新型隐球酵母U6启动子的鉴定、克隆及功能验证

Identification,cloning and functional verification of U6 promoter from Cryptococcus neoformans

【目的】鉴定及克隆新型隐球酵母(Cryptococcus neoformans)中PolⅢ型的U6启动子(Cn U6启动子),并验证CnU6启动子能否有效转录shRNA及CRISPR/Cas9系统中gRNA。【方法】结合Gen Bank数据库已公布的新型隐球酵母基因组信息和本实验室RNA-seq文库数据,利用生物信息学技术分析得到新型隐球酵母中具有高转录水平的U6RNA序列。使用重叠PCR和Easy Geno方法将预测的CnU6启动子分别克隆到sh RNA及gRNA的上游区域,通过观察shRNA对靶基因的RNAi效果及gRNA引导Cas9核酸酶对靶位点的切割结果,确定CnU6启动子能否转录短RNA。【结果】CnU6启动子能够转录形成shRNA对靶基因进行沉默,并且能转录形成g RNA引导Cas9核酸酶对靶点进行切割。【结论】新型隐球酵母的CnU6启动子被成功鉴定及克隆,它能有效驱动shRNA和gRNA的转录。

[Objective] To identify and clone the polymerase Ⅲ U6 promoter from Cryptococcus neoformans （CnU6 promoter）, and verify if CnU6 promoter can effectively transcribe shRNA and gRNA of CRISPR/Cas9 system. [Methods] Combining the C. neoformans genome information published in GenBank database and RNA-seq library data from our laboratory, we obtained the U6 RNA sequence with high transcriptional level by bioinformatics analysis. The putative CnU6 promoter was ligated upstream of shRNA and gRNA by EasyGeno and overlapping PCR respectively. Based on shRNA-mediated target gene silence phenotype by RNAi and gene mutation by gRNA-guided Cas9 nuelease mediated target sites editing by CRISPPUCas9 system, we could identify if CnU6 promoter could drive the transcription of short RNA. [Results] CnU6 promoter could drive the transcribtion of shRNA, which could silence the target gene, and gRNA, which could guide Cas9 nuclease to cut the target site. [Conclusion] The CnU6 promoter from C. neoformans was successfully identified and cloned, which could drive the transcription of shRNA and gRNA efficiently.