The complete mitochondrial genome of Chinese Bombyx mandarina(ChBm) was determined.The cir-cular genome is 15682 bp long, and contains a typical gene complement, order, and arrangement iden-tical to that of Bombyx mor...The complete mitochondrial genome of Chinese Bombyx mandarina(ChBm) was determined.The cir-cular genome is 15682 bp long, and contains a typical gene complement, order, and arrangement iden-tical to that of Bombyx mori(B.mori) and Japanese Bombyx mandarina(JaBm) except for two addi-tional tRNA-like structures:tRNASer(TGA)-like and tRNAIle(TAT)-like.All protein-coding sequences are initi-ated with a typical ATN codon except for the COI gene, which has a 4-bp TTAG putative initiator codon.Eleven of 13 protein-coding genes(PCGs) have a complete termination codon(all TAA), but the re-maining two genes terminate with incomplete codons.All tRNAs have the typical clover-leaf structures of mitochondrial tRNAs, with the exception of tRNASer(TGA)-like, with a four stem-and-loop structure.The length of the A+T-rich region of ChBm is 484 bp, shorter than those of JaBm(747 bp) and B.mori(494―499 bp).Phylogenetic analysis among B.mori, ChBm, JaBm, and Antheraea pernyi(Anpe) showed that B.mori is more closely related to ChBm than JaBm.The earliest divergence time estimate for B.mori-ChBm and B.mori-JaBm is about 1.08±0.18―1.41±0.24 and 1.53±0.20―2.01±0.26 Mya, respec-tively.ChBm and JaBm diverged around 1.11±0.16―1.45±0.21 Mya.展开更多
Gene expression quantification at mRNA level is very important for postgenomic studies, as gene expression level is the reflection of the special biological function of the target gene. Methods used for gene expressio...Gene expression quantification at mRNA level is very important for postgenomic studies, as gene expression level is the reflection of the special biological function of the target gene. Methods used for gene expression quantification, such as microarray or quantitative real-time polymerase chain reaction （qRT-PCR）, require stable expressed reference genes. Thus, finding suitable control genes is essential for gene quantification. In this study, a genome-wide survey of reference genes during metamorphism was performed on silkworm Bombyx mori. Twelve genes were chosen as putative reference genes based on a whole genome oligonucleotide microarray normalized by external controls. Then, qRT- PCR was employed for further validation and selection of potential reference gene candidates. The results were analyzed, and stable genes were selected using geNorm 3.4 and NormFinder software. Finally, considering factors from every aspect, translation initiation factor 4A, translation initiation factor 3 subunit 4, and translation initiation factor 3 subunit 5 （represented by sw22934, sw14876, and sw13956） were selected as reliable internal controls across the examined developmental stages, while cytoplasmic actin （sw22671）, the commonly used reference gene in a previous study was shown to vary drastically throughout the examined developmental stages. For future research, we recommend the use of the geometric mean of those three stable reference genes as an accurate normalization factor for data normalization of different developmental stages during metamorphism.展开更多
We analyzed 20 chemosensory protein （CSP） genes of the silkworm Bombyx mori. We found a high number of retrotransposons inserted in introns. We then analyzed expression of the 20 BrnorCSP genes across tissues using ...We analyzed 20 chemosensory protein （CSP） genes of the silkworm Bombyx mori. We found a high number of retrotransposons inserted in introns. We then analyzed expression of the 20 BrnorCSP genes across tissues using quantitative real-time polymerase chain reaction （PCR）. Relatively low expression levels of BmorCSPs were found in the gut and fat body tissues. We thus tested the effects of endectocyte insecticide abamectin （B 1 a and Blb avermectins） on BmorCSP gene expression. Quantitative real-time PCR experi- ments showed that a single brief exposure to insecticide abamectin increased dramatically CSP expression not only in the antennae but in most tissues, including gut and fat body. Furthermore, our study showed coordinate expression of CSPs and metabolic cytochrome P450 enzymes in a tissue-dependent manner in response to the insecticide. The function of CSPs remains unknown. Based on our results, we suggest a role in detecting xenobiotics that are then detoxified by cytochrome P450 anti-xenobiotic enzymes.展开更多
The long-arm and short-arm genes of fibroin light chain (L-chain) of silkworm, Bombyx Mori L., and the gene of human acidic fibroblast growth factor were cloned respectively and subsequently inserted into a transfer v...The long-arm and short-arm genes of fibroin light chain (L-chain) of silkworm, Bombyx Mori L., and the gene of human acidic fibroblast growth factor were cloned respectively and subsequently inserted into a transfer vector pVL 1392 used as a tool to target the L-chain region of the silkworm genome. Genomic DNA from their offsprings was extracted and the expected targeting was detected using polymerase chain reaction and DNA sequencing, as well as protein analysis. The results showed that positive events occurred and that the FGF gene was integrated into the L-chain locus through homologous recombination.展开更多
1-deoxynojirimycin (1-DNJ) contents in the silkworm,Bombyx mori,at different developmental stages and tissues were investigated by using reverse-phase high-performance liquid chromatography. The 1-DNJ contents of silk...1-deoxynojirimycin (1-DNJ) contents in the silkworm,Bombyx mori,at different developmental stages and tissues were investigated by using reverse-phase high-performance liquid chromatography. The 1-DNJ contents of silkworm larvae change significantly with their developmental stages. The male larvae showed higher accumulation efficiency of 1-DNJ than the females and also a significant variation was observed among the silkworm strains. The present results show that tissue distribution of 1-DNJ was significantly higher in blood,digestive juice,and alimentary canal,but no 1-DNJ was observed in the silkgland. Moreover,1-DNJ was not found in silkworms fed with artificial diet that does not contain mulberry leaf powder. This proves that silkworms obtain 1-DNJ from mulberry leaves; they could not synthesize 1-DNJ by themselves. The accumulation and excretion of 1-DNJ change periodically during the larval stage. There was no 1-DNJ in the newly-hatched larvae and 1-DNJ was mainly accumulated during the early and middle stages of every instar,while excreted at later stages of larval development. Further,it is possible to extract 1-DNJ from the larval feces and it is optimal to develop the 1-DNJ related products for diabetic auxiliary therapy.展开更多
