Bombyx mori cytoplasmic polyhedrosis virus (BmCPV) is a major pathogen of the economic insect silkworm, Bombyx mori. Virus-encoded microRNAs (miRNAs) have been proven to play important roles in host-pathogen interactions. In this study we identified a BmCPV-derived miRNA-like 21 nt small RNA, BmCPV-miR-1, from the small RNA deep sequencing of BmCPV-infected silkworm larvae by stem-loop quantitative real-time PCR (qPCR) and investigated its functions with qPCR and lentiviral expression systems. Bombyx mori inhibitor of apoptosis protein (BmIAP) gene was predicted by both target prediction software miRanda and Targetscan to be one of its target genes with a binding site for BmCPV-miR-1 at the 5 untranslated region. It was found that the expression of BmCPV-miR-1 and its target gene BmIAP were both up-regulated in BmCPV-infected larvae. At the same time, it was confirmed that BmCPV-miR-1 could up-regulate the expression of BmIAP gene in HEK293T cells with lentiviral expression systems and in BmN cells by transfecting mimics. Furthermore, BmCPV-miR-1 mimics could up-regulate the expression level of BmIAP gene in midgut and fat body in the silkworm. In the midgut of BmCPV-infected larvae, BmCPV-miR-1 mimics could be further up-regulated and inhibitors could lower the virus-mediated expression of BmIAP gene. With the viral genomic RNA segments S1 and S10 as indicators, BmCPV-miR-1 mimics could up-regulate and inhibitors down-regulate their replication in the infected silkworm. These results implied that BmCPV-miR-1 could inhibit cell apoptosis in the infected silkworm through up-regulating BmIAP expression, providing the virus with a better cell circumstance for its replication.
Flavan-3-ols are monomers of Proanthocyanidins (PAs), which are important polyphenolic compounds in grapes. Previous studies had shown that VvMYBPA2 was closely related to grape flavan-3-ol monomers biosynthesis, but its regulatory network is still unclear. Here, we found that the contents of (+)-catechin and (−)-epicatechin, the enzyme activities of anthocyanidin reductase (ANR) and leucoanthocyanidin reductase (LAR) and the expression of VvANR and VvLAR1 were increased in the VvMYBPA2 overexpression grape leaves compared to the control. It was proved that VvMYBPA2 protein interacted with VvWDR1 and VvWDR1 protein interacted with VvMYC2 by yeast two-hybrid (Y2H) and bimolecular fluorescence complementation (BiFC). The promoters of VvANR and VvLAR1 were bound by VvMYBPA2 using yeast one-hybrid (Y1H) assay. These results suggested that VvMYBPA2 could form a trimeric complex with VvWDR1 and VvMYC2 and jointly regulated the expression of flavan-3-ol monomers related genes VvANR and VvLAR1, thereby affecting the enzyme activities of ANR and LAR and ultimately regulating the contents of flavan-3-ols.
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