小菜蛾Bt抗性关键基因MAP4K4过量表达的顺式调控机制

Insufficient understanding of the molecular mechanisms of insect resistance to Bacillus thuringiensis (Bt) insecticidal proteins seriously restricts the management of Bt resistance and the exploitation and application of novel Bt insecticidal proteins, and it also threatens the lifespan of Bt transgenic crops. Previously, our study showed that high-level resistance to Bt in Plutella xylostella (L.) was associated with altered expression of multiple Bt receptor genes trans-regulated by a pivotal constitutively transcriptionally-activated MAP4K4 gene in the upstream of mitogen-activated protein kinase (MAPK) signaling pathway. Nevertheless, how the MAP4K4 gene is transcriptionally activated and overexpressed is a key fundamental scientific issue that still remains mysterious. Therefore, by further utilizing the methods and techniques of genomics, molecular biology and cytobiology, the current foundation aims to first clone and analyze the 5'-flanking sequences of MAP4K4 gene in P. xylostella, then analyze the promoter activities of the cloned 5'-flanking sequences and identify the cis-acting mutations leading to overexpression of the MAP4K4 gene in Bt-resistant P. xylostella, which will contribute to illuminating the cis-regulatory mechanism underlying overexpression of the MAP4K4 gene in P. xylostella and further revealing the novel molecular mechanism of insect resistance to Bt mediated by the MAPK signaling pathway. The results in this study will be of great theoretical and practical significance for the development and sustainable application of Bt insecticidal proteins.

对昆虫Bt杀虫蛋白抗性分子机制认识的不足正严重制约着昆虫Bt抗性治理和新型Bt杀虫蛋白的开发与应用，并威胁着转Bt基因作物的使用寿命。申请者前期研究发现小菜蛾对Bt的高抗性是由MAPK信号途径上游恒定转录激活的关键基因MAP4K4反式调控多个Bt受体基因表达变化而引起。然而，对于MAP4K4基因是如何被转录激活而过量表达的这一关键基础科学问题目前尚不清楚。因此，本项目拟进一步通过基因组学、分子生物学和细胞生物学等研究方法和技术平台，首先对小菜蛾MAP4K4基因5'-侧翼序列进行克隆和分析，然后对克隆得到的5'-侧翼序列进行启动子活性分析并对序列中导致MAP4K4基因在Bt抗性小菜蛾中过量表达的顺式作用突变进行鉴定，从而阐明MAP4K4基因过量表达的顺式调控机制，并进一步揭示MAPK信号途径介导的新型昆虫Bt抗性分子机制。研究结果对于Bt杀虫蛋白的开发与可持续应用具有重要的理论和实践意义。

基于苏云金芽胞杆菌(Bt)产生的Cry类杀虫蛋白研发的生物杀虫剂和转基因作物能有效的防治农业害虫，然而，害虫已经对这些Bt生物技术产品产生了高抗性。对害虫Bt Cry类杀虫蛋白抗性分子机制认识的不足正严重制约着害虫Bt抗性治理和新型Bt杀虫蛋白的开发与应用，并威胁着转Bt基因作物的使用寿命。小菜蛾是世界十字花科作物最主要的害虫之一，而且，它还是第一个被报道在田间对Bt生物杀虫剂产生抗性的害虫，是研究害虫Bt抗性分子机制的良好模型。申请者前期研究发现小菜蛾对Bt Cry1Ac的高抗性是由MAPK信号途径上游恒定转录激活的关键基因MAP4K4反式调控多个中肠基因表达变化而引起。然而，对于MAP4K4基因是如何被转录激活而过量表达的这一关键基础科学问题目前尚不清楚。在前期研究基础上，本项目揭示了昆虫激素含量升高及其串扰是导致MAP4K4基因过量表达和整个下游MAPK信号途径激活的关键上游信号，阐明了MAP4K4基因启动子区的关键顺式作用元件和反式调控因子及其表达调控机制，明确了MAP4K4基因下游调控的靶标中肠基因类型和数量，构建了MAPK信号途径介导的Bt Cry1Ac抗性小菜蛾中肠基因差异表达的遗传调控网络，从而最终进一步阐明了小菜蛾Bt抗性分子机制。研究结果对于我国田间重大农业害虫Bt抗性进化的监测预警和综合治理以及新型Bt生物杀虫剂/转Bt基因抗虫作物的研发推广和可持续应用均具有重要的理论和实践意义。