Extended screening by PCR for seven cry-group genes from field-collected strains of Bacillus thuringiensis
Extended screening by PCR for seven cry-group genes from field-collected strains of Bacillus thuringiensis
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DOI：
10.1128/aem.63.12.4883-4890.1997
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发表时间：
1997-12-01
影响因子：
4.4
通讯作者：
Margalith, Y
中科院分区：
文献类型：
--
作者：
BenDov, E;Zaritsky, A;Margalith, Y
建立了一种扩展的多重聚合酶链反应（PCR）方法，用于快速鉴定和分类含有对鳞翅目、鞘翅目和双翅目昆虫有毒的cry（晶体蛋白）基因的苏云金芽孢杆菌菌株。该技术丰富了现有策略，通过确定含有特定基因或具有潜在杀虫活性的独特感兴趣组合的菌株，简化了大规模筛选cry基因的初始阶段，从而有利于后续的毒性测定。设计了五对通用引物来探测高度保守的序列，并对以下几组中已知的大多数（约60个中的34个）基因进行分类：20个cry1、3个cry2、4个cry3、2个cry4、2个cry7和3个cry8基因。用为其中20个基因以及cry11A设计的一组特异性引物对每个阳性菌株的DNA进行探测。从126株田间采集的苏云金芽孢杆菌菌株中鉴定出22种不同的cry类型图谱。其中一些被发现与所有已发表的图谱不同。一些田间采集的菌株对cry2Ac呈阳性，但16株标准菌株均无此情况。3株标准菌株和38株田间采集的菌株用通用引物检测呈阳性，但用针对cry7和cry8的所有五个已知基因的特异性引物检测呈阴性。这些田间采集的菌株似乎含有一个或多个新基因，这些基因对于昆虫的生物防治和抗性管理似乎很有前景。
An extended multiplex PCR method was established to rapidly identify and classify Bacillus thuringiensis strains containing cry (crystal protein) genes toxic to species of Lepidoptera, Coleoptera, and Diptera. The technique enriches current strategies and simplifies the initial stages of large-scale screening of cry genes by pinpointing isolates that contain specific genes or unique combinations of interest with potential insecticidal activities, thus facilitating subsequent toxicity assays. Five pairs of universal primers were designed to probe the highly conserved sequences and classify most (34 of about 60) genes known in the following groups: 20 cry1, 3 cry2, 4 cry3, 2 cry4, 2 cry7, and 3 cry8 gens. The DNA of each positive strain was probed with a set of specific primers designed for 20 of these genes and for cry11A. Twenty-two distinct cry-type profiles were identified from 126 field-collected B. thuringiensis strains. Several of them were found to be different from all published profiles. Some of the field-collected strains, but none of the 16 standard strains, were positive for cry2Ac. Three standard and 38 field-collected strains were positive by universal primers but negative by specific primers for all five known genes of cry7 and cry8. These field-collected strains seem to contain a new gene or genes that seem promising for biological control of insects and management of resistance.