昆虫学报 ›› 2016, Vol. 59 ›› Issue (7): 724-731.doi: 10.16380/j.kcxb.2016.07.004

• 研究论文 • 上一篇    下一篇

家蚕miRNA Novel-31*上调溶血素基因的表达

施莉莉1, 耿涛1, 吴萍1, 潘中华2, 覃光星1,高坤1, 侯成香1, 郭锡杰1,*   

  1. (1. 江苏科技大学生物技术学院, 江苏镇江 212018; 2. 苏州大学基础医学与生物科学学院, 江苏苏州 215123)
  • 出版日期:2016-07-20 发布日期:2016-07-20

microRNA Novel-31* up-regulates the expression of hemocytin gene in the silkworm, Bombyx mori

SHI Li-Li1, GENG Tao1, WU Ping1, PAN Zhong-Hua2, QIN Guang-Xing1, GAO Kun1, HOU Cheng-Xiang1, GUO Xi-Jie1,*   

  1. (1. School of Biotechnology, Jiangsu University of Science and Technology, Zhenjiang, Jiangsu 212018, China; 2. School of Biology and Basic Medical Science, Soochow University, Suzhou, Jiangsu 215123, China)
  • Online:2016-07-20 Published:2016-07-20

摘要: 【目的】Novel-31*是在家蚕质型多角体病毒(Bombyx mori cytoplasmic polyhedrosis virus, BmCPV) 感染的家蚕中发现的一个差异表达miRNA。本研究旨在验证Novel-31*对其靶基因表达的调控作用,以便进一步研究miRNA及其靶基因在昆虫免疫调节中的作用。【方法】用生物信息学方法预测Novel-31*的靶基因,荧光定量PCR分析Novel-31*及其靶基因在家蚕感染BmCPV后不同时间点的表达变化;构建miRNA慢病毒表达载体和靶基因慢病毒表达载体,转染293T细胞,同时合成Novel-31* mimics转染家蚕培养细胞BmN,使用荧光定量PCR检测 Novel-31*对靶基因表达的调控作用。【结果】生物信息学方法预测发现,溶血素基因是Novel-31*的靶基因,其结合位点位于溶血素基因的5′ UTR 区域。荧光定量PCR分析表明,Novel-31*及溶血素基因在感染BmCPV的家蚕血淋巴细胞中呈现明显的上调表达。荧光定量PCR检测表明,在Novel-31*慢病毒表达载体和溶血素基因5′UTR慢病毒表达载体转染的293T细胞中和在转染Novel-31* mimics的家蚕BmN细胞中,溶血素基因都上调表达。【结论】溶血素基因是miRNA Novel-31*的靶基因,Novel-31*与溶血素基因5′UTR结合,上调溶血素基因的表达。  

关键词: 关键词: 家蚕, miRNA, Novel-31*, 靶基因;慢病毒载体, 溶血素

Abstract: 【Aim】 Novel-31* is one of the miRNAs identified in the silkworm (Bombyx mori ) larvae infected by B. mori cytoplasmic polyhedrosis virus (BmCPV). This study aims to predict its target genes and verify its function in regulation of target gene expression, so as to further study the roles of miRNAs and their target genes in insect immune regulation. 【Methods】 The putative target genes of miRNA Novel-31* were predicted by bioinformatics approach, and the expression patterns of Novel-31* and the predicted target gene were detected using real-time quantitative PCR at different time points in the silkworm larvae infected by BmCPV. Lentiviral expression vectors respectively expressing Novel-31* and the target gene were constructed and transfected into 293T cells. At the same time, Novel-31* mimics was synthesized and transfected into silkworm cell line BmN. The regulation of Novel-31* on target gene expression was verified by real-time quantitative PCR. 【Results】 Bioinformatics analysis predicted that hemocytin gene is one of the putative target genes of Novel-31* and its 5′ UTR region is the binding site for Novel-31*. Real-time quantitative PCR analysis indicated that the expression levels of both Novel-31* and the hemocytin gene were up-regulated obviously in the hemolymph of silkworm larvae infected with BmCPV. Lentiviral expression vectors respectively expressing Novel-31* and the 5′UTR region of hemocytin gene were successfully constructed and transfected into 293T cells. Real-time quantitative PCR detection verified that Novel-31* up-regulated the target gene expression. Furthermore, transfection of Novel-31* mimics into silkworm cell line BmN also resulted in the up-regulation of expression of the hemocytin gene. 【Conclusion】 Hemocytin gene is a target gene for miRNA Novel-31*. Novel-31* can bind to the 5′ UTR region of the target gene and up-regulate obviously its expression.    

Key words: Key words: Bombyx mori , miRNA, Novel-31*, target gene, lentiviral expression vector, hemocytin