;insecticide activity

期刊名称：ACTA BIOCHIMICA ET BIOPHYSICA SINICA （ 影响因子：3.848； 五年影响因子：3.67 ）

ISSN： 0582-9879

年卷期： 2001 年 33 卷 2 期

页码：

收录情况： SCI

摘要： Insecticidal crystal proteins, known as delta -endotoxins, from a gram-positive bacterium Bacillus thuringiensis have been used as bio-pesticides for over 3 decades. By using a successive PCR method([12]), the 1.8 kb cryIA(c)Bt gene coding for the fragment of protoxin was synthesized. Different rom B. thuringiensis subsp. kurstaki Hd 73 cry gene, the synthesized gene has the codon usage pattern of an average Pseudomonas spp gene. 614 nucleotides were changed in the synthesized cryIA(c)Bt gene and the G + C content: was increased from 37.2% to 64%. The synthesized cryIA(c)Br gene was cloned into pUT56 vector under the tac promoter and T1-T2 terminator. SDS-PAGE analysis revealed that 66 kD protein of the modified cryIA (c)Bt gene was expressed in E. coli and accounted for about 30% of total protein in the bacterial cells. Bioassays using crude expression products from host strains indicated that they had high toxicity to third instar larvae of the cabbage butterfly (Pieris brassicae). The LD50 was calculated to be 0.024 mug/cm(2).

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