The fibroin gene expression pattern and regulation of the posterior silkgland were studied by means of expressed sequence tags (ESTs) using the first and fifth day larvae of the fifth instar of silkworm, Bombyx mori L...The fibroin gene expression pattern and regulation of the posterior silkgland were studied by means of expressed sequence tags (ESTs) using the first and fifth day larvae of the fifth instar of silkworm, Bombyx mori L (strain: C 108). The results showed that there were 911 repetitive ESTs and 1950 single sequences (Singlets) among total 2861 consentient sequences, which were spliced. 1335 sequences were identified and the other 1526 were unknown. 5560 sequences (55.89%) in the posterior silkgland cell of the silkworm were new ESTs without homology with EST data published by Mita et al. The number of repetitive ESTs and single sequences from the first day larvae of the fifth instar was double more than that of the fifth day of the same instar in the silkworms. The unigenes which were more than 50 in repetitive EST size (contig size) came to only about 0.5% in total consentient sequences. There were significant differences between gene expression frequencies, and expressed genes were related to fibroin synthesis and its secretion and fibroin composition. Comparing the fifth day with the first day of the fifth instar, the genes-expressed quantity of fibroin heavy-chain gene was 18 fold higher, fibroin light-chain gene 9 fold and fibroin P52 gene 8 fold. 508 genes functioned for cellular component and 315 for enzyme after function tracing. These results implied that the gene expression of the first day was mainly for preparation for fibroin synthesis except for the growth of silkgland cells, and the gene expression of the fifth day of the fifth instar was mainly for synthesizing and excreting fibroin. Because the ratio of heavy chain, light chain and p25 of fibroin was not 6:6:1 as theoretically expected, or its special H-chain structure, the H-chain gene was not easy to detect through EST technique. Most of genes among total 2861 consentient sequences functioned for fibroin synthesis and secretion. This suggested the fibroin synthesis and secretion procedure of the posterior silkgland was more complex than the knowl展开更多
To express human insulin-like growth factor-I (hIGF-I) in transformed Bombyx mori cultured cells and silk glands, the transgenic vector pigA3GFP-hIGF-ie-neo was constructed with a neomycin resistance gene driven by th...To express human insulin-like growth factor-I (hIGF-I) in transformed Bombyx mori cultured cells and silk glands, the transgenic vector pigA3GFP-hIGF-ie-neo was constructed with a neomycin resistance gene driven by the baculovirus ie-1 promoter, and with the hIGF-I gene under the control of the silkworm sericin promoter Ser-1. The stably transformed BmN cells expressing hIGF-I were selected by using the antibiotic G418 at a final concentration of 700-800 μg/mL after the BmN cells were transfected with the piggyBac vector and the helper plasmid. The specific band of hIGF-I was detected in the transformed cells by Western blot. The expression level of hIGF-I, determined by ELISA, was about 7800 pg in 5×105 cells. Analysis of the chromosomal insertion sites by inverse PCR showed that exogenous DNA could be inserted into the cell genome randomly or at TTAA target sequence specifically for piggyBac element transposition. The transgenic vector pigA3GFP-hIGF-ie-neo was transferred into the eggs using sperm-mediated gene transfer. Finally, two transgenic silkworms were obtained after screening for the neo and gfp genes and verified by PCR and dot hybridization. The expression level of hIGF-I determined by ELISA was about 2440 pg/g of silk gland of the transgenic silkworms of the G1 generation.展开更多
Amplified inter simple sequence repeats （ISSR） markers were used to determine genetic relationships among mutant silkworm strains of Bombyx mori. Fifteen ISSR primers containing simple sequence repeat （SSR） motifs...Amplified inter simple sequence repeats （ISSR） markers were used to determine genetic relationships among mutant silkworm strains of Bombyx mori. Fifteen ISSR primers containing simple sequence repeat （SSR） motifs were used in this study. A total of 113 markers were produced among 20 mutant strains, of which 73.45% were found to be polymorphic. In selected mutant genetic stocks, the average number of observed allele was （1.7080 ± 0.4567）, effective alleles （1.5194 ± 0.3950） and genetic diversity （Ht） （0.2901 ± 0.0415）. The dendrogram produced using the unweighted pair group method with arithmetic means （UPGMA） and cluster analysis made using Nei＇s genetic distance resulted in the formation of one major group containing 6 groups separated 20 mutant silkworm strains. Therefore, ISSR amplification is a valuable method for determining the genetic variability among mutant silkworm strains. This efficient molecular marker would be useful for characterizing a considerable number of silkworm strains maintained at the germplasm center.展开更多
