SNP对褐飞虱卵黄原蛋白基因的表达调控研究

Population quantity of agricultural pests is a key factor influencing crop yield. Expression regulation of Vitellogenin (Vg) plays vital role in insect fecundity regulation. Current research on the regulation of Vg gene mainly focuses on hormone, transcription factor and nuclear receptor. In recent years, many studies have showed that Single Nucleotide Polymorphisms (SNPs) are involved in regulating gene transcription. However, no study on Vg transcription regulation by SNP has been conducted. In this study, we will investigate transcription regulation of Vg gene by SNPs in the Brown Planthopper, one of the most serious pest of rice plants. We have identified several SNPs within the Vg promoter region, which are related with population dynamics. Firstly, luciferase activities of different alleles of SNPs are detected by dual-luciferase reporter assay system. Secondly, transcription factor located in the region containing SNPs are predicted by bioinformatics methods. Furthermore, the function of transcription factor and SNPs involved in Vg gene regulation are studied by RNAi in vivo, overexpression in cell and EMSA in vitro. Finally we illuminate the molecular mechanism of Vg gene expression regulation and population regulation by interaction between SNPs and transcription factor. This study not only dissect the mechanism of Vg gene transcription at SNP level, but also make a link-up between population regulation and the function of molecular marker.

害虫的种群数量是影响农作物产量的重要因素。昆虫卵黄原蛋白（Vitellogenin, Vg）的表达调控是决定昆虫繁殖力的关键。目前，对昆虫Vg基因的表达调控主要集中在激素、转录因子和核受体上。近年来，单核苷酸多态性（SNP）参与基因转录调控的研究不断增多，但尚未有SNP参与Vg基因转录调控的报道。本项目以水稻重要害虫褐飞虱为研究对象，利用已鉴定到的Vg基因启动子上与种群动态相关的SNP，首先通过双荧光素酶报告系统鉴定SNP对Vg基因启动子活性的影响，然后用生物信息学方法预测与SNP所在区域结合的转录因子，并通过RNA干扰、细胞内过表达和体外凝胶迁移实验鉴定转录因子和SNP参与调控Vg基因的功能，初步阐明转录因子介导的SNP调控褐飞虱Vg基因表达进而影响种群动态的分子机理。本项目不仅从SNP水平深入剖析Vg基因转录调控的分子机理，而且将宏观的昆虫种群动态调节与微观的分子标记的功能联系起来。

昆虫卵黄原蛋白（Vitellogenin, Vg）基因的表达调控是决定昆虫繁殖力的关键。我们的前期工作已经鉴定出褐飞虱Vg基因上与繁殖力相关的单核苷酸多态性（Single Nucleotide Polymorphisms, SNPs）。本项目拟在此基础上，深入研究SNPs调控Vg基因表达的分子机制。主要研究包括：通过双荧光素酶报告系统鉴定SNPs对Vg基因启动子活性的功能；鉴定与上述SNPs所在区域结合的转录因子及其功能；研究转录因子如何通过SNPs调控Vg基因的表达。.我们利用定点突变技术构建携带SNPs不同基因型的Vg基因启动子报告载体，在昆虫细胞Hi5和Sf9中检测Vg基因的荧光素酶活性变化。结果显示当Vg-1191和Vg-953从高繁殖力基因型突变为低繁殖力基因型时，Vg启动子的荧光活性在两种昆虫细胞系中都显著降低，表明这两个SNPs参与了对Vg基因的转录调控。我们进一步分析发现Vg-953位于转录因子GATA-1的结合区域，然后通过在昆虫细胞中过表达GATA-1，发现过表达GATA-1可以显著提高Vg基因的转录活性，而且携带高繁殖力基因型（T等位基因）的活性显著高于携带低繁殖力基因型（A等位基因）的报告载体，进一步确认了Vg启动子上的SNPs差异造成Vg与转录因子GATA-1的结合活性变化，从而影响Vg基因的表达，最终影响褐飞虱的繁殖力。同时，我们研究发现angiotensin converting enzyme（ACE）基因不但与Vg基因的表达密切相关，而且ACE基因的启动子区存在一个转录因子E74A的结合位点，我们推测褐飞虱也可能通过ACE基因上的SNPs影响与昆虫重要转录因子E74A的结合，从而影响Vg的表达并最终影响褐飞虱繁殖力。然后我们通过RNAi技术进一步确认无论干涉E74A基因还是ACE基因，Vg基因都显著下调，而且E74A可能是通过ACE进一步调控Vg基因。.研究成果从分子水平上揭示了水稻害虫褐飞虱在适应环境过程中高繁殖力的分子进化机制，将SNPs与昆虫繁殖力表型联系起来。