›› 2018, Vol. 61 ›› Issue (10): 1145-1152.doi: 10.16380/j.kcxb.2018.10.003

• 研究论文 • 上一篇    下一篇

家蚕miR-301对化学感受蛋白基因csp9表达的调控

杨娟娟, 王远卓, 魏博帆, 邢秋婷, 阚云超, 乔惠丽*   

  1. (南阳师范学院, 河南省伏牛山昆虫生物学重点实验室, 昆虫生物反应器河南省工程实验室, 河南南阳 473061)
  • 出版日期:2018-10-20 发布日期:2018-10-20

Regulation of the expression of chemosensory protein gene csp9 by miR-301 in Bombyx mori  

YANG Juan-Juan, WANG Yuan-Zhuo, WEI Bo-Fan, XING Qiu-Ting, KAN Yun-Chao, QIAO Hui-Li*   

  1.  (Henan Provincial Key Laboratory of Insect Biology in Funiu Mountain, Henan Provincial Engineering Laboratory of Insects Bioreactor, Nanyang Normal University, Nanyang, Henan 473061, China)
  • Online:2018-10-20 Published:2018-10-20

摘要: 【目的】探索家蚕Bombyx mori miRNAs对化学感受蛋白基因表达的调控作用,以进一步研究 miRNAs及其靶基因在昆虫化学识别中的作用。【方法】利用生物信息学方法预测和筛选可能作用于家蚕化学感受蛋白CSPs基因家族成员的miRNAs;实时荧光定量PCR分析预测获得的候选miR-301和其作用的靶基因在家蚕成虫不同组织中的表达变化;构建miR-301预测靶基因3′-UTR的双荧光素酶报告载体,与合成的miR-301 mimics或阴性对照转染人胚肾细胞HEK293,通过双荧光素酶报告基因检测系统中荧光素酶活性变化检测miR-301对其靶基因表达的调控作用。【结果】生物信息学分析结果发现,家蚕化学感受蛋白基因csp9是miR-301的预测靶基因,二者的结合位点位于csp9的3′-UTR区。实时荧光定量PCR检测结果表明,miR-301在交配后家蚕雌雄成虫触角和雄成虫头部都显著上调表达,靶基因csp9在对应组织中表达则显著下调。二者共转染HEK293细胞后,双荧光素酶检测结果表明,miR-301可以通过与csp9 3′-UTR的互作,显著抑制上游荧光素酶报告基因的表达。【结论】家蚕化学感受蛋白基因csp9是miR-301的靶基因,miR-301通过与靶基因3′-UTR的结合,在翻译水平上抑制csp9的表达。

关键词: 家蚕, miRNA, 化学感受蛋白, 靶基因, 实时荧光定量PCR, 双荧光素酶

Abstract:
【Aim】 To explore the role of miRNAs in regulating the expression of chemosensory protein genes in the silkworm, Bombyx mori, so as to further study the functions of miRNAs and their target genes in insect chemoreception. 【Methods】 The miRNAs that may interact with silkworm CSPs gene family were predicted and screened using bioinformatics methods, and the expression changes of candidate miR-301 and its target gene in different tissues of silkworm adults were analyzed using RT- qPCR. Dual luciferase report vector for miR-301 target gene 3′-UTR was constructed and transfected into human embryonic kidney 293 (HEK293) cells with miR-301 mimics or the negative control. The regulation of expression of the target gene by miR-301 was detected by the change of luciferase activity in dual luciferase reporter gene assay system. 【Results】 The results of bioinformatics analysis showed that csp9, a silkworm chemosensory protein gene, is a predicted target gene of miR-301, and its binding site with miR-301 is located in the 3′ -UTR region of csp9. RT-qPCR results showed that the expression of miR-301 was significantly up-regulated in male and female adult antennae and male adult head after mating, while the expression of csp9 was significantly down-regulated in these tissues. Dual luciferase assays showed that miR-301 significantly inhibited the expression of luciferase reporter gene by interacting with csp9 3′-UTR after co-transfection into HEK293 cells. 【Conclusion】In B. mori the chemosensory protein gene csp9 is a target gene of miR-301, its expression at the translational level is inhibited by miR-301 through binding to its 3′-UTR.

Key words: Bombyx mori; miRNA, chemosensory protein, target gene, RT-qPCR, dual luciferase