家蚕bmo-miR-0031-3p体内下调丝素轻链基因<i>BmFib-L</i>的表达

doi:10.3785/j.issn.1008-9209.2018.11.261

浙江大学学报（农业与生命科学版）

2019, Vol. 45

Issue (2): 229-236 DOI: 10.3785/j.issn.1008-9209.2018.11.261

动物科学与动物医学

家蚕bmo-miR-0031-3p体内下调丝素轻链基因BmFib-L的表达

陈艳花^1,²(

),蒋涛^1,²,王雪珍^1,²,钱平^1,²,唐顺明^1,²,沈兴家²(

)

1. 江苏科技大学生物技术学院，江苏省蚕桑生物学与生物技术重点实验室，江苏镇江 212018
2. 中国农业科学院蚕业研究所，农业农村部蚕桑遗传改良重点实验室，江苏镇江 212018

Bmo-miR-0031-3p down-regulates the expression of Bombyx mori fibroin light chain gene BmFib-L in vivo

Yanhua CHEN^1,²(

),Tao JIANG^1,²,Xuezhen WANG^1,²,Ping QIAN^1,²,Shunming TANG^1,²,Xingjia SHEN²(

)

1. Jiangsu Key Laboratory of Sericultural Biology and Biotechnology, School of Biotechnology, Jiangsu University of Science and Technology, Zhenjiang 212018, Jiangsu, China
2. Key Laboratory of Silkworm and Mulberry Genetic Improvement, Ministry of Agriculture and Rural Affairs, Sericultural Research Institute, Chinese Academy of Agricultural Sciences, Zhenjiang 212018, Jiangsu, China

全文: PDF(1962 KB)

HTML ( ) HTML

摘要：

为了研究家蚕微RNA（microRNA, miRNA）对丝素轻链基因BmFib-L表达的调控作用，以BmFib-L mRNA的3′非翻译区（3′ untranslated region, 3′ UTR）为靶标，通过RNAhybrid软件分析，筛选出种子序列与BmFib-L 3′ UTR完全互补的家蚕miRNA——bmo-miR-0031-3p（简称“miR-0031-3p”）。分别构建miR-0031-3p表达载体pcDNA3.0[ie1-egfp-pre-miR-0031-3p-SV40]和BmFib-L 3′ UTR融合萤光素酶报告基因重组表达质粒pGL3.0[A3-luc-Fib-L-3′ UTR-SV40]，以海肾萤光素酶表达载体pRL-CMV为内参，共转染BmN细胞，通过检测双萤光素酶活性验证miR-0031-3p的功能；人工合成miR-0031-3p的模拟物（mimic）和抑制物（inhibitor），再进一步验证miR-0031-3p对BmFib-L的调控功能。结果显示，在BmN细胞中，miR-0031-3p显著抑制BmFib-L的表达。为了进一步验证miR-0031-3p在家蚕体内对BmFib-L表达的调控作用，在5龄第2天幼虫体腔内注射转染物，分别在体内过表达和抑制内源性miR-0031-3p，荧光定量分析靶基因表达水平。结果显示，miR-0031-3p在幼虫体内能够下调BmFib-L的表达。该研究结果有利于阐明家蚕miRNA功能和蚕丝蛋白表达调控的分子机制。

关键词： 微RNA; 家蚕; bmo-miR-0031-3p; 丝素轻链基因; 转录后调控

Abstract:

To study the regulatory function of Bombyx mori microRNAs (bmo-miRNAs) on expression of the fibroin light chain gene (BmFib-L), the 3′ untranslated region (3′ UTR) of BmFib-L mRNA was used as the target for screen of bmo-miRNAs. By using RNAhybrid software, the bmo-miR-0031-3p (abbreviated as “miR-0031-3p”) was screened out to completely bind the target gene with the seed sequence. A miR-0031-3p expression plasmid pcDNA3.0[ie1-egfp-pre-miR-0031-3p-SV40] and a BmFib-L 3′ UTR fused luciferase report plasmid pGL3.0[A3-luc-Fib-L-3′ UTR-SV40] were constructed, respectively. BmN cells were co-transfected with the above mentioned plasmids, and the pRL-CMV (contains a Renilla luciferase gene) was served as an intrinsic plasmid to validate the regulatory function of miR-0031-3p on BmFib-L by assay of dual luciferase activities, as well as artificially synthesized the mimic and inhibitor of miR-0031-3p. The results revealed that the miR-0031-3p significantly down-regulated the expression of BmFib-L in the BmN cells. To validate the regulatory function of miR-0031-3p in vivo, the day-2 5th instar larvae were injected with a transfection solution for overexpression and inhibition of endogenous expression analysis, and the BmFib-L expression was analyzed by quantitative reverse transcription polymerase chain reaction (RT-PCR) using total RNAs extracted from silk glands. The results showed that the miR-0031-3p significantly down-regulated the expression of BmFib-L in individuals. These findings are beneficial to clarify the molecular mechanism of miRNAs in regulating B. mori silk protein biosynthesis.

