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A novel vector-based RNAi method using mouse U6 promoter-driven shRNA expression in the filamentous fungus Blakeslea trispora
Biotechnology Letters ( IF 2.7 ) Pub Date : 2021-06-29 , DOI: 10.1007/s10529-021-03155-5
Ye Li 1 , Hui Feng 1 , Lihua Jin 1 , Xiulan Xin 1 , Qipeng Yuan 2
Affiliation  

Purpose

There are several studies on the use of RNA interference (RNAi) for gene function analysis in fungi. However, most studies on filamentous fungi are based on in vitro-transcribed or -synthesized small interfering RNA (siRNA), and only a few have reported the use of vector-based RNAi. Here we want to develop and evaluate a new vector-based RNAi method using the mouse U6 promoter to drive short hairpin RNA (shRNA) expression in the filamentous fungi.

Methods

Molecular techniques were employed to develop and evaluate a new vector-based RNAi method using the mouse U6 promoter to drive short hairpin RNA (shRNA) expression in the filamentous fungus Blakeslea trispora.

Results

We characterized the mouse U6 promoter and utilized it for the expression of shRNA in B. trispora. Using real-time polymerase chain reaction and western blotting analyses, we confirmed the decrease in the mRNA and protein expression of carRA, respectively, in cells transformed with the mouse U6 promoter-driven shRNA expression vector. This indicated that the shRNA was transcribed from the mouse U6 promoter and correctly processed into siRNA and that the mouse U6 promoter exhibited transcription ability in the filamentous fungi.

Conclusions

The results suggest that the mouse U6 promoter that drives the expression of shRNA vectors may serve as a novel tool for RNAi induction in filamentous fungi.



中文翻译:

一种新的基于载体的 RNAi 方法,利用小鼠 U6 启动子驱动的丝状真菌 Blakeslea trispora 中的 shRNA 表达

目的

有几项关于使用 RNA 干扰 (RNAi) 进行真菌基因功能分析的研究。然而,大多数关于丝状真菌的研究都是基于体外转录或合成的小干扰 RNA (siRNA),只有少数报道了基于载体的 RNAi 的使用。在这里,我们想开发和评估一种新的基于载体的 RNAi 方法,该方法使用小鼠 U6 启动子来驱动丝状真菌中的短发夹 RNA (shRNA) 表达。

方法

采用分子技术开发和评估一种新的基于载体的 RNAi 方法,该方法使用小鼠 U6 启动子驱动丝状真菌Blakeslea trispora中的短发夹 RNA (shRNA) 表达

结果

我们对小鼠 U6 启动子进行了表征,并将其用于在B. trispora 中表达 shRNA 。使用实时聚合酶链反应和蛋白质印迹分析,我们证实了用小鼠 U6 启动子驱动的 shRNA 表达载体转化的细胞中carRA的 mRNA 和蛋白质表达分别降低。这表明shRNA从小鼠U6启动子转录并正确加工成siRNA,并且小鼠U6启动子在丝状真菌中表现出转录能力。

结论

结果表明,驱动 shRNA 载体表达的小鼠 U6 启动子可作为丝状真菌中 RNAi 诱导的新工具。

更新日期:2021-08-29
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