昆虫学报 ›› 2020, Vol. 63 ›› Issue (3): 245-254.doi: 10.16380/j.kcxb.2020.03.001

• 研究论文 •    下一篇

家蚕miR-2769靶基因的鉴定及表达分析

孙妍妍1,2, 王景娅1,2, 王露2, 李晓哲2, 阚云超2, 乔惠丽2,*   

  1. (1. 郑州大学生命科学学院, 郑州 450001; 2. 南阳师范学院, 河南省伏牛山昆虫生物学重点实验室, 河南南阳 473061)
  • 出版日期:2020-03-20 发布日期:2020-04-16

Identification and expression analysis of miR-2769 target gene in Bombyx mori

SUN Yan-Yan1,2, WANG Jing-Ya1,2, WANG Lu2, LI Xiao-Zhe2, KAN Yun-Chao2, QIAO Hui-Li2,*   

  1.  (1. School of Life Sciences, Zhengzhou University, Zhengzhou 450001, China; 2. Henan Provincial Key Laboratory of Insect Biology in Funiu Mountain, Nanyang Normal University, Nanyang, Henan 473061, China)
  • Online:2020-03-20 Published:2020-04-16

摘要: 【目的】MiRNAs在昆虫变态发育过程中发挥非常重要的作用。对家蚕Bombyx mori miRNAs及靶基因的研究将有助于阐明miRNAs参与调控家蚕变态发育的分子机制。【方法】往家蚕5龄第2天幼虫血淋巴注射蜕皮激素20E后,qRT-PCR检测miR-2769在家蚕脂肪体中的表达;通过生物信息学方法预测家蚕miR-2769的靶基因;利用双荧光酶报告载体系统分析miR-2769与预测靶基因BmE75B的互作;qRT-PCR检测miR-2769及其靶基因BmE75不同剪接体在家蚕不同发育时期(幼虫、蛹和成虫)和幼虫不同组织(头、表皮、丝腺、脂肪体、精巢、卵巢、马氏管、中肠和血淋巴)中的表达量。【结果】研究结果表明,miR-2769可通过与家蚕BmE75B的3′UTR区结合位点的互作,显著抑制荧光素酶报告基因的表达。qRT-PCR结果表明,miR-2769和BmE75A/BmE75B在20E诱导家蚕脂肪体中表达趋势相反。时空表达分析结果表明,miR-2769与BmE75的不同剪接体在家蚕不同发育时期和不同组织中均具有特异性表达特征。在家蚕变态发育的不同阶段,miR-2769和BmE75A的表达量均较低,而BmE75B在蛹期表达量较高,BmE75C在5龄末幼虫和蛹早期有极高表达水平。此外,miR-2769与BmE75不同剪接体均存在一定程度的表达负相关。在家蚕5龄幼虫血淋巴中miR-2769可促进BmE75A的表达,在脂肪体中miR-2769可促进BmE75B的表达,而在其他组织中miR-2769抑制BmE75不同剪接体的表达。【结论】家蚕miR-2769可通过与BmE75B的3′UTR区的互作对BmE75不同剪接体的表达进行负调控。

关键词: 家蚕, 变态发育, 蜕皮激素, miRNAs, 靶基因, qRT-PCR, 表达分析

Abstract: 【Aim】 MiRNAs play very important roles in insect metamorphosis. The study of miRNAs and their target genes in Bombyx mori will help to elucidate the molecular mechanism of miRNAs involved in the metamorphosis of this insect. 【Methods】 The expression of miR-2769 in the fat body of B. mori was detected by qRT-PCR after injecting molting hormone, 20-hydroxyecdysone (20E), into the hemolymph of the day-2 5th instar larvae. The target genes of miR-2769 of B. mori were predicted by bioinformatics methods. The interaction between miR-2769 and its predicted target gene BmE75B was analyzed by dual luciferase reporter assay system. The expression levels of miR-2769 and different splice isoforms of its target gene BmE75 at different developmental stages (larva, pupa and adult) and larval tissues (head, epidermis, silk gland, fat body, testis, ovary, Malpighian tubules, midgut and hemolymph) of B. mori were detected by qRT-PCR. 【Results】 The results showed that miR-2769 could significantly inhibit the expression of luciferase reporter gene by interacting with the 3′UTR binding site of BmE75B of B. mori. The qRT-PCR results showed that miR-2769 and BmE75A/BmE75B presented the opposite expression trends in the fat body of B. mori post induction by 20E. The spatio-temporal expression analysis showed that miR-2769 and different splice isoforms of the target gene BmE75 had specific expression characteristics in different developmental stages and larval tissues of B. mori. The expression levels of miR-2769 and BmE75A were low in B. mori at different developmental stages; however, the expression level of BmE75B was relatively high in the pupal stage and that of BmE75C was extremely high in the late 5th instar larvae and early pupae. Furthermore, the expression of miR-2769 was negatively correlated with that of different splice isoforms of BmE75. miR-2769 promoted the expression of BmE75A in the hemolymph and BmE75B in the fat body of the 5th instar larvae of B. mori, while it inhibited the expression of different splice isoforms of BmE75 in other tissues. 【Conclusion】 MiR-2769 can negatively regulate the expression of different splice isoforms of BmE75 by interacting with the 3′UTR region of E75B in B. mori.

Key words: Bombyx mori, metamorphosis, molting hormone, miRNAs, target gene, qRT-PCR, expression analysis