Posttranscriptional Regulation Of Bmo-miR-305* On BmSGF-1 And BmFMBP-1

The silk glands of the silkworm(Bombyx mori) have high efficiency and distinctive capability to produce vast amounts of several silk proteins, and the silk gland transcription factors play extremely important roles in this process. MicroRNA has attracted more and more attention due to its crucial roles closely related to individual development, cell proliferation, differentiation, and metabolism process of life. The regulation network of mi RNAs is complex, that is, one miRNA can control multiple target genes, in the other face, multi-miRNAs can affect the same target. Although the binding sites and regulation factors of silk protein gene have been reported, but the precise mechanisms were still not enough being solved. In order to explore the relationship between mi RNAs, silk gland specific transcription factors and silk protein gene, it is not only need to find the regulation factors and critical points, it is important to research to miRNAs regulation on silk gland specific transcription factor gene. The project was carried out, and the following conclusions were drawed.1、Identification of bmo-miRNAs1 Bioinformatics was used to analyze the transcriptional regulation of bmo-miRNAs on SGF-1 or FMBP-1. The results showed that, there were 20 binding sites on BmSGF-1 3′UTR, and 13 binding sites on BmFMBP-1 3′UTR.Stem loop PCR was used to clone candidate bmo-miRNAs, and the results were compared with miRBase reads. The results showed that, there were six correct candidate mi RNAs that may regulate SGF-1, that is, bmo-miR-305* 、 bmo-miR-2731-1 、bmo-miR-2731-2、bmo-mi R-2775a、bmo-miR-3327* and bmo-miR-1a. There were three correct candidate miRNAs that may regulate FMBP-1, that is, bmo-miR-2b*、bmo-miR-305 and bmo-miR-2758.2、 Expression analysis of candidate bmo-miRNAs, BmSGF-1 gene and BmFMBP-1 geneSemi-quantitative PCR was used to detect the relative expression of bmo-miR-2731-1/-2 and bmo-miR-2775 a, and the result showed that the expression of bmo-miR-2731-1/-2 and bmo-miR-2775 a in PSG was lower than that in MSG, and the expression level was first decreased and then increased during the periods of larvae. Quantitative PCR was carried out to determine the relative expression of bmo-miR-305*, bmo-miR-1a and SGF-1, and the result revealed that the relative expressions of bmo-miR-305* and bmo-mi R-1a in PSG were correlated to SGF-1. Semi-quantitative PCR was used to detect the relative expression of bmo-miR-2758 and FMBP-1. The results showed that, the expression of bmo-miR-2758 was observed in both the MSG and PSG at different development stages, with expression in the PSG greater than that in the MSG, which was found to follow similar expression trends of FMBP-1.3 Regulation of bmo-miR-305* and bmo-miR-3327* on the posttranscription of BmSGF-1Recombinant vectors pcDNA3 [ie1-egfp-pri-mir-305-SV40] and pcDNA3 [ie1-egfp-pri-mir-3327-SV40] were constructed, and was co-transfected into BmN cell with constructed recombinant vectors pGL3[A3-luc-SGF-1-3′-UTR-SV40], respectively. The results showed that, the expression of a fluorescent reporter was significantly decreased when co-transfected with bmo-miR-305*(P < 0.01) or bmo-miR-3327*(P < 0.01), and increased after transfect each inhibitor(P < 0.05), which suggested that the expression of bmo-miR-305* and bmo-miR-3327*repressed the expression of SGF-1.4 Regulation of bmo-miR-2758 on the posttranscription of BmFMBP-1Recombinant vectors pcDNA3 [ie1-egfp-pri-mir-2758-SV40] were constructed, and was co-transfected into BmN cell with constructed recombinant vectors pGL3[A3-luc-SGF-1-3′-UTR-SV40]. The results showed that, luciferase activity after transfecting bmo-miR-2758 recombinant vectors was significantly reduced(P < 0.05). Meanwhile, luc expression showed a slight increase in the group transfected with bmo-miR-2758 inhibitor(P < 0.05).Thus, bmo-miR-2758 may inhibit the expression of FMBP-1.5 Regulation of bmo-miR-34 on the posttranscription of BmE74In order to identify the binding site involved in BmE74 3′UTR, bioinformatics software RNAhybrid and RNA22 were designed. The results showed that there are binding sites of bmo-miR-34 on BmE74 3′UTR. Both pcDNA3 [ie1-egfp-pri-mir-34-SV40] and pGL3[A3-luc-SGF-1-3′-UTR-SV40] recombinant vectors were constructed, and were co-transfected into BmN cell. The results showed that, the expression of a fluorescent reporter was significantly decreased when co-transfected with bmo-miR-34(P < 0.01), and ascended distinctly after transfect inhibitor(P < 0.01), which suggested that the expression of bmo-miR-34 repressed the expression of the fluorescent protein. Thus, it indicated that bmo-miR-34 inhibits E74 gene expression.