甘蔗黑穗病菌RNA干扰相关基因ssdcl的功能研究

Functional Study of ssdcl Gene involving in RNA Interference in Sporisorium scitamineum

甘蔗黑穗病菌(Sporisorium scitamineum)是典型的二型态真菌,由其引起的甘蔗黑穗病是一个全球性的病害,对甘蔗产量和含糖量造成巨大损失。RNA干扰是一种保守的小RNA介导的转录后基因沉默机制。目前甘蔗黑穗病菌的RNA干扰系统尚未明确。本研究用蛋白保守结构域对甘蔗黑穗病菌基因组进行搜索,发现了一个RNA干扰组件DICER-LIKE (SsDcl)蛋白。序列分析显示此蛋白具有剪切双链RNA所必需的RNaseⅢ结构域和双链RNA识别区域。为了研究ssdcl基因的功能,本研究以甘蔗黑穗病菌单倍体JG36为出发菌株,构建了ssdcl基因的缺失突变株。与野生型相比,Δssdcl突变体的生长速度较慢,与野生型JG35配合后形成的二倍体菌丝更为细小,且分支较多,表明ssdcl基因的缺失可能影响了黑穗病菌菌丝的生长发育过程。另外,Northern blot和qRT-PCR的结果显示,ssdcl基因缺失突变体中milR-4的表达量下调,提示ssdcl基因在甘蔗黑穗病菌milRNAs形成过程中起重要作用。

Sporisorium scitamineum(Ssc) is a typical dimorphic fungus, and the sugarcane smut caused by it has a global impact on sugarcane production, resulting in great loss of both stalk yield and sugar content. RNA interference(RNAi) is a conserved mechanism mediated by small RNAs to silence target genes at post-transcriptional level. RNAi components responsible for RNA interference in S. scitamineum are still unclear. In this study, an RNAi component DICER-LIKE(SsDcl) encoding gene was identified by searching the Ssc genome with the DICER-conserved domains. Sequence analysis showed that Ss Dcl had a RNase Ⅲ domain and a double-strain RNA recognition region which were necessary for shearing double-strain RNAs. To study the function of ssdcl,Δssdcl mutants were constructed in strain JG36. Compared with the wild-type, the Δssdcl mutants showed slower growth rate and produced thiner hyphae with more branches after mating with opposite mating type strain JG35,indicating that ssdcl was involve in the growth and development in S. scitamineum. Northern blot and qRT-PCR showed that the accumulation of milR-4 was decreased in the ssdcl mutants, suggesting that ssdcl plays an important role in milRNAs biosynthesis in S. scitamineum.