Bmo-miR-2788对家蚕丝胶蛋白基因Ser-1的表达调控

Regulation on Expression of Bombyx mori Sericin-1 Gene by BmomiR-2788

通过生物信息学分析发现,家蚕丝胶蛋白基因BmSer-1的3′非翻译区(3′-UTR)存在微小RNA Bmo-miR-2788的结合位点。半定量PCR结果显示,Bmo-miR-2788在家蚕5龄第4天幼虫的8个受试组织中均有表达,但在中部丝腺中表达水平最高;而BmSer-1仅在中部丝腺中被检测到。分别构建BmSer-13′-UTR和Bmo-miR-2788的表达载体pGL3.0(A3-luc-BmSer-1-3′-UTR-SV40)和pcDNA3.0(ie1-egfp-pri-Bmo-miR-2788-SV40),共转染BmN细胞,结果表明Bmo-miR-2788上调BmSer-1表达(P<0.01);当将上述载体与合成的Bmo-miR-2788抑制剂共转染BmN细胞时,BmSer-1表达显著下调(P<0.05)。为验证Bmo-miR-2788在蚕体内对BmSer-1表达的调控作用,将Bmo-miR-2788表达质粒与其阴性对照(pcDNA3.0)分别注射到家蚕5龄第3天幼虫体内,qRT-PCR分析结果显示,与阴性对照相比,注射Bmo-miR-2788后36 h中部丝腺中的BmSer-1表达显著上调(P<0.01)。上述结果表明Bmo-miR-2788正向调控BmSer-1基因的表达。

Integrated bioinformatics analysis revealed that there was a binding site for miRNA Bmo-miR-2788 in the 3′-untranslated region(3′-UTR)of sericin gene BmSer-1.Semi-quantitative polymerase chain reaction(PCR)results showed that Bmo-miR-2788 was expressed in 8 tissues of Bombyx mori larvae of 4 th day 5 th instar,and the highest expression was detected in the middle silk gland(MSG).BmSer-1 expression was detected only in the MSG.Then,expression vectors of BmSer-13′-UTR and Bmo-miR-2788 were constructed.After pGL3.0(A3-luc-BmSer-1-3′-UTR-SV40)and pcDNA3.0(ie1-egfp-pri-Bmo-miR-2788-SV40)were co-transfected in BmN cells,BmSer-1 expression was up-regulated by Bmo-miR-2788(P<0.01).When the above vectors and synthetic Bmo-miR-2788 inhibitor were co-transfected in BmN cells,BmSer-1 expression was significantly down-regulated(P<0.05).Additionally,Bmo-miR-2788 expressed plasmid and its negative control(pcDNA3.0)were injected into 3 rd day 5 th instar silkworm larvae to validate the function of BmomiR-2788 on BmSer-1 expression in vivo.Quantitative real-time PCR(qRT-PCR)results showed that Bmo-miR-2788 significantly up-regulated BmSer-1 expression in MSG 36 h post injection(P<0.01)as compared with the negative control.All above results demonstrate that Bmo-miR-2788 can positively regulate expression of BmSer-1 gene.