亚洲玉米螟Cry1Ab抗性相关的中肠microRNA鉴定及功能研究

It is of great significance to reveal the mechanisms of insect resistance to Bt for the application of Bt plants. The susceptibility of insect to Bt was related to the expression level of the specific receptor on BBMV. However, the regulation mechanisms of the Bt receptor expression were unclear. The discovery of miRNA, with the ability of global regulation, provided a new direction in this aspect. Our previous studies found that there were significant differences in expression of Bt receptors between Asian corn borer (ACB) resistant or susceptible to Cry1Ab. Therefore, it was proposed that miRNA-regulated Bt receptors expression might affect the susceptibility of ACB to Cry1Ab. To investigate the role of miRNA in ACB resistance to Cry1Ab, in this study, the expression difference of midgut miRNA from both Cry1Ab resistant and susceptible ACB larvae will be analyzed using Solexa depth sequencing and bioinformatics method analysis. Meanwhile, the expression of cadidate miRNA will be confirmed by q-PCR and the target genes of miRNA with different expression level will be predicted using online softwares. Moreover, the relationship between expression of miRNA and Bt receptor genes will be analyzed. The poteintional miRNA related to Cry1Ab resistance of ACB will be screened basing on the results. The effect of the expression of Bt receptors and susceptibility to Cry1Ab in vivo was analyzed through oral feeding and transfection. The results of this study will provide new avenues of exploration for mechanism of resistance to Bt and a theoretical basis for delay insect resistance.

揭示昆虫Bt抗性产生机理对Bt作物的应用意义重大，昆虫BBMV上Bt受体蛋白基因的表达水平影响昆虫对Bt的敏感性。但受体蛋白基因的表达调控机制尚不明确，具有全局调控作用的miRNA的发现为此研究提供了新的方向。申请人研究发现亚洲玉米螟Cry1Ab抗性和敏感品系幼虫中肠内Bt受体蛋白基因表达量存在显著差异，结合最新研究结果推测miRNA可能参与受体蛋白基因表达调控，进而影响亚洲玉米螟对Cry1Ab敏感水平。本项目拟通过Solexa测序和生物信息学分析，构建亚洲玉米螟Cry1Ab抗性和敏感品系幼虫中肠miRNA表达谱，获得差异表达miRNA；q-PCR方法验证表达量差异，进而预测靶标基因，筛选与Cry1Ab抗性相关的miRNA；并在活体和细胞水平研究miRNA对Bt受体蛋白基因表达的调控及其对亚洲玉米螟Cry1Ab敏感性的影响。项目的开展将为昆虫Bt抗性机理研究提供新的探索途径和理论依据。

本研究以miRNA为目标，通过Solexa深度测序、分子生物信息学分析和miRNA功能研究，探索了miRNA在亚洲玉米螟Cry1Ab蛋白抗性形成过程中发挥的作用。主要工作如下：.（1）为了开展miRNAs靶标基因预测工作，我们首先提取了亚洲玉米螟Cry1Ab抗性种群（ACB-AbR）和Bt敏感种群（ACB-BtS）总RNA，通过转录组测序，分析两种群的差异表达基因。分析发现：3983个转录本在ACB-AbR和ACB-BtS中差异表达，多个差异表达基因被标注为已知的Bt受体蛋白，如类钙粘蛋白基因的转录本在ACB-AbR中下调表达；而标注为APN的转录本在ACB-AbR中均上调表达。.（2）为了探索与亚洲玉米螟Cry1Ab抗性相关的miRNAs，我们采用与转录组测序相同的组织材料，通过Solexa测序技术对ACB-BtS和ACB-AbR两种群的miRNAs进行测序分析和表达差异比较。在ACB-BtS-1、ACB-BtS-2、ACB-AbR-1和ACB-AbR-2中分别发现了302、395、268和287个已知miRNAs，32、16、18。其中，21个miRNAs在ACB-BtS和ACB-AbR种群中显著差异表达。.（3）为了进一步研究miRNAs的功能，我们进行了差异表达miRNAs的靶标基因，预测、注释和生物信息学分析。65301基因被预测为已知差异表达miRNAs的靶基因，包括目前已报道的和昆虫Bt抗性产生相关的多个功能基因。如miR-2833b可以靶向作用于Ofapn2，miR-3737可以靶向作用于Ofapn4。功能研究发现，靶基因分别属于结构、功能和过程相关的324、658和2259个GO terms。生物通路分析发现，靶基因可被归为307个通路，其中20.62%的基因与代谢通路相关。.（4）由于miR-1和miR-31是目前研究较成熟，同时在ACB-BtS和ACB-AbR中差异表达的miRNAs，我们首先对miR-1和miR-31的功能进行了初步研究。通过转染Sf9细胞系发现，miR-31对靶标昆虫Cry1Ab的敏感性水平有一定的影响。.本项目的开展将为明确亚洲玉米螟Cry1Ab抗性产生机理提供依据，为靶标昆虫抗性治理、转Bt基因作物合理应用奠定理论基础。