Sauka Diego H, Rodriguez Sonia E, Benintende Graciela B
Insumos Bacterianos, Instituto de Microbiología y Zoología Agrícola (IMYZA), Instituto Nacional de Tecnología Agropecuaria (INTA), Castelar, Argentina.
J Mol Microbiol Biotechnol. 2012;22(6):373-80. doi: 10.1159/000345911. Epub 2013 Jan 10.
Bacillus thuringiensis is an entomopathogenic bacterium characterized by producing parasporal proteinaceous insecticidal crystal inclusions during sporulation. Many strains are capable of also expressing other insecticidal proteins called Vip during the vegetative growing phase. Particularly, Vip3A proteins have activity against certain Lepidoptera species through a unique mechanism of action which emphasized their possible use in resistance management strategies against resistant pests. The aim of the work was to develop a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method that can distinguish between vip3A genes from B. thuringiensis strains. In addition, 4 novel vip3Aa genes were cloned and sequenced. The method was originally based on amplification of a single PCR amplicon and the use of 2 restriction enzymes with recognition sites that facilitate simultaneous detection. Subsequently, a third restriction enzyme was used to distinguish between vip3A variants. Thirteen vip3Aa genes were identified in strains belonging to 10 different B. thuringiensis serovars. Three intra-subclass variants of vip3Aa genes could be differentiated. The presented method can serve as an invaluable tool for the investigation of known and novel vip3A genes in B. thuringiensis strains. To the best of our knowledge, this is the first report where variants of a same subclass of insecticidal genes could be distinguished following PCR-RFLP.
苏云金芽孢杆菌是一种昆虫病原细菌，其特征是在孢子形成过程中产生伴孢蛋白类杀虫晶体包涵体。许多菌株在营养生长阶段还能够表达其他称为Vip的杀虫蛋白。特别是，Vip3A蛋白通过独特的作用机制对某些鳞翅目物种具有活性，这突出了它们在抗药性害虫抗性管理策略中的潜在用途。这项工作的目的是开发一种聚合酶链反应-限制性片段长度多态性（PCR-RFLP）方法，该方法可以区分苏云金芽孢杆菌菌株中的vip3A基因。此外，还克隆并测序了4个新的vip3Aa基因。该方法最初基于单个PCR扩增子的扩增以及使用具有便于同时检测的识别位点的2种限制性内切酶。随后，使用第三种限制性内切酶来区分vip3A变体。在属于10种不同苏云金芽孢杆菌血清型的菌株中鉴定出13个vip3Aa基因。可以区分vip3Aa基因的3种亚类内变体。所提出的方法可作为研究苏云金芽孢杆菌菌株中已知和新的vip3A基因的宝贵工具。据我们所知，这是第一份关于通过PCR-RFLP区分同一亚类杀虫基因变体的报告。
