Identification Of MicroRNAs In Posterior Silk Gland Of The Silkworm (Bombyx Mori) And Their Regulations On Expression Of Fibroin Genes,Etc

MicroRNAs are a class of endogenous non-protein coding small RNAs of ~22 nucleotides in length that regulate expression of genes at post-transcriptional level by binding on complementary sequences of target mRNAs and play multiple roles in biological processes.So far,more than 3,000,000 miRNAs have been identified in animals,plants and viruses,etc.and the number is growing rapidly.567 miRNAs have been identified in the silkworm(Bombyx mori)(miRBase,http://www.mirbase.org/).miRNA has become one of hot spot in the reseach of gene regulation.By studying the differential expression of the miRNAs in the silk gland of different periods and their target genes,a more comprehensive understanding of the development of the silk gland and the silk protein synthesis could be obtained.We used solexa sequencing technology to detect miRNAs in the posterior silk glands of 5th-instar larvae and 4th-instar larvae respectively,and verified the regulation of miRNA on the expression of fibroin and Bm-ase genes.which provides a foundation for further study on the miRNA function and the regulation mechanism of the silk protein synthesis.The main research results are as follows.1.Characterization and profiling of microRNAs in 4th-instar day-2 and 5th-instar day-3 PSGs of the silkworm(Bombyx mori)It was well known that the silk proteins were mainly synthesized in PSGs of the fifth-instar larvae from day 3 to day 5.In order to study the differential expression of miRNAs in PSGs and provide a fundation for the study of the regulation of miRNAs on the fibroin genes,we commissioned sequencing company to detect small RNAs in the posterior silk glands of 5th-instar day-3 larvae and 4th-instar day-2 larvae(p50)by solexa sequencing,respectively.We detected 466 previously reported miRNAs,and predicted 35 novel miRNAs.By target prediction softwares,499 out of these detected miRNAs are predicted to target a different set of genes,amounting to 13,383.Additionally,we found the differential expression of 29 miRNAs between the PSG of 4th-instar day-2 larvae and PSG of 5th-instar day-3 larvae,and inferred that these differentially expressed miRNAs might involved in the regulation on the expression of silk protein genes.However,these results remain to be further verified by experiments.2.Regulation of miRNAs on expression of fibroin genes in silkwormIn order to study the regulation of miRNAs on expression of fibroin genes in silkworm,the sequences of the mature miRNAs were downloaded form the miRBase Sequence Database(http://www.mirbase.org/).RNA22(http://cbcsrv.watson.ibm.com/rna22.html)and RNAhybrid(http://bibiserv.techfak.uni-bielefeld.de/rnahybrid)softwares were used to predict the potential miRNAs targeting the 3'UTR of fibroin genes(Fib-H,Fib-L,P25).We predicted bmo-mir-2739,bmo-mir-3237,bmo-mir-3366,bmo-mir-317 etc.could target the 3'UTR of fibroin genes by the two softwares.The recombinant plasmids containing luciferase reporter gene and 3'UTR of Fib-H,Fib-L and P25 were constructed respectively.The miRNAs expression vectors(bmo-mir-2739,bmo-mir-3237,bmo-mir-3366,bmo-mir-317,etc.)were also successfully constructed respectively.Then BmN cells were co-transfected with the miRNAs expression vector and corresponding target genes fused luciferase reporter plasmids.In the transfection experiments p[FH-EGFP] was served as a negative control for the miRNAs expression vector p[FH-EGFP-miR].The activity of luciferase was detected 48 h post transfection.The results showed that bmo-mir-2739 up-regulated(not down-regulated)the luciferase activity of plasmids containing Fib-H 3'UTR;bmo-mir-3237,bmo-mir-3366 down-regulated the luciferase activity of plasmids containing Fib-P25 3'UTR.The BmN cells were co-transfected with the artificial bmo-mir-2739 mimics,bmo-miR-2998 mimics and corresponding target genes fused luciferase reporter plasmids.The results showed that bmo-mir-2739 up-regulated the luciferase activity of plasmids containing Fib-H 3'UTR;bmo-miR-2998 down-regulated the luciferase activity of plasmids containing Fib-L 3'UTR.3.Regulation of miRNAs on expression of Bm-ase gene in silkwormBm-ase mainly expresses in the neural precursor cells,and it's a homolog of asense,which is essential for the development of neural precursor cells.Bm-ase gene was predicted to be the target gene of bmo-mir-9a?bmo-mir-3223?bmo-mir-2845 and bmo-mir-745* by the same target prediction softwares.In order to verify the predicted results,we constructed the Bm-ase 3'UTR fused luciferase reporter gene plasmids and the expression vectors of bmo-mir-9a,bmo-mir-3223,bmo-mir-2845,bmo-mir-745* respectively.BmN cells were co-transfected with the miRNAs expression vectors and the Bm-ase 3'UTR fused luciferase reporter plasmids.In the transfection experiments p[FH-EGFP] was served as a negative control for the miRNAs expression vector p[FH-EGFP-miR].The activity of luciferase was detected 48 h post transfection.The results showed that the luciferase activity of was suppressed by bmo-mir-9a.In order to further verify the regulation of bmo-mir-9a on Bm-ase gene,when added the artificial bmo-mir-9a mimics into the BmN cells,the expression of the luc gene fused Bm-ase 3'UTR was also down-regulated.These indicated bmo-miR-9a down regulates the expression of Bm-ase gene.