Shunming Tang

Construction and detection of expression vectors of microRNA-9a in BmN cells

MicroRNAs (miRNAs) are small endogenous RNAs molecules, approximately 21–23 nucleotides in length, which regulate gene expression by base-pairing with 3′ untranslated regions (UTRs) of target mRNAs. However, the functions of only a few miRNAs in organisms are known. Recently, the expression vector of artificial miRNA has become a promising tool for gene function studies. Here, a method for easy and rapid construction of eukaryotic miRNA expression vector was described. The cytoplasmic actin 3 (A3) promoter and flanked sequences of miRNA-9a (miR-9a) precursor were amplified from genomic DNA of the silkworm (Bombyx mori) and was inserted into pCDNA3.0 vector to construct a recombinant plasmid. The enhanced green fluorescent protein (EGFP) gene was used as reporter gene. The Bombyx mori N (BmN) cells were transfected with recombinant miR-9a expression plasmid and were harvested 48 h post transfection. Total RNAs of BmN cells transfected with recombinant vectors were extracted and the expression of miR-9a was evaluated by reverse transcriptase polymerase chain reaction (RT-PCR) and Northern blot. Tests showed that the recombinant miR-9a vector was successfully constructed and the expression of miR-9a with EGFP was detected.

Expression of a vitelline membrane protein, BmVMP23, is repressed by bmo-miR-1a-3p in silkworm, Bombyx mori

Previous studies have shown that the 3′ UTR of BmVMP23 was destroyed by an inserted fragment, and the BmVMP23 expression was downregulated in the lethal egg of “Ming” (l‐em ). In this study, we found a miRNA (bmo‐miR‐1a‐3p) that matches the 3′ UTR sequence of BmVMP23 perfectly. The relationship between bmo‐miR‐1a‐3p and the BmVMP23 expression was examined by the co‐transfection in vitro. The luciferase assay result showed that luc expression was strongly repressed. This result inferred that bmo‐miR‐1a‐3p may downregulate BmVMP23 expression via complementary interaction with the target sites at the 3′ UTR.

Mutation of a Cuticle Protein Gene, BmCPG10, Is Responsible for Silkworm Non-Moulting in the 2nd Instar Mutant

In the silkworm, metamorphosis and moulting are regulated by ecdysone hormone and juvenile hormone. The subject in the present study is a silkworm mutant that does not moult in the 2nd instar (nm2). Genetic analysis indicated that the nm2 mutation is controlled by a recessive gene and is homozygous lethal. Based on positional cloning, nm2 was located in a region approximately 275 kb on the 5th linkage group by eleven SSR polymorphism markers. In this specific range, according to the transcriptional expression of thirteen genes and cloning, the relative expression level of the BmCPG10 gene that encodes a cuticle protein was lower than the expression level of the wild-type gene. Moreover, this gene’s structure differs from that of the wild-type gene: there is a deletion of 217 bp in its open reading frame, which resulted in a change in the protein it encoded. The BmCPG10 mRNA was detectable throughout silkworm development from the egg to the moth. This mRNA was low in the pre-moulting and moulting stages of each instar but was high in the gluttonous stage and in newly exuviated larvae. The BmCPG10 mRNA showed high expression levels in the epidermis, head and trachea, while the expression levels were low in the midgut, Malpighian tubule, prothoracic gland, haemolymph and ventral nerve cord. The ecdysone titre was determined by ELISA, and the results demonstrated that the ecdysone titre of nm2 larvae was lower than that of the wild-type larvae. The nm2 mutant could be rescued by feeding 20-hydroxyecdysone, cholesterol and 7—dehydrocholesterol (7dC), but the rescued nm2 only developed to the 4th instar and subsequently died. The moulting time of silkworms could be delayed by BmCPG10 RNAi. Thus, we speculated that the mutation of BmCPG10 was responsible for the silkworm mutant that did not moult in the 2nd instar.

Overview of research on Bombyx mori microRNA.

