Zhang Fengjiao, Shu Changlong, Crickmore Neil, Li Yanqiu, Song Fuping, Liu Chunqin, Chen Zhibao, Zhang Jie
College of Life Science and Technology, Heilongjiang Bayi Agricultural University, Daqing, People's Republic of China.
State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing, People's Republic of China.
Appl Environ Microbiol. 2016 Jun 13;82(13):3808-3815. doi: 10.1128/AEM.00862-16. Print 2016 Jul 1.
With the aim of optimizing the cloning of novel genes from a genomic pool containing many previously identified homologous genes, we designed a redundant exclusion PCR (RE-PCR) technique. In RE-PCR, a pair of generic amplification primers are combined with additional primers that are designed to specifically bind to redundant, unwanted genes that are a subset of those copied by the amplification primers. During RE-PCR, the specific primer blocks amplification of the full-length redundant gene. Using this method, we managed to clone a number of cry8 or cry9 toxin genes from a pool of Bacillus thuringiensis genomic DNA while excluding amplicons for cry9Da, cry9Ea, and cry9Eb The method proved to be very efficient at increasing the number of rare genes in the resulting library. One such rare (and novel) cry8-like gene was expressed, and the encoded toxin was shown to be toxic to Anomala corpulenta
Protein toxins from the bacterium Bacillus thuringiensis are being increasingly used as biopesticides against a wide range of insect pests, yet the search for new or improved toxins is becoming more difficult, as traditional methods for gene discovery routinely isolate previously identified clones. This paper describes an approach that we have developed to increase the success rate for novel toxin gene identification through reducing or eliminating the cloning of previously characterized genes.
为了优化从包含许多先前已鉴定的同源基因的基因组库中克隆新基因的方法，我们设计了一种冗余排除PCR（RE-PCR）技术。在RE-PCR中，一对通用扩增引物与额外的引物相结合，这些额外引物被设计用于特异性结合冗余的、不需要的基因，这些基因是由扩增引物复制的基因的一个子集。在RE-PCR过程中，特异性引物会阻止全长冗余基因的扩增。使用这种方法，我们成功地从苏云金芽孢杆菌基因组DNA库中克隆了多个cry8或cry9毒素基因，同时排除了cry9Da、cry9Ea和cry9Eb的扩增子。该方法被证明在增加所得文库中稀有基因的数量方面非常有效。其中一个这样的稀有（且新的）cry8样基因被表达，并且所编码的毒素对暗黑鳃金龟有毒。
来自苏云金芽孢杆菌的蛋白质毒素正越来越多地被用作针对多种害虫的生物杀虫剂，然而，寻找新的或改良的毒素变得更加困难，因为传统的基因发现方法通常会分离出先前已鉴定的克隆。本文描述了一种我们开发的方法，通过减少或消除先前已表征基因的克隆来提高新毒素基因鉴定的成功率。
