siRNA介导的甘蔗鞭黑粉菌基因沉默方法的建立

A Gene Silencing Method Based on siRNA inSporisorium scitamineum

甘蔗是全世界最重要的糖料作物。由甘蔗鞭黑粉菌(Sporisorium scitamineum)引起的甘蔗黑穗病是一个全球性的病害,对甘蔗生产造成巨大危害。RNA干扰技术是一种快速有效的研究基因功能的方法,然而尚没有在甘蔗鞭黑粉菌中利用RNA干扰研究基因功能的报道。本研究以荧光蛋白基因eGFP为靶标,构建了表达eGFP的菌株JG35-eGFP和含有eGFP基因回文序列的干扰菌株JG36-eGFP-RNAi,将其进行有性配合,结果显示,eGFP基因表达量及荧光强度均显著下降,表明甘蔗鞭黑粉菌的RNA干扰系统构建成功。我们用此干扰系统对甘蔗鞭黑粉菌的效应物基因g2476的功能进行研究,发现目的基因在单倍体及有性配合形成的菌丝中表达水平均明显下调,但RNA干扰单倍体的有性配合能力并无明显变化。甘蔗鞭黑粉菌中RNA干扰方法的建立为该菌基因功能研究提供了新的工具,并为培育HIGS(Host induced gene silencing)抗病植株奠定了基础。

Sugarcane is the most important sugar crop in the world.Sugarcane smut caused by Sporisorium scitamineum is a worldwide disease seriously affecting the sugar yield.RNA interference(RNAi)technology is a rapid and effective tool for studying gene function in plant.However,there has been no report on RNAi in S.scitamineum.In this study,the enhanced green fluorescent protein(eGFP)was used as a test target gene.We constructed JG35-eGFP strain expressing eGFP and JG36-eGFP-RNAi strain containing palindrome sequences of eGFP gene,respectively.After fusion of the hypae of the two transformants by mating,it was observed that the eGFP gene expression and the fluorescence intensity were significantly reduced,demonstrating the feasibility of RNA interference in S.scitamineum.We then used this interference system to study the function of the effector gene g2476.It was found that the gene g2476 was silenced in both haploid sporidium and dikaryotic hyphae but the silencing strains were indistinguishable to the wild-type strain in mating/filamentation,suggesting that this effector might not involved in mating/filamentation.Thus,this RNAi method provides another avenue for the study of gene function for S.scitamineum,and for breeding HIGS(Host induced gene silencing)disease-resistant plants.