RNAi沉默病原致病基因创制抗黑穗病甘蔗新种质

Host-induced gene silencing (HIGS), which developed on the basis of RNAi, is a new technology which has been shown to be an auspicious strategy for the development of transgenic plants to control fungal, bacterial and viral diseases. Sugarcane smut is the most important fungal disease in sugarcane production. The improvement of the smut resistance character for main cultivated sugarcane varieties is the most economic and effective measure to control this disease. ROC22 is the most prevalent sugarcane cultivar which occupies more than 60% of sugarcane cultivation in China, however it is susceptible to sugarcane smut. In the present study, based on the whole-genome sequencing of Sporisorium scitamineum, function annotation, along with the cloning and preliminary function identification of pathogenic virulence genes, firstly, gene knock-out along with phenotypic screening after inoculation by mutant isolate were applied to confirm the precise function of these smut inducing related genes and thus the ascertainment of key pathogenic virulence genes; secondly, using RNAi technology, with partial sequences of key pathogenic virulence gene as the interference sequence, RNAi expression vectors were constructed and transformed into smut susceptible sugarcane variety ROC22 by gene gun-mediated genetic transformation; finally, when transgenic lines of ROC22 were infected by S. scitamineum, siRNA (small interference RNA) entered in to S. scitamineum, through the channel of “cell wall fusion between the fungus and plant”, to inhibit or silence the targeted pathogenic virulence gene and thus smut disease resistant sugarcane germplasms were created. The above study is expected to not only provides new knowledge and experience on the breeding of smut resistant sugarcane germplasms, but also provides new ideas on the prevention and cure of fungal diseases in other crops.

寄主诱导的基因沉默（Host-Induced Gene Silencing，HIGS）是一种以RNAi技术为基础的培育转基因抗病作物的新技术。黑穗病是甘蔗生产中最重要的真菌病害，提高生产性品种的抗黑穗病性能有效控制该病害。本项目以课题组甘蔗黑穗病菌全基因组测序和功能分析结果为基础，首先，通过基因敲除技术结合突变菌株侵染甘蔗后的表型筛选，确定致病关键基因；其次，利用RNAi技术，以致病关键基因部分序列为干扰序列，构建RNAi表达载体，通过基因枪介导遗传转化甘蔗品种ROC22；最后，当黑穗病菌侵染甘蔗品种ROC22的转基因株系时，甘蔗中产生的小RNA通过“真菌细胞壁与植物细胞壁融合”过程中的小RNA交流通道，进入黑穗病菌体内，实现对靶标致病关键基因的抑制或沉默，从而创制抗黑穗病甘蔗新种质。以上研究不仅能够为培育抗黑穗病甘蔗新种质提供新的知识和经验，也有望为防治其它作物真菌病害提供新思路。

寄主诱导的基因沉默是一种以RNAi技术为基础的培育转基因抗病作物的新技术。黑穗病是甘蔗生产中最重要的真菌病害，提高生产性品种的抗黑穗病性能有效控制该病害。无论是基于RNAi沉默病原致病基因还是通过超表达寄主抗黑穗病基因创制抗黑穗病甘蔗新种质，病原致病基因和寄主抗病基因的克隆和功能鉴定、甘蔗-黑穗病菌互作中寄主和病原内参基因的筛选与评价，以及甘蔗与黑穗病菌互作机制的解析，都有助于抗病育种理论的探讨和实践的开展。遵循国家基金任务的探索属性和在经费资助下需全力推进研究的执行原则，首先，项目筛选获得7个甘蔗黑穗病菌候选致病效应蛋白基因，并对其进行了克隆和功能分析，其中包括玉米黑粉菌上有较好研究基础的同源基因-甘蔗黑穗病菌分支酸变位酶基因，为基于RNAi沉默病原致病基因创制抗黑穗病甘蔗新种质奠定基础。其次，我们开展了甘蔗抗黑穗病系列功能基因以及发挥调控作用WRKY转录因子的克隆和功能分析，包括ScJAZ、小G蛋白、AOC、CBL、CIPK、PITP和WRKY，目的是将其应用于通过超表达创制抗黑穗病新种质，一定程度上弥补实验结果不如预期时的缺憾。再次，筛选出适用于甘蔗蔗芽-黑穗病菌互作系统中基因表达定量的内参基因是黑穗病研究的基础性工作。项目筛选出在受黑穗病菌侵染后的蔗芽样品中表达水平较高、表达变异小、稳定性高的甘蔗内参基因：ACAD、SARMp1以及内参组合ACAD+SARMp1；同时，还筛选出适于病原菌基因表达分析的黑穗病菌内参基因S10、S11以及内参组合S10+S11。最后，项目开展了抑制性消减杂交文库构建及甘蔗应答黑穗病菌侵染的降解组分析及抗性相关miRNA靶标基因鉴定工作，并取得了一定进展。需要特别强调的时，我们在项目研究中发现了病原菌分泌型蛋白能够在质外体空间与寄主内源多肽竞争性结合激酶膜外结构域这一植物-病原互作新的分子机制。以上研究为培育抗黑穗病甘蔗新种质提供新知识和经验，还为利用基因工程手段定向改良甘蔗品种的抗黑穗病性提供具有自主知识产权的基因，也有望为防治其它作物真菌病害提供新思路。