基于RNAi技术创制抗二点螟甘蔗新种质材料

The sugarcane stem borer Chilo infuscatellus is one of the most serious pests in sugarcane field. Since there is no efficient way to develop an anti-borer variety for lacking of anti-insect germplasms, therefore the pest is mainly controlled by the chemical insecticide. Because of the resistance to insecticide and the difficulty of spraying insecticide during the mature stage of sugarcane, the chemical insecticide would not only make it inefficient to control borer in sugarcane, but also results in severe hazard for green production. Here our research is based on the theory of RNAi technology, first of all we get the transcriptome of Chilo infuscatellus, then we select the efficient target genes for RNAi and choose safe fragments of dsRNA. Afterwards the sequence of dsRNA is introduced into the sugarcane cells by genetic engineering method, finally we get the transgenic sugarcane that can express dsRNA. The real-time quantitative PCR and Northern blot will be used to analyze the expression level of dsRNA in the transgenic sugarcane, combined with the assessment of anti-insect ability. Based on the RNAi technology and transgenic technology we enhance the anti-Chilo infuscatellus ability of sugarcane to explore a new way of anti-insect molecular breeding. Therefore, our research will promote the advancing plant protection technology through genetic breeding, which is highly meaningful for sugarcane field integrated pest management and green production of sugarcane.

甘蔗二点螟是为害甘蔗最严重的害虫之一，由于缺乏抗性种质，生产上无法培育高效的抗螟甘蔗品种，只能利用各种农药进行化学防治。由于螟虫耐药性以及甘蔗田后期用药困难，化学防治不仅很难解决甘蔗螟害问题，还给甘蔗绿色生产带来严重隐患。本研究基于RNAi技术，在解析甘蔗二点螟转录组的基础上，筛选出安全高效的RNAi靶基因和dsRNA转入片段。以优质甘蔗品种为材料，通过转基因技术将含有这些dsRNA序列导入甘蔗细胞，培育可以稳定表达dsRNA的转基因甘蔗植株。利用实时荧光定量PCR和Northern杂交等技术分析dsRNA在转基因甘蔗植株上的表达水平，结合抗性鉴定培育出高效的抗甘蔗二点螟转基因甘蔗新种质材料。本课题基于RNAi的方法结合转基因技术改良甘蔗的抗甘蔗二点螟特性，探索一条甘蔗抗虫转基因分子育种新途径，促进植保高新技术与遗传育种的结合发展，对甘蔗螟虫的综合治理和甘蔗绿色生产具有重要的现实意义。

二点螟属于鳞翅目螟蛾科，是甘蔗上的主要害虫之一，在我国各主要蔗区普遍为害。选育抗性品种是最直接、经济有效的防御害虫的方法，但是，由于甘蔗抗虫种质的缺乏，通过常规杂交手段难以培育出高效的抗螟虫甘蔗品种。本项目首先基于对不同生育期的二点螟进行转录组测序和分析上，共获得95,921个unigene基因，平均组装长度为1542bp，N50为2591bp。其中有53815条unigenes得到注释。克隆了四个二点螟几丁质酶基因全长(CiChi1、CiChi2、CiChi11) 和一个几丁质酶结构域1 基因(CiChid1)，并对其进行时空表达分析，结合体外注射实验筛选出对二点螟生长发育具有显著致死效果的靶标基因CiChi1及其dsRNA序列，死亡率为62%。通过构建植物表达载体pUC18-CiChi1，利用基因枪介导的遗传转化体系将该载体导入甘蔗心叶组织，使甘蔗表达dsRNA序列，经过18%草铵膦的喷施筛选和PCR转基因检测，获得84株转基因甘蔗植株。此外还搜集了我国各地的甘蔗螟虫，建立了我国甘蔗螟虫的条形码数据库，为害虫的精确识别鉴定提供基础。项目研究期间还对甘蔗上另外一种重要害虫粘虫进行RNAi体系构建、靶标基因筛选及功能验证等进行研究，为将来基于RNAi技术的甘蔗害虫防治手段提供参考。