苏云金芽孢杆菌新基因cry1Ab17的克隆和生物信息学

利用高效克隆PCR产物的专用载体pMD18T,直接从苏云金芽孢杆菌WB9的PCR产物中克隆了cry1Ab17新基因.测序结果表明,该基因(GenBank登录号为AY646166)由3471个碱基组成,其编码的蛋白质含有1156个氨基酸残基,其中亲水性氨基酸占30.8%,疏水性氨基酸占45.2%,酸性氨基酸占12.9%,碱性氨基酸占11.1%.氨基酸序列的同源性分析结果表明,Cry1Ab17蛋白与已报道的Cry1Ab蛋白同源性为95.4%～99.7%,该蛋白的4个氨基酸残基———Pro170、Gly449、Gly796和Gly863与其它已报道Cry1Ab蛋白相应位置的氨基酸残基均不同.在核苷酸序列和氨基酸序列多重比较的基础上,应用PAUP4.0构建了Cry1A蛋白家族的系统发育树.SignalP分析结果显示,Cry1Ab17蛋白中不含信号肽序列.此外,对Cry1Ab17蛋白的二级结构和3个结构域也进行了预测和分析.图4表2参15

Cloning and Bioinformatics of a New Gene,cry1Ab17 from Bacillus thuringiensis

A new gene, cry1Ab17 was cloned from Bacillus thuringiensis WB9 by PCR with pMD18-T vector. Nucleotide sequences of cry1Ab17 were registered in GenBank with accession No. AY646166. It consists of 3 471 bases, which encoded a protein of 1 156 amino acids residues with 30.8% of hydrophilic amino acids, 45.2% of hydrophobic amino acids, 12.9% of acidic amino acids and 11.1% of basic amino acids. The deduced amino acid sequence of Cry1Ab17 showed 95.4% to 99.7% identity with those of the known Cry1Ab proteins. Four residues (Pro_ 170, Gly_ 449, Gly_ 796 and Gly_ 863) of Cry1Ab17 were found different from those of the respective corresponding position in all known Cry1Ab proteins. On the basis of the multiple alignment of the nucleotide and amino acid sequences of Cry1Aa, Cry1Ac, Cry1Ad, Cry1Ae, Cry1Ag and Cry1Ab, a phylogenetic tree was constructed using PAUP 4.0. SignalP analysis revealed that no signal peptide was found in Cry1Ab17. In addition, the secondary structure and three domains of Cry1Ab17 were also predicted and analysed. Fig 4, Tab 2, Ref 15