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.
Animals ; Bombyx/metabolism* ; Genes, Insect/genetics* ; Moths/metabolism* ; Insecta/metabolism* ; Drosophila ; Insect Proteins/metabolism* ; Phylogeny ; Mammals/genetics*

c-Myc protein encoded by c-Myc (cellular-myelocytomatosis viral oncogene) gene regulates the related gene expression through the Wnt/β-catenin signaling pathway, and has received extensive attention in recent years. The purpose of this study was to express Helicoverpa armigera c-Myc gene (Ha-c-Myc) by using prokaryotic expression system, prepare the polyclonal antibody, examine the spatio-temporal expression profile of Ha-c-Myc, and investigate the possible function of Ha-c-Myc in regulating H. armigera sterol carrier protein-2 (SCP-2) gene expression. The Ha-c-Myc gene was amplified by PCR and cloned into a prokaryotic expression plasmid pET-32a(+). The recombinant plasmid pET-32a-Ha-c-Myc was transformed into Escherichia coli BL21. IPTG was used to induce the expression of the recombinant protein. Protein was purified by Ni²⁺-NTA column and used to immunize New Zealand rabbits for preparing the polyclonal antibody. The Ha-c-Myc expression levels in different developmental stages (egg, larva, prepupa, pupa, and adult) of H. armigera and different tissues (midgut, fat body, head, and epidermis) of the prepupa were determined by real-time quantitative reverse transcription PCR (qRT-PCR). Ha-c-Myc siRNA was synthesized and transfected into H. armigera Ha cells. The relative mRNA levels of Ha-c-Myc and HaSCP-2 in Ha cells were detected by qRT-PCR. Results showed that the pET-32a-Ha-c-Myc recombinant plasmid was constructed. The soluble Ha-c-Myc protein of about 65 kDa was expressed in E. coli. The polyclonal antibody was prepared. Western blotting analysis suggested that the antibody had high specificity. Enzyme linked immunosorbent assay (ELISA) showed that the titer of the antibody was high. Ha-c-Myc gene expressed at all developmental stages, with high levels in the early and late instars of larva, and the prepupal stage. Tissue expression profiles revealed that Ha-c-Myc expressed in various tissues of prepupa, with high expression level in the midgut, but low levels in the epidermis and fat body. RNAi results showed that the knockdown of Ha-c-Myc expression significantly affected transcription of HaSCP-2, leading to a 50% reduction in HaSCP-2 mRNA expression level. In conclusion, the Ha-c-Myc was expressed through a prokaryotic expression system, and the polyclonal anti-Ha-c-Myc antibody was obtained. Ha-c-Myc may promote the expression of HaSCP-2 and play an important role in the lipid metabolism of H. armigera. These results may facilitate further study on the potential role and function mechanism of Ha-c-Myc in H. armigera and provide experimental data for exploring new targets of green pesticides.
Animals ; Rabbits ; Escherichia coli/metabolism* ; Enzyme-Linked Immunosorbent Assay ; Moths/genetics* ; Blotting, Western ; Larva/genetics* ; Isoantibodies/metabolism* ; Antibody Specificity

With the rapid development of transgenic technology, the safety of genetically modified products has received extensive attention. Certified reference materials for the detection of genetically modified organisms play important roles in ensuring comparability and traceability of the qualitative and quantitative detection of genetically modified products. However, the development of protein reference materials is relatively slow, and one of the difficulties is the preparation of protein candidates with high purity. The cry1Ah1 gene of Bacillus thuringiensis has been used for the development of transgenic insect-resistant crops because of its excellent insecticidal activity against lepidopteran pests such as Asian corn borer, and has obtained transgenic lines with good insect resistance traits. In order to develop Cry1Ah protein certified reference material, it is urgent to establish a preparation and purification system. In this study, a system for preparing Cry1Ah protein by Bt expression system was optimized, and a high-purity Cry1Ah protein (size exclusion chromatography purity: 99.6%) was obtained by ion-exchange chromatography and size exclusion chromatography stepwise purification. The results of biological activity assay showed that there was no significant difference in the insecticidal activity of purified Cry1Ah protein and protoxin against diamondback moths (Plutella xylostella). Finally, the amino acid sequence of the activated Cry1Ah protein was determined using Edman degradation and mass spectrometry. In summary, the obtained Cry1Ah pure protein can be used for the development of protein reference materials.
Animals ; Bacillus thuringiensis ; Bacterial Proteins ; Cryptochromes ; metabolism ; Endotoxins ; Hemolysin Proteins ; Moths ; Pest Control, Biological ; Plants, Genetically Modified