A group of lipoproteins with molecular sizes of approximately 30 kDa, referred to as 30K proteins, axe synthesized in fat body cells in the fifth instar larvae of silkworm, Bombyx mori. Analyzing the silkworm genome a...A group of lipoproteins with molecular sizes of approximately 30 kDa, referred to as 30K proteins, axe synthesized in fat body cells in the fifth instar larvae of silkworm, Bombyx mori. Analyzing the silkworm genome and its expressed sequence tags （ESTs）, we found 10 genes encoding 30K proteins, which are mainly distributed in three subfamilies. Of these, seven coding proteins were found to harbor the degrading sites of 30kP protease A, although the number of degrading sites may be different. As some potential core promoters and regulatory elements were supposed to be essential for gene transcription, the expression profiles of these genes were examined by semi-quantitative reverse transcription polymerase chain reaction. Eight 30K protein genes were detected to express luxuriantly in the fat body, while two were hardly expressed. Such results suggest that these 30K proteins may have different functions, and their adjacent regulatory elements play a crucial role in regulating their transcription.展开更多
Pupae inside cocoons rarely suffer from disease. It is apparent that some factors in the cocoon exert antimicrobial effects whereby the pupae inside can be protected from microbial infection. In the present study, we ...Pupae inside cocoons rarely suffer from disease. It is apparent that some factors in the cocoon exert antimicrobial effects whereby the pupae inside can be protected from microbial infection. In the present study, we investigated the expression of cocoon protease inhibitors using immunoblotting and activity staining. Enzymatic hydrolysis of cocoon proteins in vitro was performed to characterize their roles in protecting the cocoon from microbial proteases. We found that some protease inhibitors, particularly trypsin inhibitor-like （TIL）-type protease inhibitors, can be secreted into the cocoon layer during the spinning process, thereby providing effective protection to the cocoon and pupa by inhibiting the extracellular proteases that can be secreted by pathogens.展开更多
Silkworm powder containing 1-deoxynojirimycin (DNJ) has α-glucosidase inhibitory activity and is promising as a complementary and alternative medicine (CAM) agent in Japan. Silkworm powder produced in Korea was extra...Silkworm powder containing 1-deoxynojirimycin (DNJ) has α-glucosidase inhibitory activity and is promising as a complementary and alternative medicine (CAM) agent in Japan. Silkworm powder produced in Korea was extracted with 75% ethanol. The extract was derivatized with 9-fluorenylmethyl chloroformate (FMOC-Cl), and DNJ-FMOC content was measured by HPLC. Then, alfa-glucosidase inhibition by silkworm and mulberry powder in pig liver crude enzyme was assayed using 4-nitrophenyl-alfa-D-glucopyranoside as a substrate. Silkworm powder DNJ content (0.39 to 0.58%) was higher than that in mulberry powder (0.08 to 0.12%). The alfa-glucosidase inhibitory activity of silkworm powder was more potent than that of mulberry leaves and green tea. Silkworm powder DNJ was stable upon heating to 121?C for up to 15 min.展开更多
The insect exoskeleton is mainly composed of chitin filaments linked by cuticle proteins. When insects molt, the cuticle of the exoskeleton is renewed by degrading the old chitin and cuticle proteins and synthesizing ...The insect exoskeleton is mainly composed of chitin filaments linked by cuticle proteins. When insects molt, the cuticle of the exoskeleton is renewed by degrading the old chitin and cuticle proteins and synthesizing new ones. In this study, chitin-binding activity of the wing disc cuticle protein BmWCP4 in Bombyx mori was studied. Sequence analysis showed that the protein had a conservative hydrophilic ＂R＆R＂ chitin-binding domain （CBD）. Western blotting showed that BmWCP4 was predominately expressed in the wing disc-containing epidermis during the late wandering and early pupal stages. The immunohistochemistry result showed that the BmWCP4 was mainly present in the wing disc tissues containing wing bud and trachea blast during day 2 of wandering stage. Recombinant full-length BmWCP4 protein, ＂R＆R＂ CBD peptide （CBD）, non-CBD peptide （BmWCP4-CBD^-）, four single site-directed mutated peptides （M1, M2, M3 and M4） and four-sites-mutated peptide （MF） were generated and purified, respectively, for in vitro chitin-binding assay. The results indicated that both the full-length protein and the ＂R＆R＂ CBD peptide could bind with chitin, whereas the BmWCP4-CBD- could not bind with chitin. The single residue mutants M1, M2, M3 and M4 reduced but did not completely abolish the chitin-binding activity, while four-sites-mutated protein MF completely lost the chitin-binding activity. These data indicate that BmWCP4 protein plays a critical role by binding to the chitin filaments in the wing during larva-to-pupa transformation. The conserved aromatic amino acids are critical in the interaction between chitin and the cuticle protein.展开更多
Serine proteases play important roles in digestion and immune responses during insect development. In the present study, the serine protease gene BmSP36, which encodes a 292-residue protein, was cloned from the midgut...Serine proteases play important roles in digestion and immune responses during insect development. In the present study, the serine protease gene BmSP36, which encodes a 292-residue protein, was cloned from the midgut cells ofBombyx mori. BmSP36 contains an intact catalytic triad （H57, D102 and S195） and a conserved substrate-binding site （G189, H216 and G226）, suggesting that it is a serine protease with chymotrypsin- like specificity. The temporal and spatial expression patterns of BmSP36 indicated that its messenger RNA and protein expression mainly occurred in the midgut at the feeding stages. Western blotting, immunofluorescence and liquid chromatography-tandem mass spectrometry analyses revealed secretion of BmSP36 protein from epithelial cells into the midgut lumen. The transcriptional and translational expression of BmSP36 was down- regulated after starvation but up-regulated after refeeding. Moreover, expression of the BmSP36 gene could be up-regulated by a juvenile hormone analogue. These results enable us to better define the potential role of BmSP36 in dietary protein digestion at the feeding stages during larval development.展开更多
