siRNA干涉技术在黄曲霉毒素抑制中的研究

doi:10.11924/j.issn.1000-6850.casb18100089

中国农学通报 ›› 2020, Vol. 36 ›› Issue (5): 30-36.doi: 10.11924/j.issn.1000-6850.casb18100089

siRNA干涉技术在黄曲霉毒素抑制中的研究

焦健, 杜鹏()

中国科学院科技战略咨询研究院,北京 100190

收稿日期:2018-10-24 修回日期:2018-12-28 出版日期:2020-02-15 发布日期:2020-02-21
通讯作者: 杜鹏
作者简介:焦健,男,1983年出生,助理研究员,博士,主要从事遗传学研究。通信地址：100190 北京市海淀区中关村北一条15号,Tel：010-59358428,E-mail：jiaojian@casipm.ac.cn。
基金资助:
中国科学院科技战略咨询研究院院长青年基金“基于技术预见的科研议题遴选方法研究”(Y8X1261Q01)

The Application of siRNA Interference Technique in Aflatoxin Inhibition

Jiao Jian, Du Peng()

Institutes of Science and Development, Chinese Academy of Sciences, Beijing 100190

Received:2018-10-24 Revised:2018-12-28 Online:2020-02-15 Published:2020-02-21
Contact: Du Peng

摘要/Abstract

摘要：

构建真菌基因敲除突变体的传统方法周期较长,为了快速检测和高通量筛选具有特定功能的目标基因,本研究利用小分子RNA干涉技术,沉默了黄曲霉毒素生物合成关键基因,有效降低了黄曲霉毒素B₁(AFB₁)的生物合成。通过设计合成人工小干涉RNA(artificial siRNA),特异靶向黄曲霉毒素合成路径的关键基因aflR,将siRNA直接转化黄曲霉原生质体,有效沉默了黄曲霉毒素合成的关键调控基因aflR,并导致AFB₁的合成明显减少。利用特异靶向黄曲霉毒素合成路径中其他11个靶标基因的siRNAs,通过原生质体转化方法可直接沉默这些基因,沉默后这些基因的转录水平均显著降低,其中hypC和aflW两个基因沉默后AFB₁的生物合成量与对照相比下降显著。此外,利用特异靶向黄曲霉毒素合成基因簇外基因的siRNA,如pks-nrps基因,通过siRNA干涉技术沉默该基因同样可显著降低AFB₁的合成。因此,本研究借助小分子RNA干涉技术,建立了一种快速检测和高通量筛选黄曲霉毒素生物合成路径相关基因的反向遗传学方法。

关键词: 小分子RNA, 基因沉默, 黄曲霉毒素, RNA干涉, 反向遗传学技术

Abstract:

Traditional methods for constructing fungal gene knockout mutants would take a long experimental period. In order to rapidly detect and high-throughput screen target genes with specific functions, small molecule RNA interference technique was used to silence the key genes of aflatoxin biosynthesis in this study, which effectively reduced the biosynthesis of aflatoxin B₁ (AFB₁). By designing and synthesizing artificial small interfering RNAs (artificial siRNAs) which specifically targeted the key gene aflR in aflatoxin synthesis pathway, artificial siRNAs were directly transformed into the protoplast of Aspergillus flavus, which effectively silenced the key regulator aflR gene of aflatoxin synthesis, and led to significantly decreasing AFB₁ biosynthesis. The siRNAs of the other 11 target genes in the aflatoxin synthesis pathway were used in this study, protoplast transformation could directly silence these genes, and the transcription levels of these genes were significantly reduced, and among them, the amount of AFB₁ biosynthesis decreased significantly after silencing hypC and aflW gene, compared to the control. In addition, using siRNA specifically targeting aflatoxin synthesize gene clusters, such as the pks-nrps gene, silencing of pks-nrps gene by siRNA interference could also significantly reduce the synthesis of AFB₁. Therefore, this study relies on small molecule RNA interference technique and established a reverse genetics method for rapid detection and high-throughput screening of aflatoxin biosynthesis pathway-related genes.

Key words: small RNA, gene silencing, aflatoxin, RNA interference, reverse genetics technology

中图分类号:

焦健, 杜鹏. siRNA干涉技术在黄曲霉毒素抑制中的研究[J]. 中国农学通报, 2020, 36(5): 30-36.

Jiao Jian, Du Peng. The Application of siRNA Interference Technique in Aflatoxin Inhibition[J]. Chinese Agricultural Science Bulletin, 2020, 36(5): 30-36.

图/表 9

图1. 靶向aflR的siRNAs示意图

图2. 半定量RT-PCR检测siRNAs_aflR

图3. 半定量RT-PCR检测各种siRNAs

图4. Real-time RT-PCR检测各基因沉默效果

图5. HPLC检测靶标基因的沉默对AFB1合成的影响

图6. 半定量RT-PCR检测3种siRNAs

图7. Real-time RT-PCR检测3个基因沉默效果

图8. HPLC检测靶标基因的沉默对AFB1合成的影响

图9. Real-time RT-PCR检测pks-nrps基因沉默效果及HPLC检测沉默对AFB1合成的影响

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siRNA干涉技术在黄曲霉毒素抑制中的研究

The Application of siRNA Interference Technique in Aflatoxin Inhibition

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