Establishment of a suitable control reporter plasmid of a dual luciferase reporter gene system for hormone research in silkworm cell lines / 生物工程学报

The dual luciferase reporter gene system provides sensitive readout, while it relies on a constitutively-expressed control gene for readout normalization. However, most standard control reporter genes are not constitutively expressed under all conditions. Here, we report an effective method to construct a control reporter plasmid for the dual luciferase reporter gene system that would be suitable for hormone research in silkworm cell lines. First, we modified BmVgP78M, a stably-expressed constitutive promoter in silkworm cells by mutating its hormone-related element. Then, we constructed the pRL-VgP78M control reporter plasmid by replacing the SV40 promoter and chimeric intron sequences in pRL-SV40 with the BmVgP78M sequence. Finally, we confirmed that the pRL-VgP78M control reporter plasmid could be stably expressed in silkworm cell lines via cell transfection experiments, and it was unresponsive to the induction of ecdysone, juvenile hormone, or their transcription factors. We thus obtained a control reporter plasmid pRL-VgP78M that could be expressed stably and moderately in silkworm cells. It can be readily used as the control reporter plasmid of the dual luciferase reporter gene system for hormone research in silkworm cell lines. It will also provide a reference for construction of control reporter plasmids of dual luciferase reporter gene systems that are adaptable to cell lines isolated from other species.

Interaction of Bombyx mori aminopeptidase N and cadherin-like protein with Bacillus thuringiensis Cry1Ac toxin / 生物工程学报

Bacillus thuringiensis (Bt) produces Cry toxins that are widely used as insecticides in agriculture and forestry. Receptors are important to elucidate the mode of interaction with Cry toxins and toxicity in lepidopteran insects. Here, we purified the Cry toxin from Bt and identified this toxin by flight mass spectrometry as Cry1Ac, and then recombinantly expressed aminopeptidase N (BmAPN6) and repeat domains of cadherin-like protein (CaLP) of B. mori. Using co-immunoprecipitation (co-IP), Far-Western blotting, and enzyme-linked immunosorbent assays (ELISAs), we identified the interaction between Cry1Ac and BmAPN6. Furthermore, analysis of the cytotoxic activity of Cry1Ac toxin in Sf9 cells showed that BmAPN6 directly interacted with Cry1Ac toxin to induce morphological aberrations and cell lysis. We also used co-IP, Far-Western blotting and ELISAs to analyze the interactions of Cry1Ac with three binding sites corresponding to cadherin repeat (CR) 7 CR11, and CR12 of CaLP. Notably, the three repeat domains were essential Cry1Ac binding components in CaLP. These results indicated that BmAPN6 and CaLP served as a functional receptor involved in Bt Cry1Ac toxin pathogenicity. These findings represent an important advancement in our understanding of the mechanisms of Cry1Ac toxicity and provide promising candidate targets for gene editing to enhance resistance to pathogens and increase the economic value of B. mori.

Identification and characterization of epidermal growth factor receptor ligands in Bombyx mori / 生物工程学报

Epidermal growth factor receptor (EGFR) is a multi-functional receptor distributed throughout the metazoa. Study on its ligands so far remained mainly on mammals, including how ligands are processed into active forms, their interaction with EGFR, and the signaling pathway they induce. However, in invertebrates, ligands are more divergent among species. Currently, except for Drosophila, less is known about the insect EGFR ligands. Here, we identified two EGFR ligands in Bombyx mori by homology search, domain prediction, analysis of the potential translation initiation sequence and construction of phylogenetic tree, termed as BmEGF-1 and BmEGF-2. BmEGF-1 shows the greatest similarity to Drosophila Spitz and their Rhomboid-recognition motifs are highly identical. BmEGF-2 is a homolog to Drosophila Vein. Then we purified BmEGF-1 extracellular domain expressed in E. coli, and performed pull-down assay with BmEGFR extracellular domain secreted by Sf9 cells. The result confirmed their interaction. Lastly, we found the phosphorylation level of ERK and p38 MAPK was elevated after expression of BmEGF-1 in BmE cells, which suggested that BmEGF-1 is not only able to activate the canonical ERK signaling pathway, but may participate in other cellular processes by inducing p38 MAPK signaling pathway. Our study provides reference to further study of the biological function of BmEGF in silkworm.