Abstract MicroRNAs (miRNAs) constitute some of the most significant regulatory factors involved at the post-transcriptional level after gene expression, contributing to the modulation of a large number of physiological processes such as development, metabolism, and disease occurrence. This review comprehensively and retrospectively explores the literature investigating silkworm, Bombyx mori L. (Lepidoptera: Bombicidae), miRNAs published to date, including discovery, identification, expression profiling analysis, target gene prediction, and the functional analysis of both miRNAs and their targets. It may provide experimental considerations and approaches for future study of miRNAs and benefit elucidation of the mechanisms of miRNAs involved in silkworm developmental processes and intracellular activities of other unknown non-coding RNAs.

Molecular cloning and characterization of hatching enzyme-like geneII (BmHELII) in the silkworm, Bombyx mori.

Hatching enzyme (HE) is an enzyme that digests an egg envelop at the time of embryo hatching. Previously, we have reported a kind of Bombyx mori hatching enzyme-like gene (BmHEL). In this paper, the full length of another BmHEL cDNA sequence (BmHELII, GenBank ID: JN627443) was cloned from bluish-silkworm-eggs. The cDNA was 977 bp in length with an open reading frame of 885 bp which encodes a polypeptide of 294 amino acids including a putative signal peptide of 16 amino acid residues and a mature protein of 278 amino acids. The deduced BmHELII had a predicted molecular mass of 33.62 kDa, isoelectric point of 5.44 and two conserved signature sequences of astacin family. Bioinformatic analysis results showed that the deduced protease domain amino acid sequence of BmHELII had 29.5-87.0% identities to that of HE identified in the other species. The BmHELII gene structure was 6-exon-5-intron, and the promoter region harbored some basal promoter elements and some embryo development related transcription factor binding sites. Semi-quantitative RT-PCR analysis revealed that the relative level of BmHELII transcripts at different stages during egg incubation increased with the development of embryos and reached to a maximum just before hatching, hence declined gradually after hatching. The spatio-temporal expression pattern of BmHELII basically resembled that of hatching enzyme gene. Moreover, the BmHELII transcript was detected in testis of the silkworm, and semi-quantitative RT-PCR analysis showed that it kept at the high level in testis of silkworm from larvae to moth, which suggested that BmHELII might take part in the development of sperm. These results will be helpful to provide a molecular basis for understanding the mechanism underlying silkworm hatching as well as spermatogenesis.

Transcriptome analysis of the epidermis of the purple quail-like (q-lp) mutant of silkworm, Bombyx mori

A new purple quail-like (q-lp) mutant found from the plain silkworm strain 932VR has pigment dots on the epidermis similar to the pigment mutant quail (q). In addition, q-lp mutant larvae are inactive, consume little and grow slowly, with a high death rate and other developmental abnormalities. Pigmentation of the silkworm epidermis consists of melanin, ommochrome and pteridine. Silkworm development is regulated by ecdysone and juvenile hormone. In this study, we performed RNA-Seq on the epidermis of the q-lp mutant in the 4th instar during molting, with 932VR serving as the control. The results showed 515 differentially expressed genes, of which 234 were upregulated and 281 downregulated in q-lp. BLASTGO analysis indicated that the downregulated genes mainly encode protein-binding proteins, membrane components, oxidation/reduction enzymes, and proteolytic enzymes, whereas the upregulated genes largely encode cuticle structural constituents, membrane components, transport related proteins, and protein-binding proteins. Quantitative reverse transcription PCR was used to verify the accuracy of the RNA-Seq data, focusing on key genes for biosynthesis of the three pigments and chitin as well as genes encoding cuticular proteins and several related nuclear receptors, which are thought to play key roles in the q-lp mutant. We drew three conclusions based on the results: 1) melanin, ommochrome and pteridine pigments are all increased in the q-lp mutant; 2) more cuticle proteins are expressed in q-lp than in 932VR, and the number of upregulated cuticular genes is significantly greater than downregulated genes; 3) the downstream pathway regulated by ecdysone is blocked in the q-lp mutant. Our research findings lay the foundation for further research on the developmental changes responsible for the q-lp mutant.

