PgCad基因特异转录因子的鉴定及其DNA结合位点突变介导的Cry1Ac抗性机制

Pest resistance to Bt restricts the sustainable utilization of Bt crops. The occurrence of resistance is mainly caused by the mutation or the expression alteration of Bt receptor gene in the midgut of insects, while the molecular mechanism of expression regulation of Bt receptor gene still remains to be unveiled. In our previous study, a Cry1Ac-resistant strain JL25 of pink bollworm was obtained, which was mediated by a significant down-regulation of the midgut cadherin receptor (PgCad) gene expression. Gene mutation was found in the promoter of PgCad but there was no decrease in transcriptional activity in insect cell lines, and the activity of both mutant promoter and non-mutant promoter of PgCad was very low. In view of insect cell lines are different from midgut tissue cells, we infer that there may be a specific transcription factor to regulate the expression of PgCad in the midgut of pink bollworm. In this proposal, we plan to screen and identify the specific transcription factor that regulates the expression of PgCad in the midgut of pink bollworm using yeast one-hybrid technique and the dual luciferase reporter gene assay system, and identify transcription factor binding sites using site-directed mutagenesis and electrophoretic mobility shift assays, and verify the regulation of this transcription factor on PgCad expression through RNAi and other technologies to clarify the molecular mechanism of JL25 resistance to Cry1Ac. This research will promote the understanding of the interaction mechanism between target insects and Bt proteins, and provide a theoretical basis for pest resistance monitoring and resistance management to Bt crops.

害虫对Bt的抗性制约着Bt作物的持续应用。抗性的产生主要由昆虫中肠Bt受体基因突变或表达量变化导致，而Bt受体基因表达调控的分子机理尚待解析。本团队获得一个红铃虫抗Cry1Ac品系JL25，其抗性由中肠钙粘蛋白受体基因（PgCad）表达量显著下调介导；PgCad启动子发生突变，但昆虫细胞系中未检测到其转录活性下降，且与未突变启动子的活性都很低。鉴于昆虫细胞系不同于中肠组织细胞，推测红铃虫中肠可能存在某种特异转录因子调控PgCad的表达。本项目拟通过酵母单杂交与双荧光素酶报告基因检测系统，筛选并鉴定调控红铃虫中肠PgCad表达的特异转录因子；利用定点突变与凝胶阻滞，明确该转录因子的结合位点；通过RNAi等技术验证该转录因子对PgCad表达的调控，阐明JL25对Cry1Ac抗性的分子机制。本研究将有助于加深靶标昆虫与Bt蛋白之间互作机制的认识，为害虫对Bt作物的抗性监测与抗性治理提供理论依据。

害虫对Bt的抗性制约着Bt作物的持续应用。抗性的产生主要由昆虫中肠Bt受体基因突变或表达量变化导致，而Bt受体基因表达调控的分子机理尚待解析。前期本团队获得一个红铃虫抗Cry1Ac品系JL25，其抗性由中肠钙粘蛋白受体基因（PgCad）表达量显著下调介导；PgCad启动子发生突变，但昆虫细胞系中未检测到其转录活性下降，且与未突变启动子的活性都很低。鉴于昆虫细胞系不同于中肠组织细胞，推测红铃虫中肠可能存在某种特异转录因子调控PgCad的表达。本项目通过酵母单杂交筛选、中肠转录因子的克隆与双荧光素酶报告基因检测，筛选并鉴定出了9个对红铃虫PgCad基因启动子转录活性具有显著正调控作用的转录因子，其中GATAe对PgCad基因启动子的调控最显著，其次是GATA-4-like，但两种转录因子对抗、感品系PgCad启动子的转录活性均没有显著差异；与敏感品系相比，抗性品系中9个转录因子均没有发生基因突变，但AP-2-epsilon的基因表达水平显著降低；利用凝胶阻滞，明确了转录因子GATAe和GATA-4-like的DNA结合位点；通过RNA干扰实验明确了GATAe的沉默显著降低了红铃虫PgCad受体基因的表达。结果表明：GATAe是调控红铃虫中肠PgCad基因表达的关键转录因子；转录因子AP-2-epsilon的基因表达水平显著降低很可能是导致JL25抗性品系PgCad表达显著下调的因素之一。本研究有助于加深靶标昆虫与Bt蛋白之间互作机制的认识，为害虫对Bt作物的抗性监测与抗性治理提供理论依据。