Interaction of aminopeptidase (BmAPN5) and parasporal crystal (PC) toxin isolated from Bacillus bombysepticus / 生物工程学报

Aminopeptidase N (APN) belonging to zinc-dependent metalloproteinase, not only catalyzes protein proteolytic process, but also is involved in the pathogenic process as the receptor of pathogenic toxin. In Bombyx mori, APN gene family consists of 16 members, of which BmAPN4 binds trypsin-activated parasporal crystal (PC) toxin isolated from Bacillus bombysepticus (Bb). In order to verify whether or not other APNs interact with PC toxin during the pathogenesis of Bb, we cloned BmAPN5, a member of aminopeptidase family, from the silkworm midgut. The full length of BmAPN5 is 3313 bp, encoding 953 amino acids, containing a zinc peptidase_M1 and ERAP1_C domains. A recombinant GST-BmAPN5 was purified by a prokaryotic expression system. Far-Western blotting, co-immunoprecipitation and ELISA. Binding saturation assays demonstrated that PC after activated by trypsin could be bound by BmAPN5. Additionally, cytotoxic activity of trypsin-activated PC in Sf9 cells transfected with BmAPN5 showed that cells exhibited dramatic cytological changes, including swelling and lysis, revealing BmAPN5 serves as a functional receptor that participates in Bb and PC pathogenicity. These provide some clues for further exploring the pathogenesis relationships of Bb and host.

Analysis of molting fluid in silkworm (Bombyx mori) by two-dimensional electrophoresis and mass spectrometry / 生物工程学报

Molting fluid, a liquid between the old epidermis and new epidermis, plays an important role in the process of ecdysis and metamorphosis for insect. In order to explore the function of molting fluid, we used two-dimensional electrophoresis to study the molting fluid during the prepupal stage and pre-eclosion stage. More than 200 protein spots were found in the molting fluid of the 2 stages, which distributed in the 4-9 of pI and 10-180 kDa of molecular weight. We selected 42 spots to be analyzed by the matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI TOF/TOF) from the molting fluid of pre-eclosion stage, of which 34 proteins were identified successfully, including apolipoprotein, protease and protease inhibitors, chitin-binding protein and protein involved in immunity. Some of the proteins demonstrated differential expression between the two stages of metamorphosis. In order to validate the result from proteomics analysis, we studied expression of the apolipoprotein D by Q-PCR from the developmental stages. The results showed that the gene encoding apolipoprotein D had the high expression from the 1st day to the 4th day of the pupa stage, which indicated they could be involved in eclosion due to the abundant accumulation in the late pupa. Our results offered more clues for understanding the mechanism of ecdysis and metamorphosis in insect and could give reference for further study of molting fluid.

Cloning and expression profile of Bmlin-41 and its regulation by the silkworm microRNA let-7 / 生物工程学报

The heterochronic genes regulate cell proliferation and switch development stage transitions. Heterochronic genes might also play important roles in regulating the development of silkworm, but very few of their expression profiles, functions and their relationship with microRNAs are available so far. Firstly, in this work, the primers for cloning Bmlin-41 were designed based on the homologous sequence of known Drosophila melanogaster lin-41, which was used as the query to blast against SilkDB. The obtained full CDS (2 166 bp) of Bmlin-41 encodes 721 amino acids and contains B-box and NHL domains. Then, the spatiotemporal expression patterns of Bmlin-41 were characterized by RT-PCR, quantitative real time PCR as well as our lab's previous silkworm genome microarray data. Bmlin-41 was increasingly expressed from embryonic to adult stage. In diverse tissues of day-3 fifth instar, Bmlin-41 showed the highest accumulation in ovary, secondly in testis and midgut, but very low expression was observed in other tissues. Finally, 3'UTR of Bmlin-41 1 434 bp was cloned by rapid-amplification of cDNA ends (3'RACE) and was predicted to bare two binding sites of bmo-let-7 by using online RNAhybrid. To verify the binding effect, 3'UTR was cloned into psi-CHECK-2 vector and submitted to dual luciferase assay in the S2 cells in vitro. The dual luciferase assay demonstrated that Bmlin-41 was down-regulated by bmo-let-7 mimics and upregulated by bmo-let-7 antagomir, thus confirming the Bmlin-41 is negatively regulated by bmo-let-7. Our work might help further study on the roles of Bmlin-41 and bmo-let-7 and their regulation relationship involved in controlling metamorphosis of silkworm.