Key words: microRNA Bombyx mori bmo-miR-0031-3p fibroin light chain gene BmFib-L post-transcriptional regulation

收稿日期: 2018-11-26 出版日期: 2019-04-25

CLC:

S 881.2

基金资助: 国家自然科学基金(31672490，31172266);江苏省自然科学基金(BK20151322);江苏省高校自然科学研究重大项目(15KJA180001)

通讯作者: 沈兴家 E-mail: 2317965026@qq.com;shenxjsri@163.com

作者简介: 陈艳花（https://orcid.org/0000-0002-5002-8736），E-mail： 2317965026@qq.com|沈兴家（https://orcid.org/0000-0002-7436-1760），Tel：+86-511-85616543，E-mail： shenxjsri@163.com

	服务
	把本文推荐给朋友
	加入引用管理器
	E-mail Alert
	RSS
	作者相关文章
	陈艳花
	蒋涛
	王雪珍
	钱平
	唐顺明
	沈兴家

引用本文:

陈艳花,蒋涛,王雪珍,钱平,唐顺明,沈兴家. 家蚕bmo-miR-0031-3p体内下调丝素轻链基因BmFib-L的表达[J]. 浙江大学学报（农业与生命科学版）, 2019, 45(2): 229-236.

Yanhua CHEN,Tao JIANG,Xuezhen WANG,Ping QIAN,Shunming TANG,Xingjia SHEN. Bmo-miR-0031-3p down-regulates the expression of Bombyx mori fibroin light chain gene BmFib-L in vivo. Journal of Zhejiang University (Agriculture and Life Sciences), 2019, 45(2): 229-236.

链接本文:

http://www.zjujournals.com/agr/CN/10.3785/j.issn.1008-9209.2018.11.261 或 http://www.zjujournals.com/agr/CN/Y2019/V45/I2/229

表1 相关基因引物序列

图1 miR-0031-3p对 BmFib - L mRNA 3´ UTR潜在结合位点的生物信息学预测

图2 半定量RT-PCR检测miR-0031-3p在不同组织中的表达

图3 Pre-miR-0031-3p和 BmFib - L 3´ UTR表达载体的构建

图4 miR-0031-3p在BmN细胞中对 BmFib - L 的表达调控

图5 在体内过表达和抑制表达miR-0031-3p后 BmFib-L 的表达检测

1	JUNICHI T , HIROYUKI K , KIYOSHI Y , et al . Reduced expression of the let-7 microRNAs in human lung cancers in association with shortened postoperative survival.Cancer Research, 2004,64(11):3753-3756.
2	LI H , XIE H , LIU W , et al . A novel microRNA targeting HDAC5 regulates osteoblast differentiation in mice and contributes to primary osteoporosis in humans.Journal of Clinical Investigation, 2009,119(12):3666-3677.
3	KARA M , YUMRUTAS O , OZCAN O , et al . Differential expressions of cancer-associated genes and their regulatory miRNAs in colorectal carcinoma. Gene, 2015,567(1):81-86.
4	FRANKEL L B , CHRISTOFFERSEN N R , JACOBSEN A , et al . Programmed cell death 4 (PDCD4) is an important functional target of the microRNA miR-21 in breast cancer cells. Journal of Biological Chemistry, 2008,283(2):1026-1033.
5	PILLAI R S , BHATTACHARYYA S N , FILIPOWICZ W . Repression of protein synthesis by miRNAs: how many mechanisms? Trends in Cell Biology , 2007,17(3):118-126.
6	BARTEL D P . MicroRNAs: target recognition and regulatory functions. Cell, 2009,136(2):215-233.
7	HUANG Y , ZOU Q , SONG F , et al . The regulation of silkworm fibroin L chain production by miRNA-965 and miRNA-1926 in insect cells. Russian Journal of Bioorganic Chemistry, 2012,38(4):417-421.
8	YU F Y , YAO H R , ZHU P C , et al . let-7 regulates self renewal and tumorigenicity of breast cancer cells. Cell, 2007,131(6):1109-1123.
9	FORMAN J J , LEGESSE-MILLER A , COLLER H A . A search for conserved sequences in coding regions reveals that the let-7 microRNA targets Dicer within its coding sequence.Proceedings of the National Academy of Sciences of the USA, 2008,105(39):14879-14884.
10	DUURSMA A M , KEDDE M , SCHRIER M , et al . miR-148 targets human DNMT3b protein coding region. RNA, 2008,14(5):872-877.
11	ZHOU H L , RIGOUTSOS I . MiR-103a-3p targets the 5′ UTR of GPRC5A in pancreatic cells. RNA, 2014,20(9):1431-1439.
12	JULIEN E , BORDEAUX M C , GAREL A , et al . Fork head alternative binding drives stage-specific gene expression in the silk gland of Bombyx mori .Insect Biochemistry & Molecular Biology, 2002,32(4):377-387.
13	DURAND B , DREVET J , COUBLE P . P25 gene regulation in Bombyx mori silk gland: two promoter-binding factors have distinct tissue and developmental specificities.Molecular & Cellular Biology, 1992,12(12):5768-5777.
14	范洋洋,陈晨,王欣,等 .Bmo-miR-2755对丝素蛋白基因BmFib-L和BmP25表达的调控作用.蚕业科学,2015,41(5):847-853. FAN Y Y , CHEN C , WANG X , et al . Expressional regulation of Bombyx mori* fibroin genes Fib-L and P25 by Bmo-miR-2755*. Science of Sericulture, 2015,41(5):847-853. (in Chinese with English abstract)
15	WANG X , TANG S M , SONG F , et al . Bmo-miR-2758 targets BmFMBP-1 (Lepidoptera: Bombycidae) and supp-resses its expression in BmN cells. Journal of Insect Science, 2016,16(1):28.
16	SONG F , WANG X , CHEN C , et al . Characterization and profiling of microRNAs in posterior silk gland of the silkworm (Bombyx mori). Genes & Genomics, 2015,37(8):703-712.
17	CAO J , TONG C Z , WU X J , et al . Identification of conserved microRNAs in Bombyx mori (silkworm) and regulation of fibroin L chain production by microRNAs in heterologous system.Insect Biochemistry and Molecular Biology, 2008,38(12):1066-1071.
18	汪生鹏,孙霞 .家蚕蚕丝蛋白基因表达调控研究进展.安徽农业科学,2009,37(28):13514-13518. WANG S P , SUN X . Research progress of gene expression regulation of silkworm silk protein. Journal of Anhui Agricultural Sciences, 2009,37(28):13514-13518. (in Chinese with English abstract)
19	SHALGI R , LIEBER D , OREN M , et al . Global and local architecture of the mammalian microRNA-transcription factor regulatory network.PLoS Computational Biology, 2007,3(7):e131.