The yellow color of the cocoon of the silkworm Bombyx mori is controlled by three genes, Y （Yellow haemolymph）, 1 （Yellow inhibitor） and C （ Outer-layer yellow cocoon）, which are located on linkage groups 2, 9 a...The yellow color of the cocoon of the silkworm Bombyx mori is controlled by three genes, Y （Yellow haemolymph）, 1 （Yellow inhibitor） and C （ Outer-layer yellow cocoon）, which are located on linkage groups 2, 9 and 12, respectively. Taking advantage of a lack of crossing over in females, reciprocal backcrossed F1 （BC1） progeny were used for linkage analysis and mapping of the C gene using silkworm strains C 108 and KY, which spin white and yellow cocoons, respectively. DNA was extracted from individual pupae and analyzed for simple sequence repeat （SSR） markers. The C gene was found to be linked to seven SSR markers. All the yellow cocoon individuals from a female heterozygous backcross （BC1F） showed a heterozygous profile for SSR markers on linkage group 12, whereas individuals with light yellow cocoons showed the homozygous profile of the strain C108. Using a reciprocal heterozygous male backcross （BC1M）, we constructed a linkage map of 36.4 cM with the C gene located at the distal end, and the closest SSR marker at a distance of 13.9 cM.展开更多
Molecular systematic studies on mandarina silkworm (Bombyx mandarina M.) in 11 regions in China and 25 representative strains of domestic silkworm (Bombyx mori L.) were conducted using molecular biology techniques. Re...Molecular systematic studies on mandarina silkworm (Bombyx mandarina M.) in 11 regions in China and 25 representative strains of domestic silkworm (Bombyx mori L.) were conducted using molecular biology techniques. Results obtained from the analysis of DNA polymorphism and clustering of all the silkworm samples provide new evidence for the view that the domestic silkworm originated from the Chinese mandarina silkworm. On the basis of literature reviewing, a new hypothesis on the origin of the domestic silkworm was put forward. It was thought that the domestic silkworm was most probably domesticated from the Chinese mandarina silkworm of different ecotypes including monovoltinism, bivoltinism and multivoltinism; and that the domestic silkworm had the genetic background of monovoltinism, bivoltinism and multivoltinism at the very beginning of the domestication. The current strains of the domestic silkworm of different voltinism are the evolutionary results of thousands of years of rearing and artificial selections.展开更多
We report the thermotolerance of new bivoltine silkworm, Bombyx mori strains NB4D2, KSO1, NP2, CSR2 and CSR4 and differential expression of heat shock proteins at different instars. Different instars of silkworm larva...We report the thermotolerance of new bivoltine silkworm, Bombyx mori strains NB4D2, KSO1, NP2, CSR2 and CSR4 and differential expression of heat shock proteins at different instars. Different instars of silkworm larva were subjected to heat shock at 35℃, 40℃ and 45℃ for 2 hours followed by 2 hours recovery. Heat shock proteins were analyzed by SDS-PAGE. The impact of heat shock on commercial traits of cocoons was analyzed by following different strategies in terms of acquired thermotolerance over control. Comparatively NP2 exhibited better survivability than other strains. Resistance to heat shock was increased as larval development proceeds in the order of first instar 〉 second instar 〉 third instar 〉 fourth instar 〉 fifth instar in all silkworm strains. Expression of heat shock proteins varies in different instars. 90 kDa in the first, second and third instars, 84 kDa in the fourth instar and 84, 62, 60, 47 and 33 kDa heat shock proteins in fifth instar was observed in response to heat shock. Relative influence of heat shock on commercial traits that correspond to different stages was significant in all strains. In NB4D2, cocoon and shell weight significantly increased to 17.52% and 19.44% over control respectively. Heat shock proteins as molecular markers for evaluation and evolution of thermotolerant silkworm strains for tropics was discussed.展开更多
In silkworms, the white egg 1 （w-1） mutant, which is characterized by white eyes and white eggs, is deficient in Bombyx kynurenine 3-monooxygenase （KMO） activity. To investigate whether the w-1 mutant phenotype is...In silkworms, the white egg 1 （w-1） mutant, which is characterized by white eyes and white eggs, is deficient in Bombyx kynurenine 3-monooxygenase （KMO） activity. To investigate whether the w-1 mutant phenotype is rescued by introducing the wild-type KMO gene, we constructed transgenic silkworms with the wild-type Bombyx KMO gene under the control of either the cytoplasmic actin gene promoter （A3KMO） or the native KMO gene promoter （KKMO）. We created two transgenic lines with A3KMO and one line with KKMO constructs. The eyes of adults in these lines were brown, and the eggs laid by the transgenic females were also brown. Reverse transcription-polymerase chain reaction（RT-PCR） analysis showed that the A3KMO silkworm lines expressed the transcript in the mid-gut, fat bodies, and Malpighian tubules. The KKMO line expressed the transcript only in the fat bodies and Malpighian tubules. The intensity of eye and egg color in the transgenic lines was proportional to the KMO expression level. Interestingly, transgenic larvae with the A3KMO construct had a light brown larval cuticle, but the KKMO line did not. These results indicate that the wild-type KMO gene can be used as a marker gene for visually screening transgenic silkworms.展开更多
The embryonic diapause of the silkworm, Bombyx mori, is induced by the diapause hormone (DH) which is secreted from the suboesophageal ganglion of pupae. The diapause nature of bivoltine strains uses environmental sti...The embryonic diapause of the silkworm, Bombyx mori, is induced by the diapause hormone (DH) which is secreted from the suboesophageal ganglion of pupae. The diapause nature of bivoltine strains uses environmental stimuli as the initial signal to determine the diapause nature. The experiments showed that DH gene expression is a direct response to the environmental stimulus, such as high incubation temperature. The cDNA from the embryonic stage was cloned and sequence analysis showed the cDNA encoding DH. Expression patterns of the DH gene in embryonic stage are different at incubation temperatures 15℃ and 25℃, suggesting that the incubation temperature as an environmental signal is kept within the body to control the DH gene expression at the pupal stage, so that the embryonic diapause of next generation can be determined.展开更多
