【摘要】 为了研究家蚕miRNA对丝素蛋白基因表达的调控作用,采用生物信息学方法筛选获得对家蚕丝素轻链基因(BmFib-L)和P25蛋白基因(BmP25)有潜在调控作用的Bmo-miR-2755*。利用半定量RT-PCR技术分析Bmo-miR-2755*及其靶基因BmFib-L、BmP25在家蚕4~5龄期幼虫后部丝腺和5龄3 d幼虫不同组织器官中的表达水平,结果显示三者在后部丝腺组织中的表达水平都较高,呈现严格的时空特异性。以pcDNA3质粒为载体,构建Bmo-miR-2755*的重组表达载体pcDNA3[ie1-egfpprimiR-2755*-SV40];以pGL3.0-Basic质粒为载体,分别构建BmFib-L 3’UTR、BmP25 3’UTR与荧光素酶报告基因luc融合的重组报告质粒pGL3.0[A3-luc-Fib-L-3’UTR-SV40]和pGL3.0[A3-luc-P25-3’UTR-SV40]。以海肾荧光素酶表达载体pRL-CMV为内参,分别将构建的Bmo-miR-2755*重组表达载体和2个重组报告质粒共转染BmN细胞,通过检测细胞中的荧光素酶活性,明确Bmo-miR-2755*可显著下调BmFib-L基因的表达,并能上调BmP25基因的表达,但上调作用未达显著水平。上述结果为研究家蚕miRNA的功能和阐明蚕丝蛋白基因表达调控分子机制提供了新的实验数据。更多还原

【Abstract】 In order to study the regulation function of Bombyx mori miRNA on expression of silk fibroin genes,we identified a miRNA,named Bmo-miR-2755*,by bioinformatics,which is predicted to have potential regulatory function on the expression of silk fibroin light chain gene( BmFib-L) and BmP25, and we analyzed the expression level of Bmo-miR-2755* and its target genes BmFib-L and BmP25 in posterior silk gland of the 4th-5th instar larvae and differenttissues of day 3 larvae of the 5th instar by semiquantitative RT-PCR. The results showed that their expressions in posterior silk gland were at higher level,showing strict temporal and spatial specificity. The recombinant expression plasmid pcDNA3 [ie1-egfp-pri-miR-2755*-SV40] carrying Bmo-miR-2755* was constructed by using plasmid pcDNA3 as vector, and luciferase reporter gene luc was fused with 3’UTR of BmFib-L andBmP25 respectively to construct the recombinant reporter plasmids pGL3. 0 [A3-luc-Fib-L-3’UTR-SV40] and pGL3. 0[A3-luc-P25-3’UTR-SV40] by using plasmid pGL3. 0-Basic as vector. Then BmN cells were cotransfected with the recombinant expression vector of Bmo-miR-2755* and two fused recombinant reporter plasmids using Renilla reniformis luciferase expression vector pRL-CMV as internal reference. Luciferase activity determination indicated that Bmo-miR-2755* significantly down-regulated the expression of BmFib-L gene,and the expression of BmP25 gene was up-regulated but had no significant difference. It provides new data for the study of miRNA function and regulation mechanism of silk fibroin genes.更多还原