Li Ying, Shu Changlong, Zhang Xuewen, Crickmore Neil, Liang Gemei, Jiang Xingfu, Liu Rongmei, Song Fuping, Zhang Jie
State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing 100193, PR China.
Northeast Agricultural University, Harbin 150030, PR China.
J Invertebr Pathol. 2014 Oct;122:6-9. doi: 10.1016/j.jip.2014.07.006. Epub 2014 Aug 6.
There has been considerable effort made in recent years for research groups and other organizations to build up large collections of strains of Bacillus thuringiensis in the search for genes encoding novel insecticidal toxins, or encoding novel metabolic pathways. Whilst next generation sequencing allows the detailed genetic characterization of a bacterial strain with relative ease it is still not practicable for large strain collections. In this work we assess the practicability of mining a mixture of genomic DNA from a two thousand strain collection for particular genes. Using PCR the collection was screened for both a rare (cry15) toxin gene as well as a more commonly found gene (vip3A). The method was successful in identifying both a cry15 gene and multiple examples of the vip3A gene family including a novel member of this family (vip3Aj). A number of variants of vip3Ag were cloned and expressed, and differences in toxicity observed despite extremely high sequence similarity.
近年来，研究团队和其他组织付出了巨大努力，建立了大量苏云金芽孢杆菌菌株库，以寻找编码新型杀虫毒素或新型代谢途径的基因。虽然新一代测序技术能相对轻松地对细菌菌株进行详细的基因特征分析，但对于大型菌株库来说，这仍然不可行。在这项研究中，我们评估了从一个包含两千个菌株的文库中提取基因组DNA混合物以寻找特定基因的可行性。利用聚合酶链反应（PCR）对该文库进行筛选，以寻找一种罕见的（cry15）毒素基因以及一种更常见的基因（vip3A）。该方法成功鉴定出了一个cry15基因以及vip3A基因家族的多个实例，包括该家族的一个新成员（vip3Aj）。克隆并表达了多个vip3Ag变体，尽管它们的序列相似度极高，但仍观察到了毒性差异。