Cathepsin B belongs to lysosomal cysteine protease of the papain family. Temporal and spatial expression analysis of cathepsin B of Bombyx mori （BmCtB） was carried out based on Expression Sequence Tags （ESTs） data...Cathepsin B belongs to lysosomal cysteine protease of the papain family. Temporal and spatial expression analysis of cathepsin B of Bombyx mori （BmCtB） was carried out based on Expression Sequence Tags （ESTs） data, oligonucleotide microarray, reverse transcription polymerase chain reaction （RT-PCR） and quantitative real-time PCR. Expression of BmCtB was observed in all of the tissues and stages. Among the 10 tested tissues, the fat body and posterior silk gland are the two most enriched tissues with BmCtB. During Bombyx development, there was an expression fastigium of BmCtB during metamorphosis. RNA interference was used to suppress the expression of cathepsin B during metamorphosis. Significant developmental defective phenotypes were obtained in the RNAi treated group. The dramatically reduced expression of BmCtB was con^rmed by Northern blot and quantitative real-time PCR. These evidences strongly suggest cathepsin B proteinase was predominantly involved in the metabolism process of fat body and the posterior silk gland and was critical for metamorphism and development of silkworm, Bombyx mori.展开更多
基金the National Basic Research Program of China (Grant No. 2005cb121000)National Natural Science Foundation of China (Grant No. 30771630)
文摘The complete mitochondrial genome of Chinese Bombyx mandarina(ChBm) was determined.The cir-cular genome is 15682 bp long, and contains a typical gene complement, order, and arrangement iden-tical to that of Bombyx mori(B.mori) and Japanese Bombyx mandarina(JaBm) except for two addi-tional tRNA-like structures:tRNASer(TGA)-like and tRNAIle(TAT)-like.All protein-coding sequences are initi-ated with a typical ATN codon except for the COI gene, which has a 4-bp TTAG putative initiator codon.Eleven of 13 protein-coding genes(PCGs) have a complete termination codon(all TAA), but the re-maining two genes terminate with incomplete codons.All tRNAs have the typical clover-leaf structures of mitochondrial tRNAs, with the exception of tRNASer(TGA)-like, with a four stem-and-loop structure.The length of the A+T-rich region of ChBm is 484 bp, shorter than those of JaBm(747 bp) and B.mori(494―499 bp).Phylogenetic analysis among B.mori, ChBm, JaBm, and Antheraea pernyi(Anpe) showed that B.mori is more closely related to ChBm than JaBm.The earliest divergence time estimate for B.mori-ChBm and B.mori-JaBm is about 1.08±0.18―1.41±0.24 and 1.53±0.20―2.01±0.26 Mya, respec-tively.ChBm and JaBm diverged around 1.11±0.16―1.45±0.21 Mya.
文摘Gene expression quantification at mRNA level is very important for postgenomic studies, as gene expression level is the reflection of the special biological function of the target gene. Methods used for gene expression quantification, such as microarray or quantitative real-time polymerase chain reaction （qRT-PCR）, require stable expressed reference genes. Thus, finding suitable control genes is essential for gene quantification. In this study, a genome-wide survey of reference genes during metamorphism was performed on silkworm Bombyx mori. Twelve genes were chosen as putative reference genes based on a whole genome oligonucleotide microarray normalized by external controls. Then, qRT- PCR was employed for further validation and selection of potential reference gene candidates. The results were analyzed, and stable genes were selected using geNorm 3.4 and NormFinder software. Finally, considering factors from every aspect, translation initiation factor 4A, translation initiation factor 3 subunit 4, and translation initiation factor 3 subunit 5 （represented by sw22934, sw14876, and sw13956） were selected as reliable internal controls across the examined developmental stages, while cytoplasmic actin （sw22671）, the commonly used reference gene in a previous study was shown to vary drastically throughout the examined developmental stages. For future research, we recommend the use of the geometric mean of those three stable reference genes as an accurate normalization factor for data normalization of different developmental stages during metamorphism.
文摘We analyzed 20 chemosensory protein （CSP） genes of the silkworm Bombyx mori. We found a high number of retrotransposons inserted in introns. We then analyzed expression of the 20 BrnorCSP genes across tissues using quantitative real-time polymerase chain reaction （PCR）. Relatively low expression levels of BmorCSPs were found in the gut and fat body tissues. We thus tested the effects of endectocyte insecticide abamectin （B 1 a and Blb avermectins） on BmorCSP gene expression. Quantitative real-time PCR experi- ments showed that a single brief exposure to insecticide abamectin increased dramatically CSP expression not only in the antennae but in most tissues, including gut and fat body. Furthermore, our study showed coordinate expression of CSPs and metabolic cytochrome P450 enzymes in a tissue-dependent manner in response to the insecticide. The function of CSPs remains unknown. Based on our results, we suggest a role in detecting xenobiotics that are then detoxified by cytochrome P450 anti-xenobiotic enzymes.
基金Project supported by the ScientificResearch Foundation forReturned Overseas Chinese Scholars,Education Ministry of Chinaand the Natural Science Foundation of Zhejiang Province (No.301306), China
文摘The long-arm and short-arm genes of fibroin light chain (L-chain) of silkworm, Bombyx Mori L., and the gene of human acidic fibroblast growth factor were cloned respectively and subsequently inserted into a transfer vector pVL 1392 used as a tool to target the L-chain region of the silkworm genome. Genomic DNA from their offsprings was extracted and the expected targeting was detected using polymerase chain reaction and DNA sequencing, as well as protein analysis. The results showed that positive events occurred and that the FGF gene was integrated into the L-chain locus through homologous recombination.
