基于基因组水平的细胞色素P450基因农药响应元件的预测和验证

The over-expression of cytochrome P450 gene is one of the important reasons conferring insecticide resistance. However, the mechanism of insecticide induced over-expression of P450 genes remains unclear. In fruit fly, butterflies and mosquitoes, it has been shown that plant secondary metabolites induce the over-expression of P450 genes by the Cis-elements in the promoter regions. This proposal intends to find insecticide response element (IRE) in the resistance associated P450 genes in rice stem borer. First, P450 gene sequences will be obtained from the genome sequences. Then, we will use promoter prediction software, such as NNPP, to predict the promoter regions. Second, we will collect pests in the fields from different place. The bioassay analysis will be carried out to determine the resistance ratio of the pests to conventional used insecticides. Third, the expression levels of P450 genes will be detected by using microarray. The enzyme activities will be measured by using kinetic method. By these methods, we can determine the resistance associated P450 genes at large scale level. Fourth, we will use MDScan to find the motifs in the promoters of resistance-associated or non-resistance associated P450 genes. The insecticide response elements will be found by further bioinformatics analysis. Fifth, we will use site-directed mutagenesis technique and insect cell expression system to validate the reliability of identified IRE. Finally, a new algorithm will be developed to predict IRE in the promoter of P450 genes at genome level and an online prediction service platform will be provided. This project has an important scientific significance in understanding the metabolic mechanism of insecticide resistance and has a very important application value in chemical control of insect pests.

细胞色素P450基因增强表达是导致农业害虫对农药产生代谢抗性的重要机制，但P450基因响应农药诱导的机制尚不清楚。研究表明，果蝇、蝴蝶、蚊子等昆虫P450基因的启动子区存在植物次生代谢物质的响应调控元件。本项目拟对农业害虫二化螟基因组序列进行挖掘，获得完整P450基因序列，用NNPP等软件获得P450基因启动子序列；综合生测数据、P450基因表达谱芯片数据和P450基因酶活力数据，确定抗药性相关的P450基因；用MDScan等软件和关联分析方法，筛选出P450基因启动子区与农药诱导相关的顺式作用元件（简称农药响应元件）；采用定点突变技术和表达载体技术对这些农药响应元件进行验证。在此基础上，开发基因组层面的P450基因农药响应元件预测算法，并提供在线预测服务平台。本项目的完成不仅在害虫抗药性机理深入研究方面有重要的科学意义，而且在害虫化学防治方面具有重要的应用价值。

P450基因的高表达是导致抗药性产生的一个重要原因。本项目以农业害虫二化螟为试虫，全面筛查P450基因，构建相关在线服务数据库及软件，并根据P450基因组基因序列寻找抗性产生机制。本项目于2013年立项，开展了如下研究：组装并注释二化螟基因组序列，在此基础上全面筛查P450基因，对敏感品系和抗性品系害虫进行转录组测序和生物信息学分析，获得抗性相关P450基因，分析P450基因的基因组序列，找到与抗性相关的元件。项目经过三年实施，完成了如下预定的研究内容，并且取得了一些重要的成果，主要包括以下几个方面：设计开发了基因组注释分析平台（OMIGA/EGAS），可以准确可靠地预测昆虫基因，收集整理二化螟基因组、转录组等各种相关数据，构建了在线二化螟数据库（ChiloDB），筛查P450基因并进行家族分类和验证，比较分析抗氯虫苯甲酰胺品系和敏感品系转录组表达数据，筛选出可能与抗性相关的2893个基因，其中17个P450基因具有显著表达差异；对差异表达P450基因序列的启动子元件进行分析，没有发现显著相关的农药响应元件。本项目较好地完成了计划任务，为二化螟研究提供了大量的基因数据，为二化螟研究奠定了数据基础；此外，抗性品系和敏感品系间的比较分析结果为抗性深入研究提供了重要的线索。本项目已发表标注论文5篇，其中SCI论文4篇，1个在线服务数据库平台、1个基因组分析软件，较好地完成了项目预期目标。