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Received: 2023-10-17

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Journal of Zhejiang University SCIENCE B 2016 Vol.17 No.2 P.127-135

http://doi.org/10.1631/jzus.B1500078


bmo-miR-0001 and bmo-miR-0015 down-regulate expression of Bombyx mori fibroin light chain gene in vitro


Author(s):  Chen Chen, Yang-yang Fan, Xin Wang, Fei Song, Tao Jiang, Ping Qian, Shun-Ming Tang, Xing-Jia Shen

Affiliation(s):  Jiangsu Key Laboratory of Sericultural Biology and Biotechnology, School of Biotechnology, Jiangsu University of Science and Technology, Zhenjiang 212003, China; more

Corresponding email(s):   shenxjsri@163.com

Key Words:  Bombyx mori, MicroRNA, bmo-miR-0001, bmo-miR-0015, BmFib-L, Regulation of expression


Chen Chen, Yang-yang Fan, Xin Wang, Fei Song, Tao Jiang, Ping Qian, Shun-Ming Tang, Xing-Jia Shen. bmo-miR-0001 and bmo-miR-0015 down-regulate expression of Bombyx mori fibroin light chain gene in vitro[J]. Journal of Zhejiang University Science B, 2016, 17(2): 127-135.

@article{title="bmo-miR-0001 and bmo-miR-0015 down-regulate expression of Bombyx mori fibroin light chain gene in vitro",
author="Chen Chen, Yang-yang Fan, Xin Wang, Fei Song, Tao Jiang, Ping Qian, Shun-Ming Tang, Xing-Jia Shen",
journal="Journal of Zhejiang University Science B",
volume="17",
number="2",
pages="127-135",
year="2016",
publisher="Zhejiang University Press & Springer",
doi="10.1631/jzus.B1500078"
}

%0 Journal Article
%T bmo-miR-0001 and bmo-miR-0015 down-regulate expression of Bombyx mori fibroin light chain gene in vitro
%A Chen Chen
%A Yang-yang Fan
%A Xin Wang
%A Fei Song
%A Tao Jiang
%A Ping Qian
%A Shun-Ming Tang
%A Xing-Jia Shen
%J Journal of Zhejiang University SCIENCE B
%V 17
%N 2
%P 127-135
%@ 1673-1581
%D 2016
%I Zhejiang University Press & Springer
%DOI 10.1631/jzus.B1500078

TY - JOUR
T1 - bmo-miR-0001 and bmo-miR-0015 down-regulate expression of Bombyx mori fibroin light chain gene in vitro
A1 - Chen Chen
A1 - Yang-yang Fan
A1 - Xin Wang
A1 - Fei Song
A1 - Tao Jiang
A1 - Ping Qian
A1 - Shun-Ming Tang
A1 - Xing-Jia Shen
J0 - Journal of Zhejiang University Science B
VL - 17
IS - 2
SP - 127
EP - 135
%@ 1673-1581
Y1 - 2016
PB - Zhejiang University Press & Springer
ER -
DOI - 10.1631/jzus.B1500078


Abstract: 
Based on bioinformatic analysis, we selected two novel microRNAs (miRNAs), bmo-miR-0001 and bmo-miR-0015, from high-throughput sequencing of the Bombyx mori larval posterior silk gland (PSG). Firstly, we examined the expression of bmo-miR-0001 and bmo-miR 12 different tissues of the 5th instar Day-3 larvae of the silkworm. The results showed that the expression levels of both bmo-miR-0001 and bmo-miR-0015 were obviously higher in the PSG than in other tissues, implying there is a spatio-temporal condition for bmo-miR-0001 and bmo-miR-0015 to regulate the expression of BmFib-L. To test this hypothesis, we constructed pri-bmo-miR-0001 expressing the plasmid pcDNA3.0 and pri-bmo-miR-0015 expressing the plasmid pcDNA3.0 [ie1-egfp-pri-bmo-miR-0015-SV40]. Finally, the BmN cells were harvested and luciferase activity was detected. The results showed that luciferase activity was reduced significantly (P<0.05) in BmN cells co-transfected by pcDNA3.0 [ie1-egfp-pri-bmo-miR-0001-SV40] or pcDNA3.0 with pGL3.0 [A3-luc-Fib-L-3'UTR-SV40], suggesting that both bmo-miR-0001 and bmo-miR-0015 can down-regulate the expression of BmFib-L in vitro.

家蚕miR-0001和miR-0015体外下调BmFib-L基因的表达

目的:探究新发现的两个miRNA对家蚕丝素轻链基因BmFib-L的调控作用。
创新点:在家蚕后部丝腺中发现两个新的miRNA,并首次证明它们对家蚕丝素蛋白轻链基因BmFib-L有负调控作用。
方法:本实验通过生物信息学分析,从家蚕后部丝腺miRNA高通量测序获得的新miRNA中,筛选出两个可能对家蚕丝素蛋白轻链基因BmFib-L有调控作用的miRNA,即bmo-miR-0001和bmo-miR-0015。设计茎环引物,采用反转录聚合酶链反应(RT-PCR)方法对家蚕5龄3 d头部、表皮、脂肪体、马氏管、精巢/卵巢、丝腺(前、中、后)、气管、中肠和血淋巴细胞等12个不同组织的bmo-miR-0001和bmo-miR-00015进行半定量表达分析。并采用双荧光报告基因检测系统进一步在细胞水平上验证bmo-miR-0001和bmo-miR-0015对BmFib-L表达的调控作用。
结论:本实验中RT-PCR结果显示,这两个新miRNA在家蚕后部丝腺中表达量最高(图4)。双荧光报告基因检测结果显示,报告基因荧光素酶活性明显低于阳性对照组(图7),转染bmo-miR-0001和bmo-miR-0015表达载体的细胞,报告基因和荧光素酶活性分别只及对照组60%和69%。t检验分析结果显示两个实验组与对照组之间差异都达到显著水平(P<0.05)。由此可见,bmo-miR-0001和bmo-miR-0015在体外对BmFib-L的表达具有显著的抑制作用。

关键词:家蚕;miRNA;bmo-miR-0001;bmo-miR-0015;BmFib-L;功能验证

Darkslateblue:Affiliate; Royal Blue:Author; Turquoise:Article

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