研究野生型苏云金芽孢杆菌Cry1Aa和Cry1C的毒性变化发现,不同的pH不但影响这些蛋白质的毒性,而且影响它们在跨膜过程中形成孔洞的能九将 Cry1 Aa α4螺旋中的15个氨基酸突变后与BBMV结合,进行光散射分析,与野生型Cry1Aa相比较,发现有3...研究野生型苏云金芽孢杆菌Cry1Aa和Cry1C的毒性变化发现,不同的pH不但影响这些蛋白质的毒性,而且影响它们在跨膜过程中形成孔洞的能九将 Cry1 Aa α4螺旋中的15个氨基酸突变后与BBMV结合,进行光散射分析,与野生型Cry1Aa相比较,发现有3个突变体几乎完全失去毒性,7个突变体毒性明显降低,5个突变体保持野生型毒性.采用计算机模拟方法研究了苏云金芽孢杆菌Cry1Aa毒蛋白α4螺旋的三维空间结构,通过观察15个不同残基定点突变对其功能的影响,解释了突变体毒性变化的原因,说明了参与膜孔洞形成氨基酸残基对Cry1Aa昆虫毒杀性的重要作用.展开更多
采用两步 PCR法成功地克隆了一个全长的 c DNA.首先 ,用差式分析法克隆得到差别表达的 c DNA片段 ,再分别用这些片段内部的特异序列及 c DNA两端不同接头的序列为引物进行第一步 PCR扩增 ,得到差别 c DNA片段的上游和下游序列 .然后 ,...采用两步 PCR法成功地克隆了一个全长的 c DNA.首先 ,用差式分析法克隆得到差别表达的 c DNA片段 ,再分别用这些片段内部的特异序列及 c DNA两端不同接头的序列为引物进行第一步 PCR扩增 ,得到差别 c DNA片段的上游和下游序列 .然后 ,根据第一步 PCR扩增得到的上游和下游序列设计基因特异的引物进行第二步 PCR,从而得到全长的 c DNA.展开更多
Development of targeted biological agents against agricultural insect pests is of prime importance for the elaboration and implementation of integrated pest management strategies that are environment-friendly, respect...Development of targeted biological agents against agricultural insect pests is of prime importance for the elaboration and implementation of integrated pest management strategies that are environment-friendly, respectful of bio-diversity and safer to human health through reduced use of chemical pesticides. A major goal to understand how Bt toxins work is to elucidate the functions of their three domains. Domains II and III are involved in binding specificity and structural integrity, but the function of Domain I remains poorly understood. Using a Manduca sexta BBMV (brush border membrane vesicles) system, we analyzed its responses to Cry1Aa 15 single-point mutations with altered Domain I helix 4 residues. Light scattering assay showed that toxicity was almost lost in 3 mutants, and we observed significantly reduced toxicity in other 7 mutants. However, 5 mutants retained wild-type toxicity. Using computer software, we simulated the three-dimensional structures of helix 4. Both experimental and bioinformatic analysis showed that residues in Cry1Aa Domain I helix 4 were involved in the formation of ion channels that is critical for its insect toxicity.展开更多
文摘研究野生型苏云金芽孢杆菌Cry1Aa和Cry1C的毒性变化发现,不同的pH不但影响这些蛋白质的毒性,而且影响它们在跨膜过程中形成孔洞的能九将 Cry1 Aa α4螺旋中的15个氨基酸突变后与BBMV结合,进行光散射分析,与野生型Cry1Aa相比较,发现有3个突变体几乎完全失去毒性,7个突变体毒性明显降低,5个突变体保持野生型毒性.采用计算机模拟方法研究了苏云金芽孢杆菌Cry1Aa毒蛋白α4螺旋的三维空间结构,通过观察15个不同残基定点突变对其功能的影响,解释了突变体毒性变化的原因,说明了参与膜孔洞形成氨基酸残基对Cry1Aa昆虫毒杀性的重要作用.
文摘采用两步 PCR法成功地克隆了一个全长的 c DNA.首先 ,用差式分析法克隆得到差别表达的 c DNA片段 ,再分别用这些片段内部的特异序列及 c DNA两端不同接头的序列为引物进行第一步 PCR扩增 ,得到差别 c DNA片段的上游和下游序列 .然后 ,根据第一步 PCR扩增得到的上游和下游序列设计基因特异的引物进行第二步 PCR,从而得到全长的 c DNA.
基金This work was supported by the China-Canada 3x3 Cooperation Project
文摘Development of targeted biological agents against agricultural insect pests is of prime importance for the elaboration and implementation of integrated pest management strategies that are environment-friendly, respectful of bio-diversity and safer to human health through reduced use of chemical pesticides. A major goal to understand how Bt toxins work is to elucidate the functions of their three domains. Domains II and III are involved in binding specificity and structural integrity, but the function of Domain I remains poorly understood. Using a Manduca sexta BBMV (brush border membrane vesicles) system, we analyzed its responses to Cry1Aa 15 single-point mutations with altered Domain I helix 4 residues. Light scattering assay showed that toxicity was almost lost in 3 mutants, and we observed significantly reduced toxicity in other 7 mutants. However, 5 mutants retained wild-type toxicity. Using computer software, we simulated the three-dimensional structures of helix 4. Both experimental and bioinformatic analysis showed that residues in Cry1Aa Domain I helix 4 were involved in the formation of ion channels that is critical for its insect toxicity.
