Regulation Of Bmo-miR-0001 And Bmo-miR-0015 On Expression Of Fibrion Light Chain Gene Of Bombyx Mori

MicroRNAs(miRNAs) are approximately 20 to 25 nucleotides non-coding small RNA molecules which mostly inhibit the expression or translation of target genes by complementary base pairing to its target mRNA 3’-untranslated region(3’-UTR). The silkworm(Bombyx mori) is an insect that undergoes complete metamorphosis. It not only has the common biological traits of other insects but has its own representative properties making it to be an excellent model for study of insect genetics and molecular biology. Therefore, studing on miRNA of silkworm will help to reveal the role of miRNAs in the process of growth and development of silkworm. In the previous high-throughput sequencing of PSG sRNA in our laboratory, 35 novel bmo-miRNAs were obtained. To validate the regulation of miRNA on the expression of fibroin, the BmFib-L as target genes be used to screen the miRNA. Then we examined the expression of these miRNA in 12 different tissues of the 5th instar day-3larvae of the silkworm by semi-quantitative reverse transcription polymerase chain reaction(RT-PCR). Finally, Dual fluorescence reporter system was applied to validate the regulation of these two miRNAs on expression of BmFib-L in cells. The main results are as follows:1. Bmo-miR-0001 and bmo-miR-0015 were predicted to potentially target BmFib-L3’UTR.By using RNAhybrid target gene prediction software and based on our preliminary results of Solexa sequencing, we obtained 2 novel bmo-miRNA(bmo-miR-0001 and bmo-miR-0015) that possess potential binding sites for 3′UTR of BmFib-L mRNA and we done the experimental verification. We also predict their secondary structure, the sequence analysis shows that the precursor of bmo-miR-0001 and bmo-miR-0015 could form a typical stem-loop structure, and include a complete mature-miRNA sequences and sequences are in the arms of the hairpin structure, these results in line with the secondary structure of miRNAs.2. Bmo-miR-0001 and bmo-miR-00015 have the tissue specificity conditions of the regulation of BmFib-L expression.Semi-quantitative expression analysis of tissues were carried out in 12 different tissues(head, skin, fat body, Malpighian tubules, testis / ovary, silk gland(ASG, MSG,PSG), trachea, intestines and blood lymphocytes) of the 5th instar day-3 by stem-loopprimer RT-PCR. The results show that both the relative expression of miRNA in the posterior silk gland were significantly higher than other tissues, indicating that they exist the tissue specificity conditions of regulating BmFib-L gene expression.3. Bmo-miR-0001 and bmo-miR-0015 down regulate the expression of BmFib-L gene.In order to further validate the BmFib-L regulation effect of bmo-miR-0001 and bmo-miR-0015, pcDNA3.0 [ie1-egfp-pri-mir-0001-SV40] /pcDNA3.0[ie1-egfp-pri-mir-0015-SV40] and pGL3 [A3-luc-Fib-L-3’UTR-SV40] that express BmFib-L 3′UTR were constructed. These plasmids co-expressed in Silkworm BmN cells and Renilla luciferase expression vector pRL-CMV as internal control, the luciferase activity as a index to evaluated the regulation of mir-0001 and mir-0015.The results show that the luciferase activity of experimental group significantly decreased than that in positive control group, indicating that the two miRNA gene have a negative regulatory role in regulating BmFib-L.These results may provide some experimental data on miRNA regulatory mechanisms of silkworm silk protein.