阿维链霉菌CRISPR-Cas系统的功能研究及基于该系统的抗噬菌体菌株的构建

Avermectins, the secondary metabolites of Streptomyces avermitilis, are effective pesticides which are mainly used in the agriculture in China. However, the reduction or complete loss of avermectins productivity caused by bacteriophages occurs in fermentation production frequently year by year .To avoid the failure caused by pollution of phage, a series of studies should be done. In the last few years, bacteriophages have been separated and purified from phage pollution fermentation liquor of avemectins by our lab. The founction of genes leading to host lysis such as endolysin and holin must be identified. CRISPR-Cas (clustered regularly interspaced short palindromic repeats, and associated proteins) described recently was an immune system providing resistance against phage. Three CRISPR loci flanked by eight cas genes have been found in S.avermitilis. The characterizations of CRISPR-Cas of S.avermitilis including the interference of genetic elements and acquisition of new spacers from intruding foreign genetic nucleic acid will be identified. Based on these studies，the bacteriophage-resistance strains conferred by CRISPR-Cas system will be constructed. The strategies to make sure the high production of avermecins will also be considered.

阿维链霉菌是高效杀虫剂阿维菌素的产生菌，在阿维菌素发酵工业中，因噬菌体污染造成的经济损失逐年加重。CIRSPR-Cas系统是存在于大部分细菌中抵抗外源核酸的免疫系统，它包含的CRISPR序列可以特异性识别外源核酸如质粒和噬菌体，在cas基因编码的Cas蛋白的协助下将外源核酸降解。根据预测阿维链霉菌中也存在CRISPR-Cas系统。本项目将从两方面进行研究：一方面着重分析阿维链霉菌中CRISPR-Cas系统的作用机制，通过质粒干扰实验研究该系统抵抗外源核酸的功能以及获取外源核酸片段成为新的间隔序列的能力；另一方面将对实验室已分离测序的阿维链霉菌噬菌体与裂解相关的功能基因进行研究，包括编码裂解酶、内溶素、穿孔素基因功能的鉴定。最后，针对噬菌体功能基因，构建基于CRISPR-Cas系统特点的抗噬菌体菌株。

阿维链霉菌是高效杀虫剂阿维菌素的产生菌，在阿维菌素发酵工业中，因噬菌体污染造成了严重的经济损失。CIRSPR-Cas系统是存在于大部分细菌中抵抗外源核酸的免疫系统，根据预测阿维链霉菌中也存在CRISPR-Cas系统。项目研究阿维链霉菌中CRISPR-Cas系统的特点，并根据其特点构建抗噬菌体菌株，取得如下研究成果：①检测了阿维链霉菌中CRISPR-Cas系统抵抗质粒的能力和从外源核酸中获取新的间隔序列的能力，明确了阿维链霉菌中CRISPR-Cas系统的基本特点；②完成了阿维链霉菌噬菌体phiSASD1中重要功能基因——内溶素基因和穿孔素基因的功能验证；③针对阿维链霉菌噬菌体的CRISPR序列，构建了依赖于CRISPR-Cas系统的特异性抗噬菌体的阿维链霉菌，得到了产量稳定且具有抗噬菌体活性的工程菌株。在完成上述工作的基础上，①还对另外一株阿维链霉菌噬菌体phiSAJS1进行了基因测序，并完成了功能注释。②构建另一株杀虫抗生素——多杀菌素的基因工程菌，提高了多杀菌素产量。