Analyzing the evolution of insect TMED gene and the expression pattern of silkworm TMED gene / 生物工程学报

Transmembrane emp24 domain (TMED) gene is closely related to immune response, signal transduction, growth and disease development in mammals. However, only the Drosophila TMED gene has been reported on insects. We identified the TMED family genes of silkworm, Tribolium castaneum, tobacco moth and Italian bee from their genomes, and found that the TMED family gene composition patterns of one α-class, one β-class, one δ-class and several γ-classes arose in the common ancestor of pre-divergent Hymenoptera insects, while the composition of Drosophila TMED family members has evolved in a unique pattern. Insect TMED family γ-class genes have evolved rapidly, diverging into three separate subclasses, TMED6-like, TMED5-like and TMED3-like. The TMED5-like gene was lost in Hymenoptera, duplicated in the ancestors of Lepidoptera and duplicated in Drosophila. Insect TMED protein not only has typical structural characteristics of TMED, but also has obvious signal peptide. There are seven TMED genes in silkworm, distributed in six chromosomes. One of seven is single exon and others are multi-exons. The complete open reading frame (ORF) sequences of seven TMED genes of silkworm were cloned from larval tissues and registered in GenBank database. BmTMED1, BmTMED2 and BmTMED6 were expressed in all stages and tissues of the silkworm, and all genes were expressed in the 4th and 5th instar and silk gland of the silkworm. The present study revealed the composition pattern of TMED family members, their γ class differentiation and their evolutionary history, providing a basis for further studies on TMED genes in silkworm and other insects.

Comparison of chemical constituents of wild silkworm cocoon and domestic silkworm cocoon by UHPLC-MS technology / 生物工程学报

Identifying and comparing the chemical constituents of wild silkworm cocoon and silkworm cocoon is of great significance for understanding the domestication of silkworm. In this study, we used high temperature and high pressure and methanol-water system to extract cocoon chemical constituents. We used UHPLC-MS to identify and compare cocoon chemical constituents of wild silkworm and domestic silkworm Dazao and Haoyue strains. The cocoon metabolic fingerprints of wild silkworm and domestic silkworm Dazao and Haoyue strains were obtained by using the UHPLC-MS in the positive ion mode and negative ion mode. By annotation, we found that cocoon chemical compounds with high abundances contained amino acids, flavonoids, alkaloids, terpenes, organic acids, and lignans. PLS-DA showed that the cocoon components were significantly different among the wild silkworm and two domestic silkworm strains Dazao and Haoyue. Proline, leucine/isoleucine and phenylalanine showed significantly higher abundances in the cocoon of domestic silkworm Dazao strain than in those of wild silkworm and domestic silkworm Haoyue strain. The flavonoid secondary metabolites are abundant in the Dazao cocoon, including quercetin, isoquercetin, quercetin 3-O-sophoroside, quercetin-3-O-α-L-rhamnoside, quercetin-3-O- rutinoside, and kaempferol. The other secondary metabolites, alkaloids, terpenes and lignans, showed higher abundances in the wild silkworm cocoon than in the domestic silkworm cocoon, including neurine, candicine, pilocarpidine, artemisiifolin, eupassopin, and eudesobovatol. By exposing cocoons to UV light and observing the green fluorescence of flavonoids, we found that Dazao cocoon had the most flavonoids, and Haoyue cocoon had least flavonoids and wild silkworm cocoon had mediate flavonoids. Alkaloids and organic acids are good anti-insect and antimicrobial agents, which have high abundance in the wild silkworm cocoon and could enhance the defense ability of wild silkworm cocoon. Flavonoids are abundant in the cocoon of domestic silkworm Dazao strain, which the main factors are leading to the yellow-green cocoon of Dazao.

Gene cloning and expression characteristics of vacuolar-type ATPase subunit B in Bombyx mori / 生物工程学报

Vacuolar-type ATPase (V-ATPase), located in the membrane and organelle membrane, is one of important H⁺-transporting proteins. It keeps the proton balance by transporting H⁺ into vacuole, vesicle, or extracellular using the energy from ATP hydrolysis. The subunit B of the vacuolar-type ATPase (BmV-ATPase B) contains the ATP catalytic site, and plays an important role in this process. To study the function of V-ATPase B in Bombyx mori (BmV-ATPase B), we cloned its coding gene from the midgut of the 5th instar silkworm larvae. Then we constructed prokaryotic expression vector and produced the recombinant protein in E. coli. The recombinant protein was identified as BmV-ATPase B by mass spectrometry and purified using Ni-NTA affinity chromatography. This purified protein was used to immunize rabbit to generate polyclonal antibodies of BmV-ATPase B. Finally, the expression patterns of BmV-ATPase B in the silk gland were analyzed by western blotting and immunofluorescence. The full length CDS sequence of BmV-ATPase B was 1 473 bp. BmV-ATPase B was 55 kDa with a PI of 5.3. We analyzed the expression patterns of BmV-ATPase B in different sections of silk gland from the silkworm on the 3rd day of 5th instar and 1st day of wander stage by western blotting. BmV-ATPase B was expressed in all sections of the silk gland and it was abundant in the anterior silk gland (ASG) both in these two developmental stages. Furthermore, immunofluorescence indicated that BmV-ATPase B was located in the silk gland cells. Laser confocal scanning microscopy analysis revealed that BmV-ATPase B was mainly expressed in the cytomembrane of silk gland cells. These data elucidated the expression patterns of BmV-ATPase B in the silk gland of silkworm, which provides a good basis for further studies on the function of V-ATPase B in silk fiber formation.

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.

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.