[1]	史利利，蒋彩英，于威，陈琛，蒋磊，巩成见，童富淡. BmNPV orf98对家蚕核型多角体杆状病毒复制、转录及包装的影响[J]. 浙江大学学报(农业与生命科学版), 2015, 41(6): 623-630.
[2]	沈恩惠，刘扬，叶楚玉，樊龙江. 植物非编码小RNA研究进展（英文）[J]. 浙江大学学报(农业与生命科学版), 2014, 40(4): 370-378.
[3]	尹晓辉　朱国念　庄惠生　吴慧明　李少南　. 除草剂炔草酯对家蚕血细胞DNA损伤的研究[J]. 浙江大学学报(农业与生命科学版), 2008, 34(4): 361-366.
[4]	顾燕燕　贺伟强　时连根. 家蚕雌蛾体中孕酮含量的测定方法[J]. 浙江大学学报(农业与生命科学版), 2008, 34(1): 93-97.
[5]	陈金娥　杨惠娟　李建营等　. 家蚕幼虫关联基因在胚胎期的表达（英文）[J]. 浙江大学学报(农业与生命科学版), 2008, 34(1): 38-44.
[6]	朱立成　陈健　林蓉　金勇丰　张耀洲. 融合6个组氨酸的人粒细胞-巨噬细胞集落刺激因子在家蚕幼虫中的表达[J]. 浙江大学学报(农业与生命科学版), 2005, 31(5): 613-616.
[7]	卢觅佳　于涟　谢荣辉　张朝政. 家蚕生物反应器表达传染性法氏囊病病毒多聚蛋白的免疫原性研究[J]. 浙江大学学报(农业与生命科学版), 2004, 30(5): 545-552.
[8]	丁农　钟伯雄　陶京亚　姚耀涛　沈建华. 家蚕杂交率抽样检验的判别方法[J]. 浙江大学学报(农业与生命科学版), 2004, 30(1): 53-56.
[9]	钟伯雄　张金卫　丁农　陶京亚　陆云翀. RAPD技术在家蚕杂交率检验中的应用[J]. 浙江大学学报(农业与生命科学版), 2003, 29(3): 321-324.
[10]	吕顺霖　顾国达　丁春阳. 萘胺类化合物对氟中毒家蚕体内氧自由基水平的影响[J]. 浙江大学学报(农业与生命科学版), 2001, 27(5): 565-568.
[11]	杨瑞丽　吴玉澄　金勇丰　张耀洲　吴祥甫. 利用家蚕生物反应器生产有用蛋白的研究[J]. 浙江大学学报(农业与生命科学版), 2001, 27(2): 173-178.
[12]	鲁兴萌　吴忠长. 家蚕微粒子虫（Nosema bombycis）感染家蚕的病理学研究[J]. 浙江大学学报(农业与生命科学版), 2000, 26(5): 547-550.
[13]	时连根. 常用消毒药剂对不同存在状态微孢子虫孢子的消毒效果[J]. 浙江大学学报(农业与生命科学版), 2000, 26(5): 551-554.
[14]	吕顺霖　顾国达　丁春阳. 萘胺类化合物对氟中毒家蚕幼虫血淋巴中GSH-Px活性的影响[J]. 浙江大学学报(农业与生命科学版), 2000, 26(5): 543-546.
[15]	吕顺霖　杜孟达. 萘胺类化合物对氟中毒家蚕幼虫血液H₂O₂酶活性的影响[J]. 浙江大学学报(农业与生命科学版), 2000, 26(3): 259-260.

Viewed

Full text

Abstract

Cited

Shared

Discussed