Bile acid is a type of metabolite degraded from cholesterol in liver. Its accumulation in liver could cause liver diseases, liver damage and liver fibrosis. In this experiment, dimethyl nitrosamine (DMN) liver fibrosis was established in rats. The rats were delivered into the normal group, the model group and four treated groups. After the four-week modeling, the treated groups were orally administered with drugs for 2 weeks, whereas the model and normal groups were given equal amount of sterile water at the same time. In the experiment, serum bile acid was taken the as marker, and liver function indexes and changes in bile acid metabolism were detected and observed to identify liver damage-related bile acid targets. It was the first time to evaluate the reverse effect of artificial CsB and its components on liver fibrosis in rats with bile acid metabolic level, and discuss its potential mechanism. The main study contents and results are as follows: a quantitative analysis was made on totally 17 endogenous bile acids, including taurocholic acid conjugated bile acid, glycine conjugated bile acid and free bile acid, and a liver damage evaluation was made for the model according to the detection of serum biochemical indexes and the pathological biopsy. After modeling, ALT, AST activity and TBil content significantly increased, whereas Alb significantly decreased. According to the pathological biopsy HE staining, the model group showed damage in normal hepatic lobule structure, liver cell edema and connective tissue proliferation in portal area; The treated groups showed mitigation in pathological changes to varying degrees. Cordyceps sinensis and its components may impact the bile acid metabolism in rats by activating HDCA, TCA, TCDCA, TLCA, TUDCA, UDCA, THDCA metabolim-related receptors or blocking relevant signaling pathway.
Animals ; Bile Acids and Salts ; metabolism ; Biological Factors ; administration & dosage ; Cordyceps ; chemistry ; physiology ; Humans ; Liver Cirrhosis ; drug therapy ; metabolism ; Male ; Moths ; chemistry ; microbiology ; Rats ; Rats, Wistar

OBJECTIVEThis study aims to reveal the effect of total ginsenoside on the protein content and digestive enzyme activities of 4th-instar Mythimna separata larvae, including alpha-amylase and cellulose, and explore the ecological function of total ginsenoside.

METHODWhile simulating natural growing condition indoors, 4th-instar M. separata larvae were fed by poison leaf disk method. The protein content was tested by Lowry Protein Assay Kit method, the activity of alpha-amylase was measured by dinitrosalicylic acid test, and the activity of cellulase was determined by the filter paper method.

RESULTThe total ginsenoside could reduce the content of protein of 4th-instar M. separata larvae significantly, and the activity of digestive enzyme, including alpha-amylase and cellulase. The protein content, alpha-amylase and cellulase activity of treatments were obviously lower than that of the control. Inhibition ratio of alpha-amylase and cellulase activity was positively correlated with total ginsenoside concentration: i. e. 20 g x L(-1) > 10 g x L(-1) > 5 g x L(-1).

CONCLUSIONThe results suggest that the inhibition effect of total ginsenoside on protein content and digestive enzymes may be one of the causes to antifeedant and dysplasia of M. separata larvae.

Animals ; Digestion ; Ginsenosides ; pharmacology ; Insect Proteins ; metabolism ; Larva ; drug effects ; enzymology ; growth & development ; Moths ; drug effects ; enzymology ; growth & development

Plutella xylostella granulovirus (PlxyGV) contains homologs of 15 Autographa californica MNPV (AcMNPV) late expression factor (lef) genes. The prospective products of 14 PlxyGV lef genes (ie-0 is not included) share 13%-53% amino acid similarity with their corresponding homologs of AcMNPV, among which LEF-9, LEF-8 and P47, three subunits of the virus-encoded RNA polymerase, share 49%, 53% and 46% sequence identity, respectively. In this study, an established transient expression system was used to test the ability of the PlxyGV LEFs to activate an AcMNPV vp39 promoter-driven reporter gene in SF9 cells. It was shown that PlxyGV le f-2 replaced the corresponding AcMNPV gene and exhibited partial activity in the context of the remaining set of AcMNPV le fs. PlxyGV LEF-2 was found to contain additional 100aa and 70aa at the C-terminus in comparison with the LEF-2 of other GVs and lepidopteran NPVs respectively.
Amino Acid Sequence ; Animals ; Gene Expression Regulation, Viral ; Granulovirus ; chemistry ; genetics ; metabolism ; Molecular Sequence Data ; Moths ; virology ; Nucleopolyhedrovirus ; genetics ; metabolism ; Sequence Alignment ; Viral Proteins ; genetics ; metabolism

ENHANCIN is an enhancing protein chiefly found in insect baculoviruses. One ENHANCIN homologue was identified, by blast method, in Agrotis Segetum granulovirus (AgseGV) genome, named enhancin-like. Sequence analysis indicated that this gene includes the conserved domains, conserved in other ENHANCIN, and it has no signal peptide or a-transmembrane helix. A proline-rich domain, which is similar to those of mammals, is present at its C-terminal. To analyze the synergistic function of AgseGV enhancin-like gene, prokaryotic expression vectors of its whole gene and the 5'-truncated fragment (1, 017bp) were constructed. Expression product of truncated fragment was purified by chromatography, and then it was used to prepare antibody. The expression product of whole gene was identified by Western blot with specific antibody and anti-His-Tag antibody. Bioassay proved that the expression product of whole gene can increase the mortality with 16.25% to 3th instar larvae of Helicoverpa armigera (HaNPV: 1.17 x 10(2) PIBS/mL), while the truncated fragment has no obvious synergistic function.
Amino Acid Sequence ; Animals ; Baculoviridae ; genetics ; metabolism ; Gene Expression ; Insect Control ; Larva ; drug effects ; growth & development ; Molecular Sequence Data ; Moths ; drug effects ; growth & development ; Pest Control, Biological ; Viral Proteins ; genetics ; isolation & purification ; metabolism ; toxicity

OBJECTIVETo clarify the relevance of amino acid content in cultivated Cordyceps and cultivation materials.