文摘1-deoxynojirimycin (1-DNJ) contents in the silkworm,Bombyx mori,at different developmental stages and tissues were investigated by using reverse-phase high-performance liquid chromatography. The 1-DNJ contents of silkworm larvae change significantly with their developmental stages. The male larvae showed higher accumulation efficiency of 1-DNJ than the females and also a significant variation was observed among the silkworm strains. The present results show that tissue distribution of 1-DNJ was significantly higher in blood,digestive juice,and alimentary canal,but no 1-DNJ was observed in the silkgland. Moreover,1-DNJ was not found in silkworms fed with artificial diet that does not contain mulberry leaf powder. This proves that silkworms obtain 1-DNJ from mulberry leaves; they could not synthesize 1-DNJ by themselves. The accumulation and excretion of 1-DNJ change periodically during the larval stage. There was no 1-DNJ in the newly-hatched larvae and 1-DNJ was mainly accumulated during the early and middle stages of every instar,while excreted at later stages of larval development. Further,it is possible to extract 1-DNJ from the larval feces and it is optimal to develop the 1-DNJ related products for diabetic auxiliary therapy.
文摘The fibroin gene expression pattern and regulation of the posterior silkgland were studied by means of expressed sequence tags (ESTs) using the first and fifth day larvae of the fifth instar of silkworm, Bombyx mori L (strain: C 108). The results showed that there were 911 repetitive ESTs and 1950 single sequences (Singlets) among total 2861 consentient sequences, which were spliced. 1335 sequences were identified and the other 1526 were unknown. 5560 sequences (55.89%) in the posterior silkgland cell of the silkworm were new ESTs without homology with EST data published by Mita et al. The number of repetitive ESTs and single sequences from the first day larvae of the fifth instar was double more than that of the fifth day of the same instar in the silkworms. The unigenes which were more than 50 in repetitive EST size (contig size) came to only about 0.5% in total consentient sequences. There were significant differences between gene expression frequencies, and expressed genes were related to fibroin synthesis and its secretion and fibroin composition. Comparing the fifth day with the first day of the fifth instar, the genes-expressed quantity of fibroin heavy-chain gene was 18 fold higher, fibroin light-chain gene 9 fold and fibroin P52 gene 8 fold. 508 genes functioned for cellular component and 315 for enzyme after function tracing. These results implied that the gene expression of the first day was mainly for preparation for fibroin synthesis except for the growth of silkgland cells, and the gene expression of the fifth day of the fifth instar was mainly for synthesizing and excreting fibroin. Because the ratio of heavy chain, light chain and p25 of fibroin was not 6:6:1 as theoretically expected, or its special H-chain structure, the H-chain gene was not easy to detect through EST technique. Most of genes among total 2861 consentient sequences functioned for fibroin synthesis and secretion. This suggested the fibroin synthesis and secretion procedure of the posterior silkgland was more complex than the knowl
基金Supported by the National Basic Research Program of China (Grant No. 2005CB121000)the National Natural Science Foundation of China (Grant No. 30671590)
文摘To express human insulin-like growth factor-I (hIGF-I) in transformed Bombyx mori cultured cells and silk glands, the transgenic vector pigA3GFP-hIGF-ie-neo was constructed with a neomycin resistance gene driven by the baculovirus ie-1 promoter, and with the hIGF-I gene under the control of the silkworm sericin promoter Ser-1. The stably transformed BmN cells expressing hIGF-I were selected by using the antibiotic G418 at a final concentration of 700-800 μg/mL after the BmN cells were transfected with the piggyBac vector and the helper plasmid. The specific band of hIGF-I was detected in the transformed cells by Western blot. The expression level of hIGF-I, determined by ELISA, was about 7800 pg in 5×105 cells. Analysis of the chromosomal insertion sites by inverse PCR showed that exogenous DNA could be inserted into the cell genome randomly or at TTAA target sequence specifically for piggyBac element transposition. The transgenic vector pigA3GFP-hIGF-ie-neo was transferred into the eggs using sperm-mediated gene transfer. Finally, two transgenic silkworms were obtained after screening for the neo and gfp genes and verified by PCR and dot hybridization. The expression level of hIGF-I determined by ELISA was about 2440 pg/g of silk gland of the transgenic silkworms of the G1 generation.
文摘Amplified inter simple sequence repeats （ISSR） markers were used to determine genetic relationships among mutant silkworm strains of Bombyx mori. Fifteen ISSR primers containing simple sequence repeat （SSR） motifs were used in this study. A total of 113 markers were produced among 20 mutant strains, of which 73.45% were found to be polymorphic. In selected mutant genetic stocks, the average number of observed allele was （1.7080 ± 0.4567）, effective alleles （1.5194 ± 0.3950） and genetic diversity （Ht） （0.2901 ± 0.0415）. The dendrogram produced using the unweighted pair group method with arithmetic means （UPGMA） and cluster analysis made using Nei＇s genetic distance resulted in the formation of one major group containing 6 groups separated 20 mutant silkworm strains. Therefore, ISSR amplification is a valuable method for determining the genetic variability among mutant silkworm strains. This efficient molecular marker would be useful for characterizing a considerable number of silkworm strains maintained at the germplasm center.
基金This work was supported by the National Basic Research Program of China, the National Scientific and Technological Project and the National Natural Science Foundation under grant No. 2005 CB 121000, 2005 B A711A07 and 30471313, respectively.