bmo-miR-0001 and bmo-miR-0015 down-regulate expression of Bombyx mori fibroin light chain gene in vitro

Based on bioinformatic analysis, we selected two novel microRNAs (miRNAs), bmo-miR-0001 and bmomiR-0015, from high-throughput sequencing of the Bombyx mori larval posterior silk gland (PSG). Firstly, we examined the expression of bmo-miR-0001 and bmo-miR-0015 in 12 different tissues of the 5th instar Day-3 larvae of the silkworm. The results showed that the expression levels of both bmo-miR-0001 and bmo-miR-0015 were obviously higher in the PSG than in other tissues, implying there is a spatio-temporal condition for bmo-miR-0001 and bmo-miR-0015 to regulate the expression of BmFib-L. To test this hypothesis, we constructed pri-bmo-miR-0001 expressing the plasmid pcDNA3.0 [ie1-egfp-pri-bmo-miR-0001-SV40] and pri-bmo-miR-0015 expressing the plasmid pcDNA3.0 [ie1-egfp-pribmo-miR-0015-SV40]. Finally, the BmN cells were harvested and luciferase activity was detected. The results showed that luciferase activity was reduced significantly (P<0.05) in BmN cells co-transfected by pcDNA3.0 [ie1-egfp-pri-bmomiR-0001-SV40] or pcDNA3.0 [ie1-egfp-pri-bmo-miR-0015-SV40] with pGL3.0 [A3-luc-Fib-L-3′UTR-SV40], suggesting that both bmo-miR-0001 and bmo-miR-0015 can down-regulate the expression of BmFib-L in vitro.中文概要目 的探究新发现的两个miRNA 对家蚕丝素轻链基因BmFib-L 的调控作用。创新点在家蚕后部丝腺中发现两个新的miRNA, 并首次证明它们对家蚕丝素蛋白轻链基因BmFib-L 有负调控作用。方 法本实验通过生物信息学分析, 从家蚕后部丝腺 miRNA 高通量测序获得的新miRNA 中, 筛选出 两个可能对家蚕丝素蛋白轻链基因BmFib-L 有 调控作用的miRNA, 即bmo-miR-0001 和bmomiR-0015。设计茎环引物, 采用反转录聚合酶链 反应(RT-PCR)方法对家蚕5 龄3 d 头部、表皮、 脂肪体、马氏管、精巢/卵巢、丝腺(前、中、 后)、气管、中肠和血淋巴细胞等12 个不同组 织的bmo-miR-0001 和bmo-miR-00015 进行半定 量表达分析。并采用双荧光报告基因检测系统进 一步在细胞水平上验证bmo-miR-0001 和 bmo-miR-0015 对BmFib-L 表达的调控作用。结 论本实验中RT-PCR 结果显示, 这两个新miRNA 在家蚕后部丝腺中表达量最高(图4)。双荧光 报告基因检测结果显示, 报告基因荧光素酶活性 明显低于阳性对照组(图7), 转染bmo-miR-0001 和bmo-miR-0015 表达载体的细胞, 报告基因和 荧光素酶活性分别只及对照组60%和69%。t 检 验分析结果显示两个实验组与对照组之间差异 都达到显著水平(P<0.05) 。由此可见, bmo-miR-0001 和bmo-miR-0015 在体外对 BmFib-L 的表达具有显著的抑制作用。

Bmo-miR-2758 Targets BmFMBP-1 (Lepidoptera: Bombycidae) and Suppresses Its Expression in BmN Cells