Cloning and expression profile of Bmyan in the silkworm (Bombyx mori) and experimental validation as one target of microRNA 7 / 生物工程学报

microRNAs (miRNAs) are an extensive class of -22-nucleotide (nt) endogenous noncoding RNAs regulating life activities ofmetazoans through binding to 3'-untranslated regions (3'-UTRs) of their target genes. This work aimed to identify yan gene in the silkworm, reveal its expression profile and confirm if it is one target of bmo-miR-7 and, as such, have potential for contributing to better understanding of the molecular mechanisms involved in the metamorphosis of silkworm. Based on homolog searching and PCR amplification, we cloned the coding sequence (CDS) of Bmyan, which encodes 476 amino acid residues and contains SAM-PNT and ETs domains. Quantitative PCR (q-PCR), RT-PCR and microarray data revealed high expression of Bmyan in the head, body wall and ovary of day-3 fifth instar larval silkworm, low or no expression in other tissues. It was lowly expressed in the early larval stages, but highly expressed from late spinning to day 4 pupa. The 3'-UTR of Bmyan was obtained by rapid-amplification of cDNA ends (3'RACE) and predicted to contain two potential recognition sites of bmo-miR-7. The luciferase reporter vector containing the 3'-UTR of Bmyan was constructed and co-transfected into BmE cell line with the mimic of bmo-miR-7 and the decreased relative activity of luciferase showed that Bmyan is one target of bmo-miR-7. This work helps further functional analysis of bmo-miR-7 and Bmyan in the silkworm.

Abundance and Distribution of Microsatellites in The Entire Mosquito Genome / 生物化学与生物物理进展

Microsatellite is a genetic marker, explored recently. In order to improve related studies on genetics of Anopheles gambiae, simple sequence repeats of the entire mosquito genome with 1～6 bp nucleotide motifs were analyzed.Abundance and distribution of microsatellites across the A.gambiae genome were analyzed and compared between various (exons, introns and intergenic) regions of all the chromosomes. About 2.14% of the mosquito genome was occupied by SSRs. Chromosome X had the maximum density of SSRs. Abundance orA repeats was similar to C repeats. AC was a little more than two times as abundant much as AG. However, AT and CG repeats were rare. For tri- and tetramer repeats, AGC,AAAC and AAAT predominated while ACG, ACT, AGG, CCG, ATGC, CCCG, ACTG, AACT, ACGT, AGAT, CCGG,ACCT and AGCT were rare. For some pentamer repeats, one was completely absent on a certain chromosome, even on several chromosomes. SSRs in exons of all chromosomes were less abundant than in introns and intergenic regions except for mono- and dimer repeats in exons of chromosome 2L. Abundance and distribution of SSRs on the two arms of each chromosome showed much in common.

Analysis of a Novel Transcription Mode of Fhx/P25 Gene in Bombyx mori / 生物化学与生物物理进展

Fhx/P25 in silkworm, Bombyx mori, one of the main components of silk fibroin, is presumed in previous reports to be expressed exclusively in the posterior silk gland (PSG) of the animal with strict territorial and developmental specificities. On the basis of a large-scale analysis ofthe silkworm EST data, it was found that Fhx/P25 gene is transcribed not only in the posterior silk gland, but in the ovary and in other tissues of the larvae at day 3 of the fifth-instar as well and that this gene has distinct transcription start sites (TSSs) in the posterior silk gland and the ovary. The TSS in the ovary is located about 115 bp upstream sequence of that in the posterior silk gland. Subsequent RT-PCR, FQ-PCR and sequencing have verified the validity of this presumption. In addition, alternative splicing is predicted in pre-mRNA of Fhx/P25 gene and confirmed by RT-PCR. In conclusion, Fhx/P25 gene is not a gene with strictly tissue-specific transcription.Complicated regulation mechanisms may exist for its transcription and expression and it may have other functions to perform.

Analysis on frequency and density of microsatellites in coding sequences of several eukaryotic genomes / 基因组蛋白质组与生物信息学报·英文版

Microsatellites or simple sequence repeats (SSRs) have been found in most organisms during the last decade. Since large-scale sequences are being generated, especially those that can be used to search for microsatellites, the development of these markers is getting more convenient. Keeping SSRs in viewing the importance of the application, available CDS (coding sequences) or ESTs (expressed sequence tags) of some eukaryotic species were used to study the frequency and density of various types of microsatellites. On the basis of surveying CDS or EST sequences amounting to 66.6 Mb in silkworm, 37.2 Mb in fly, 20.8 Mb in mosquito, 60.0 Mb in mouse, 34.9 Mb in zebrafish and 33.5 Mb in Caenorhabditis elegans, the frequency of SSRs was 1/1.00 Kb in silkworm, 1/0.77 Kb in fly, 1/1.03 Kb in mosquito, 1/1.21 Kb in mouse, 1/1.25 Kb in zebrafish and 1/1.38 Kb in C. elegans. The overall average SSR frequency of these species is 1/1.07 Kb. Hexanucleotide repeats (64.5%-76.6%) are the most abundant class of SSR in the investigated species, followed by trimeric, dimeric, tetrameric, monomeric and pentameric repeats. Furthermore, the A-rich repeats are predominant in each type of SSRs, whereas G-rich repeats are rare in the coding regions.