文摘A group of lipoproteins with molecular sizes of approximately 30 kDa, referred to as 30K proteins, axe synthesized in fat body cells in the fifth instar larvae of silkworm, Bombyx mori. Analyzing the silkworm genome and its expressed sequence tags （ESTs）, we found 10 genes encoding 30K proteins, which are mainly distributed in three subfamilies. Of these, seven coding proteins were found to harbor the degrading sites of 30kP protease A, although the number of degrading sites may be different. As some potential core promoters and regulatory elements were supposed to be essential for gene transcription, the expression profiles of these genes were examined by semi-quantitative reverse transcription polymerase chain reaction. Eight 30K protein genes were detected to express luxuriantly in the fat body, while two were hardly expressed. Such results suggest that these 30K proteins may have different functions, and their adjacent regulatory elements play a crucial role in regulating their transcription.
文摘Pupae inside cocoons rarely suffer from disease. It is apparent that some factors in the cocoon exert antimicrobial effects whereby the pupae inside can be protected from microbial infection. In the present study, we investigated the expression of cocoon protease inhibitors using immunoblotting and activity staining. Enzymatic hydrolysis of cocoon proteins in vitro was performed to characterize their roles in protecting the cocoon from microbial proteases. We found that some protease inhibitors, particularly trypsin inhibitor-like （TIL）-type protease inhibitors, can be secreted into the cocoon layer during the spinning process, thereby providing effective protection to the cocoon and pupa by inhibiting the extracellular proteases that can be secreted by pathogens.
文摘Silkworm powder containing 1-deoxynojirimycin (DNJ) has α-glucosidase inhibitory activity and is promising as a complementary and alternative medicine (CAM) agent in Japan. Silkworm powder produced in Korea was extracted with 75% ethanol. The extract was derivatized with 9-fluorenylmethyl chloroformate (FMOC-Cl), and DNJ-FMOC content was measured by HPLC. Then, alfa-glucosidase inhibition by silkworm and mulberry powder in pig liver crude enzyme was assayed using 4-nitrophenyl-alfa-D-glucopyranoside as a substrate. Silkworm powder DNJ content (0.39 to 0.58%) was higher than that in mulberry powder (0.08 to 0.12%). The alfa-glucosidase inhibitory activity of silkworm powder was more potent than that of mulberry leaves and green tea. Silkworm powder DNJ was stable upon heating to 121?C for up to 15 min.
基金This study was supported by research grants of the National Natural Science Foundation of China （31172265 31330071 31301918） and the National Basic Research Program of China （2012CB 114602）.
文摘The insect exoskeleton is mainly composed of chitin filaments linked by cuticle proteins. When insects molt, the cuticle of the exoskeleton is renewed by degrading the old chitin and cuticle proteins and synthesizing new ones. In this study, chitin-binding activity of the wing disc cuticle protein BmWCP4 in Bombyx mori was studied. Sequence analysis showed that the protein had a conservative hydrophilic ＂R＆R＂ chitin-binding domain （CBD）. Western blotting showed that BmWCP4 was predominately expressed in the wing disc-containing epidermis during the late wandering and early pupal stages. The immunohistochemistry result showed that the BmWCP4 was mainly present in the wing disc tissues containing wing bud and trachea blast during day 2 of wandering stage. Recombinant full-length BmWCP4 protein, ＂R＆R＂ CBD peptide （CBD）, non-CBD peptide （BmWCP4-CBD^-）, four single site-directed mutated peptides （M1, M2, M3 and M4） and four-sites-mutated peptide （MF） were generated and purified, respectively, for in vitro chitin-binding assay. The results indicated that both the full-length protein and the ＂R＆R＂ CBD peptide could bind with chitin, whereas the BmWCP4-CBD- could not bind with chitin. The single residue mutants M1, M2, M3 and M4 reduced but did not completely abolish the chitin-binding activity, while four-sites-mutated protein MF completely lost the chitin-binding activity. These data indicate that BmWCP4 protein plays a critical role by binding to the chitin filaments in the wing during larva-to-pupa transformation. The conserved aromatic amino acids are critical in the interaction between chitin and the cuticle protein.
文摘Serine proteases play important roles in digestion and immune responses during insect development. In the present study, the serine protease gene BmSP36, which encodes a 292-residue protein, was cloned from the midgut cells ofBombyx mori. BmSP36 contains an intact catalytic triad （H57, D102 and S195） and a conserved substrate-binding site （G189, H216 and G226）, suggesting that it is a serine protease with chymotrypsin- like specificity. The temporal and spatial expression patterns of BmSP36 indicated that its messenger RNA and protein expression mainly occurred in the midgut at the feeding stages. Western blotting, immunofluorescence and liquid chromatography-tandem mass spectrometry analyses revealed secretion of BmSP36 protein from epithelial cells into the midgut lumen. The transcriptional and translational expression of BmSP36 was down- regulated after starvation but up-regulated after refeeding. Moreover, expression of the BmSP36 gene could be up-regulated by a juvenile hormone analogue. These results enable us to better define the potential role of BmSP36 in dietary protein digestion at the feeding stages during larval development.
基金Acknowledgments This project was supported by the National Basic Research （973） Program （grant no. 2005CB121000）, Natural Science Foundation of Jiangsu Province （grant no. BK2006508）, Hi-tech Research and Development Program （863） of China （2006AA10A119）.
