Cloning, Expression And Function Analysis Of Endogenous Noncoding MicroRNA MD5 From Antheraea Pernyi Nucleo Polyhedrovirus

Nucleopolyhedrovirus is the pathogenic microorganism which can cause purulent disease threatening a large number of productions of Antheraea pernyi, the Chinese oak silkmoth to death. It is related to the molecular level for pathogenicity of the insect virus, but it is still in the initial stage for studying interaction between insects and pathogens on miRNA. MicroRNAs (miRNAs) are a class of endogenous, noncoding RNAs of 21-25 nucleotides (nt) and widely exist in eukaryocyte, which plays vital roles in the biological process of growth, development, differentiation, proliferation, metabolism, immune responses and cell apoptosis.In this paper, miRNA was predicted from ApNPV genome by Vmir software and 7 novel miRNAs (as well as their corresponding miRNA*s) were screened. The target genes of the mature VmiRNA were found by using RNAhybrid software. Based on the results of target genes prediction, among the 12 target genes of VmiRNA,2 genes were related to the process of immune response,1 was involved in immune regulation, and 1 participates in the development of the immune system. GO analysis was performed and the result showed that the target genes of VmiRNA MD5 mainly involved in cell composition, molecular function, and biological process pathway, and which are related to molecular binding, catalytic and immune system. Real-time quantitative PCR was performed to detect the expression pattern of MD5 and its target genes after infected by ApNPV. The results showed that the expression level of MD5 was obviously up-regulated in fat body and target genes (MAPKK4/7-. Ap-1-like, IRAK4) were down-regulated, which suggest that MD5 may be involved in immune response and participate in regulation of expression of related genes.For further elucidate the function, MD5 was cloned from ApNPV genome and over expression vector used for transformation was constructed. Transient expression and transformation in Tn-Hi5 cell suggested that overexpression MD5 inhibited the expression of its target genes. Chemical mimics and dsRNA of MD5 were synthetized in vitro and to dected the fluorescence intensity of enhance green fluorescent protein (EGFP) in Tn-Hi5 cells which was infected by ApNPV-Aph/egfp+. The results showed that overexpression of MD5 inhibited the expression of the antiviral genes, which was conducive to the proliferation of the virus in the early stage of the infection. The average fluorescence intensity of transfected cells by dsRNA wass lower than that of normal Tn-Hi5 cells transfected by ApNPV-Aph/egfp+ cell and the average fluorescence intensity of transfected cells by MD5 mimics was higher than that of normal cells.In a word, VmiRNA MD5 is involved in the interference of antiviral immune response in Antheraea pernyi.