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Journal of Zhejiang University SCIENCE B
ISSN 1673-1581(Print), 1862-1783(Online), Monthly
2016 Vol.17 No.2 P.127-135
bmo-miR-0001 and bmo-miR-0015 down-regulate expression of Bombyx mori fibroin light chain gene in vitro
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.
Key words: Bombyx mori, MicroRNA, bmo-miR-0001, bmo-miR-0015, BmFib-L, Regulation of expression
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。
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DOI:
10.1631/jzus.B1500078
CLC number:
S881.26
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On-line Access:
2016-02-01
Received:
2015-04-06
Revision Accepted:
2015-11-07
Crosschecked:
2016-01-13