中国生物防治学报Issue(5)：696-702,7.
基于聚合酶链式反应-高通量测序（PCR-HTS）联用的cry2A基因鉴定方法
Identification of cry2 Gene Based on Polymerase Chain Reaction-High Throughput Sequencing (PCR-HTS)
摘要
Abstract
cry2 genes, distributed widely in natural strains of Bacillus thuringiensis, are important and valuable due to their high activity against several lepidopteran pests and no cross-resistance with known cry1A genes. In order to improve the efficiency of screening and identification of cry2 insecticidal genes from B. thuringiensis, with analysis of the conserved blocks of cry2A, four pairs of universal primers were designed, and PCR was conducted with genomic DNA mixture from 2000 Bt isolates as the templates. These PCR product mixtures were sequenced by 454 High-throughput Sequencer. A total of 28024 Reads were obtained. Analysis of sequencing data showed that these Bt strains contain all the nine known cry2 genes and two unknown genes in China (cry2Ae and cry2Ai). Meanwhile, fragments of novel cry2 genes were revealed. With partial-length gene fragments cry2-1 and cry2-2 obtained by amplification with specific primers, the full length novel gene cry2X were gained by over lapping PCR. cry2X gene is composed of 1902 bp, and encodes 633 amino acid residues. Similarity of amino acid sequence of Cry2X is 93%with that of Cry2Ab13, which indicates cry2X is a third holo-type novel gene. These results indicate that the established new method for cry gene identification based on polymerase chain reaction-high throughput sequencing (PCR-HTS) is of many advantages:rapid, sensitive, accurate, high-throughput, and wide coverage. This approach can be used to identify known genes and discover novel gene from B. thuringiensis wild isolates, and will play a significant role in insecticidal gene identification and discovery from B. thuringiensis.关键词
苏云金芽胞杆菌/聚合酶链式反应/454高通量测序/cry2基因Key words
Bacillus thuringiensis/polymerase chain reaction/454 high throughput sequencing/cry2 gene分类
农业科技引用本文复制引用
陈桂花,束长龙,李颖,宋福平,郭媛媛,李国勋,张杰..基于聚合酶链式反应-高通量测序（PCR-HTS）联用的cry2A基因鉴定方法[J].中国生物防治学报,2014,(5):696-702,7.基金项目
国家“863”计划（2011AA10A203、2013AA102701）；转基因生物新品种培育重大专项 （）