MicroRNAs (miRNAs) are an abundant family of endogenous noncoding small RNA molecules. They play crucial roles on regulation of life processes both in plants and animals. Fibroin modulator binding protein-1 (FMBP-1) is a silk gland transcription factor of Bombyx mori, which is considered as a trans-activator of fibroin genes. And bioinformatics prediction showed that at the 3′ untranslated region (3′ UTR) of BmFMBP-1 there were binding sites for three bmo-miRNAs, bmo-miR-2b*, bmo-miR-305, and bmo-miR-2758, separately. In order to validate whether these bmo-miRNAs involved in the regulation of BmFMBP-1 expression, the expression levels of three bmo-miRNAs and BmFMBP-1 in the middle silk gland (MSG) and posterior silk gland (PSG) during the fourth- and fifth-larval stages of B. mori were measured by semi-quantitative reverse transcription polymerase chain reaction. The results revealed that the expression level of bmo-miR-2758 was the highest in the three, and it expressed higher in the PSG than in the MSG with a similar expression pattern as BmFMBP-1, implying that bmo-miR-2758 may involved in regulation of BmFMBP-1. To validate the regulation function of bmo-miR-2758 on BmFMBP-1, recombinant plasmids pcDNA3 [ie1-egfp-pri-bmo-miR-2758-SV40] and pGL3 [A3-luc-FMBP-1 3′ UTR-SV40] were constructed and co-transfected in BmN cells. The dual-luciferase reporter assay system was used for assay of transient expression. The results showed that the expression of the luciferase reporter was significantly decreased when pGL3 [A3-luc-FMBP-1 3′ UTR-SV40] co-transfected with pcDNA3 [ie1-egfp-pri-bmo-miR-2758-SV40] (P < 0.01). Furthermore, when the artificial antisense RNA of bmo-miR-2758 (inhibitor) was added to the above co-transfection, the expression of the luciferase reporter was recovered significantly (P < 0.01). These results suggest that bmo-miR-2758 represses the expression of BmFMBP-1 in vitro.

Characterization and profiling of MicroRNAs in posterior silk gland of the silkworm ( Bombyx mori )

MicroRNAs (miRNAs) regulate expression of genes at post-transcriptional level by binding on complementary sequences of target mRNAs and play multiple roles in biological processes. To investigate the differential expression of miRNAs in posterior silk gland (PSG) of silkworm (Bombyx mori) in different periods and regulation of miRNAs on the expression of fibroin genes, Solexa sequencing technology was used to detect miRNAs in PSGs of fourth-instar day-2 larvae and fifth-instar day-3 larvae, respectively. As a result, 466 previously reported miRNAs, and 35 novel miRNAs were detected, and 499 of these detected miRNAs are predicted to target 13,383 genes by target prediction softwares. Additionally, 29 miRNAs expressed differently between the PSG of fourth-instar day-2 larvae and fifth-instar day-3 larvae were found, and the differential expression of these miRNAs may play an important role in the expression of fibroin genes.

Inhibition of expression of BmNPV cg30 by bmo-miRNA-390 is a host response to baculovirus invasion

Bombyx mori larvae exhibit in vivo defensive reactions immediately after invasion by a virus. One of these defense systems is to express appropriate microRNAs (miRNAs) to respond to the infection. A novel Bombyx mori–encoded miRNA, bmo-miR-390, was identified previously by high-throughput sequencing. Based on bioinformatic predictions, the Bombyx mori nuclear polyhedrosis virus cg30 gene (BmNPV-cg30) is one of the target genes of bmo-miR-390. In this study, expression vectors with an enhanced green fluorescence protein (EGFP) or a luciferase (luc) reporter gene together with bm-miR-390 or the cg30 3’ UTR were constructed and used to co-transfect BmN cells. Using a dual luciferase reporter (DLR) assay, we found that bmo-miR-390 significantly downregulates the expression of BmNPV-cg30 (P < 0.05) in vitro. Moreover, artificially synthesized bmo-miR-390 mimics enhanced the regulatory effect of bmo-miR-390, while an inhibitor eliminated the inhibitory effect. These results show for the first time that bmo-miR-390 can effectively downregulate the expression of BmNPV-cg30 in BmNPV-infected BmN cells.