文摘The yellow color of the cocoon of the silkworm Bombyx mori is controlled by three genes, Y （Yellow haemolymph）, 1 （Yellow inhibitor） and C （ Outer-layer yellow cocoon）, which are located on linkage groups 2, 9 and 12, respectively. Taking advantage of a lack of crossing over in females, reciprocal backcrossed F1 （BC1） progeny were used for linkage analysis and mapping of the C gene using silkworm strains C 108 and KY, which spin white and yellow cocoons, respectively. DNA was extracted from individual pupae and analyzed for simple sequence repeat （SSR） markers. The C gene was found to be linked to seven SSR markers. All the yellow cocoon individuals from a female heterozygous backcross （BC1F） showed a heterozygous profile for SSR markers on linkage group 12, whereas individuals with light yellow cocoons showed the homozygous profile of the strain C108. Using a reciprocal heterozygous male backcross （BC1M）, we constructed a linkage map of 36.4 cM with the C gene located at the distal end, and the closest SSR marker at a distance of 13.9 cM.
基金supported by the National Natural Science Foundation of China(No.39870603)the Special Funding Program of the Ministry of Education for Ph.D.Degree Awarding Units.
文摘Molecular systematic studies on mandarina silkworm (Bombyx mandarina M.) in 11 regions in China and 25 representative strains of domestic silkworm (Bombyx mori L.) were conducted using molecular biology techniques. Results obtained from the analysis of DNA polymorphism and clustering of all the silkworm samples provide new evidence for the view that the domestic silkworm originated from the Chinese mandarina silkworm. On the basis of literature reviewing, a new hypothesis on the origin of the domestic silkworm was put forward. It was thought that the domestic silkworm was most probably domesticated from the Chinese mandarina silkworm of different ecotypes including monovoltinism, bivoltinism and multivoltinism; and that the domestic silkworm had the genetic background of monovoltinism, bivoltinism and multivoltinism at the very beginning of the domestication. The current strains of the domestic silkworm of different voltinism are the evolutionary results of thousands of years of rearing and artificial selections.
文摘We report the thermotolerance of new bivoltine silkworm, Bombyx mori strains NB4D2, KSO1, NP2, CSR2 and CSR4 and differential expression of heat shock proteins at different instars. Different instars of silkworm larva were subjected to heat shock at 35℃, 40℃ and 45℃ for 2 hours followed by 2 hours recovery. Heat shock proteins were analyzed by SDS-PAGE. The impact of heat shock on commercial traits of cocoons was analyzed by following different strategies in terms of acquired thermotolerance over control. Comparatively NP2 exhibited better survivability than other strains. Resistance to heat shock was increased as larval development proceeds in the order of first instar 〉 second instar 〉 third instar 〉 fourth instar 〉 fifth instar in all silkworm strains. Expression of heat shock proteins varies in different instars. 90 kDa in the first, second and third instars, 84 kDa in the fourth instar and 84, 62, 60, 47 and 33 kDa heat shock proteins in fifth instar was observed in response to heat shock. Relative influence of heat shock on commercial traits that correspond to different stages was significant in all strains. In NB4D2, cocoon and shell weight significantly increased to 17.52% and 19.44% over control respectively. Heat shock proteins as molecular markers for evaluation and evolution of thermotolerant silkworm strains for tropics was discussed.
文摘In silkworms, the white egg 1 （w-1） mutant, which is characterized by white eyes and white eggs, is deficient in Bombyx kynurenine 3-monooxygenase （KMO） activity. To investigate whether the w-1 mutant phenotype is rescued by introducing the wild-type KMO gene, we constructed transgenic silkworms with the wild-type Bombyx KMO gene under the control of either the cytoplasmic actin gene promoter （A3KMO） or the native KMO gene promoter （KKMO）. We created two transgenic lines with A3KMO and one line with KKMO constructs. The eyes of adults in these lines were brown, and the eggs laid by the transgenic females were also brown. Reverse transcription-polymerase chain reaction（RT-PCR） analysis showed that the A3KMO silkworm lines expressed the transcript in the mid-gut, fat bodies, and Malpighian tubules. The KKMO line expressed the transcript only in the fat bodies and Malpighian tubules. The intensity of eye and egg color in the transgenic lines was proportional to the KMO expression level. Interestingly, transgenic larvae with the A3KMO construct had a light brown larval cuticle, but the KKMO line did not. These results indicate that the wild-type KMO gene can be used as a marker gene for visually screening transgenic silkworms.
文摘The embryonic diapause of the silkworm, Bombyx mori, is induced by the diapause hormone (DH) which is secreted from the suboesophageal ganglion of pupae. The diapause nature of bivoltine strains uses environmental stimuli as the initial signal to determine the diapause nature. The experiments showed that DH gene expression is a direct response to the environmental stimulus, such as high incubation temperature. The cDNA from the embryonic stage was cloned and sequence analysis showed the cDNA encoding DH. Expression patterns of the DH gene in embryonic stage are different at incubation temperatures 15℃ and 25℃, suggesting that the incubation temperature as an environmental signal is kept within the body to control the DH gene expression at the pupal stage, so that the embryonic diapause of next generation can be determined.
文摘Cathepsin B belongs to lysosomal cysteine protease of the papain family. Temporal and spatial expression analysis of cathepsin B of Bombyx mori （BmCtB） was carried out based on Expression Sequence Tags （ESTs） data, oligonucleotide microarray, reverse transcription polymerase chain reaction （RT-PCR） and quantitative real-time PCR. Expression of BmCtB was observed in all of the tissues and stages. Among the 10 tested tissues, the fat body and posterior silk gland are the two most enriched tissues with BmCtB. During Bombyx development, there was an expression fastigium of BmCtB during metamorphosis. RNA interference was used to suppress the expression of cathepsin B during metamorphosis. Significant developmental defective phenotypes were obtained in the RNAi treated group. The dramatically reduced expression of BmCtB was con^rmed by Northern blot and quantitative real-time PCR. These evidences strongly suggest cathepsin B proteinase was predominantly involved in the metabolism process of fat body and the posterior silk gland and was critical for metamorphism and development of silkworm, Bombyx mori.
