家蚕核型多角体病毒编码miR-364对DNA聚合酶基因表达的调控作用研究

Analysis on Expressional Regulation of DNA Polymerase Gene by Bom byx mori Nucleopolyhedrovirus-encoded miR-364

MicroRNA(miRNA)在调控病毒与宿主相互作用方面起着重要作用。为了研究家蚕核型多角体病毒(Bm NPV)编码的miRNA对其自身靶基因的调控作用,通过高通量测序得到Bm NPV编码的一条miRNA——Bm NPV-miR-364,利用在线靶基因预测程序RNAhybrid与RNA22预测Bm NPV基因组中有6个基因可能是Bm NPV-miR-364的靶基因。分别构建Bm NPVmiR-364与6个侯选靶基因3'-UTR的表达载体,共转染Bm N细胞后检测其双荧光素酶活性,结果表明Bm NPV-miR-364对Bm NPV DNA聚合酶(DNA polymerase)基因的抑制作用显著(P<0.05),对其余5个靶基因的抑制作用不显著。进一步采用Bm NPV-miR-364 mimic与Bm NPV-miR-364 inhibitor分别进行过表达和缺失表达分析,显示Bm NPV-miR-364 mimic能显著抑制DNA polymerase基因的表达,而Bm NPV-miR-364 inhibitor能显著解除Bm NPV-miR-364对DNA polymerase基因的抑制作用。将Bm NPV-miR-364与DNA polymerase基因的表达载体共转染Bm N细胞,qRT-PCR分析发现实验组细胞中病毒DNA polymerase基因的表达水平显著低于对照组。qRT-PCR检测家蚕经口接种Bm NPV后24 h内,血淋巴中Bm NPV-miR-364和病毒DNA polymerase基因的表达量很低,但在24 h之后表达量急剧上升。研究结果提示:Bm NPV编码的miR-364可以靶向抑制自身复制过程中的重要酶基因,调节DNA的复制。

MicroRNAs （miRNAs） were reported as playing important roles in the regulation of virus-host interactions. This experiment focuses on the study of regulatory effect of Bombyx mori nucleopolyhedrovirus （BmNPV）-encoded miRNAs on its own target genes. We obtained a BmNPV-encoded miRNA, named BmNPV-miR-364, by high-throughput sequen- cing. Using the online target gene prediction program RNAhybrid and RNA22, we found 6 potential target genes of BmN- PV-miR-364 in BmNPV genome. The expression vectors carrying BmNPV-miR-364 and 3＇-UTR of 6 candidate target genes were constructed and then co-transfected into BmN cells. Determination of luciferase activity revealed that BmNPV- miR-364 could inhibit the expression of DNA polymerase gene significantly, but had no significant effect on the other 5 target genes. Furthermore, BmNPV-miR-364 mimic and BmNPV-miR-364 inhibitor vectors were constructed for the over-expression and deficiency-expression of BmNPV-miR-364. The results showed that the expression of BmNPV DNA polymerase gene was inhibited by BmNPV-miR-364 mimic significantly, while BmNPV-miR-364 inhibitor could relieve the inhibition of BmNPV-miR-364 on DNA polymerase gene. After the expression vector carrying BmNPV-miR-364 and DNA polymerase gene was co-transfected into BmN cells, qRT-PCR analysis showed that the expression of viral DNA polymerase gene in experimental group was significantly lower than that in control group, qRT-PCR analysis indicated that the expression of BmNPV-miR-364 and viral DNA polymerase gene was very low in silkworm larval hemolymph within 24 h after BmNPV infection, and after the ex- pression of both of them increased sharply. These results demonstrated that BmNPV-miR-364 can interact with its target genes which involves in self-replication of BmNPV to regulate the replication of BmNPV genomic DNA.