METHODThe content of amino acid was determined with L-8800 amino acid analyzer, and the relevance of amino acid content was analyzed with SPSS.

RESULT AND CONCLUSIONExcept mycelium of the C. sinensis or the blood-lymph of the larva, the cultivated Cordyceps and the main relevant cultivation materials had detected to contain all kinds of amino acids. Except among the mycelium, the blood-lymph of the larva, the part of the larva or of the stroma of cultivated Cordyceps, there was distinct relevance of amino acid contents in cultivated Cordyceps and the cultivation materials (P<0.01).

Amino Acids ; analysis ; metabolism ; Animals ; Cordyceps ; chemistry ; metabolism ; Larva ; chemistry ; microbiology ; Moths ; chemistry ; microbiology ; Mycelium ; chemistry ; metabolism

OBJECTIVETo explore the intervening and therapeutic effect of Cordyceps mycelia extract (CME) on liver cirrhosis induced by dimethylnitrosamine (DMN) in rats.

METHODSRat liver cirrhosis model was established by peritoneal injection of DMN at a dose of 10 microg/kg, once daily in the first 3 days of every week for 4 successive weeks. Experimental study on CME-intervention was conducted from the beginning of modeling to the end of the 4th week, while the CME-treatment experiment was carried out from the 4th week of modeling, when terminating the modeling factor, to the end of the 8th week, by administering CME at a dose of 0. 74 g/( kg d) once a day. Animals were killed in batches on the 3rd day, the 2nd (T1), 4th (T2), 6th (T3) and 8th (T4) week after modeling, to observe the histopathologic change in liver and the immunohistochemical staining of alpha-smooth muscle actin (alpha-SMA) and collagen type I (Col I), determine the content of hydroxyproline (Hyp) in liver, and the liver function was tested as well.

RESULTSCME-intervention experiment showed that as compared to those in the modeled rats at corresponding time points, in rats at T1 and T2, serum alanine aminotransferase (ALT), aspartate aminotransferase (AST) activity and total bilirubin (TBIL) content were significantly lower, and albumin (Alb) obviously higher; while at T2, Hyp content, ct-SMA and Col I positive expression were significantly lower (P < 0.05), the proliferation of collagen fibre attenuated. CME-treatment experiment showed that as compared to those in the modeled rats at corresponding time points, lower serum ALT, AST activity and TBIL content, and higher serum level of Alb were shown in rats at T1; and lower Hyp content, liver collagen fibre, and alpha-SMA positive expression were shown at T1 and T2; while less Col I positive expression at T2 was also shown in them (all P < 0.05).

CONCLUSIONCME could not only prevent the development of liver cirrhosis induced by DMN in rats, but also effectively promote the reversion of already formed liver cirrhosis, having a favourable prospect of clinical application.

Alanine Transaminase ; genetics ; metabolism ; Animals ; Aspartate Aminotransferases ; genetics ; metabolism ; Biological Factors ; therapeutic use ; Cordyceps ; chemistry ; Dimethylnitrosamine ; adverse effects ; Humans ; Liver ; metabolism ; Liver Cirrhosis ; chemically induced ; drug therapy ; enzymology ; genetics ; Male ; Moths ; chemistry ; Mycelium ; chemistry ; Random Allocation ; Rats ; Rats, Wistar ; Treatment Outcome

The knowledge of the relationship between sequence characteristics of insecticidal crystal proteins (ICP) and their inhibitory against Plutella xylostella provided helpful information for the rational design of ICP with desirable activity against Plutella xylostella. The four key loops of ICP with determined activities against Plutella xylostella were selected to study the quantitative relationship between sequence characteristics and insecticidal activity. The first principle components' score vectors for 20 amino acids were assigned to converting amino acids into data. The six key sites X3, X9, X12, X13, X14 and X19 were predicted by stepwise regression method. The amino acids L/ X3, S/ X9, S/ X12, T/ X13, A/ X14 and G/ X19 found by partial least squares regression and second order polynomial models were predicted to increase the activity of ICP against Plutella xylostella.
Amino Acid Sequence ; Animals ; Bacterial Proteins ; genetics ; pharmacology ; Endotoxins ; genetics ; pharmacology ; Hemolysin Proteins ; genetics ; pharmacology ; Insecticides ; metabolism ; pharmacology ; Models, Biological ; Molecular Sequence Data ; Moths ; genetics ; metabolism ; Pest Control, Biological ; Sequence Analysis, Protein