Identification Of MicroRNA In Middle Silk Gland Of Bombyx Mori 5^th Instar Larva And Analysis Of Regulation Founction Of Them On Expression Of Sericin Gene

MicroRNA(miRNA), widely existing in eukaryotes, is approximately 21 to 24 nucleotides single-stranded noncoding RNAs encoded by endogenous genes. Over the years many publications have reported that the main function of miRNA is regulation of gene expression at the post-transcriptional level. It has become clear that miRNA has diverse roles in fundamental biological processes such as embryonic development, cell differentiation, proliferation, neural development, metabolism and apoptosis. Almost 50% of protein-coding genes are regulated by miRNAs in animals. As the typical representative of Lepidoptera, Bombyx mori sericin genes including BmSer-1 and BmSer-2 are specifically expressed in the middle silk gland of B. mori larvae, and they are also the main gene encoding silk-fibroin. Previous studies have shown that the expression of fibroin gene is regulated by miRNAs, however, there are few reports about miRNAs regulating B.mori sericin gene.To systematically explore mi RNAs involved in expressional regulation of sericin gene in B. mori, Solexa sequencing technology was used to identify miRNAs in the middle silk gland(MSG) from 5th-instar larvae of P50 and a naked pupa mutant(Nd). Some conservative mi RNAs and novel ones were identified. Several miRNAs such as bmo-miR-275 were predicted to participate the post-transcriptional regulation of BmSer-1 and BmSer-2. By construction of mi RNA and target gene 3′UTR recombinant vectors separately, the regulation function of bmo-miR-2771 and bmo-miR-275 were verified. The main results are as follows.1. Identification of miRNAs in MSGs of P50 and Nd 5th-instar day-3 larvaeThe small RNAs in MSG of P50 and Nd 5th-instar day-3 larvae were respectively high-throughput sequenced by Solexa. A total of 272 known miRNAs were identified and 333 novel miRNAs were predicted. In the novel miRNAs, 86 ones exhibited significant difference between P50 and Nd.2. Identification of novel miRNA and prediction of target genesTo verify the predicted data, quantitative RT-PCR(qRT-PCR) was performed on 10 novel miRNAs. The results were identical to the prediction. Target genes were predicted using software. A total of 2522 putative target genes were predicted for 272 conservative miRNAs, while 1376 putative target genes for 331 novel miRNAs.3. Expression patterns of BmSer-1, BmSer-2 and correlated miRNAsTarget prediction of miRNAs showed that BmSer-1 and BmSer-2 were the potential target genes of bmo-miR-2771 and bmo-miR-275, respectively. Semi-quantitative PCR was used to analyze their expression patterns. The results showed that bmo-miR-2771 was specifically expressed in the middle silk gland.4. Regulation Role function of bmo-mi R-2771 on expression post-transcriptional control of BmSer-1Recombinant vectors pcDNA3.0[ie1-egfp-pri-mir-2771-SV40] and pGL3[A3-luc-Ser-1-3′-UTR-SV40] were constructed, and o-transfected into BmN cells. The results showed that the expression of a luciferase reporter was significantly decreased, suggesting that bmo-mi R-2771 down-regulated the expression of BmSer-1.5. Regulation function Role of bmo-miR-275 on post-transcriptional controlexpression of BmSer-2Recombinant vectors pcDNA3.0[ie1-egfp-pri-mir-275-SV40] and pGL3[A3-luc-Ser-2-3′-UTR-SV40] were constructed and co-transfected into BmN cells. The results showed that the expression of a luciferase reporter was significantly decreased. When cells were co-transfected with bmo-miR-275 inhibitor, luciferase reporter was ascended slightly, suggesting that bmo-miR-275 down-regulated the expression of BmSer-2.These results provided new experimental data for demonstrating the mechanism of mi RNA on regulating the expression of silk-fibroin genes and the function of miRNAs as well.