The sericulture industry plays a very important role in our national economy. Silkworm (Bombyx mori) is always regarded as a model animal and biological reactor. There have been detailed studies on the structure, expr...The sericulture industry plays a very important role in our national economy. Silkworm (Bombyx mori) is always regarded as a model animal and biological reactor. There have been detailed studies on the structure, expression and control and molecular evolution of silk genes. However, few, if any, reports are available on the localization of structural genes in silkworm by molecular cytogenetics. The present experiment has tentatively localized the Fib-H gene at the distal end of the 25th linkage group, namely at the 25-0.0 position, and verified that Fib-H has only one locus, thus providing a temporary solution to the problem about its localization.展开更多
To provide materials used in investigating the relationship between amino acid compositions of silk-like protein, structure, and functions, especially the biological functions, the motif genes encoding the silk fibroi...To provide materials used in investigating the relationship between amino acid compositions of silk-like protein, structure, and functions, especially the biological functions, the motif genes encoding the silk fibroin amorphous domain, SGFGPVANGGSGEASSESDFGSSGFGPVANASSGEASSESDFAG(F) were designed and extended using a "head-to-tail" construction strategy. The designed genes were cloned into PSLFA1180FA and multimerized to form structures containing a two-timer, a four-timer, an eight-timer, and a twelve-timer. All the resulting plasmids were digested using the restriction enzyme BamHI and the double-enzymes BglII/HindIII. Restriction enzyme analysis and DNA sequencing revealed the motif was successfully cloned into PSLFA1180FA and multimerized to form a twelve-timer without gene deletion or mutation.展开更多
The resorption of the transplanted fat over time limited the use of autologous fat for the reconstruction of soft tissue defect. Tissue engineering (TE) adipose with silk fibroin scaffold could be a promising substitu...The resorption of the transplanted fat over time limited the use of autologous fat for the reconstruction of soft tissue defect. Tissue engineering (TE) adipose with silk fibroin scaffold could be a promising substitute for soft tissue filling. In this study, we try to develop a tissue engineering adipose in vitro by seeding silk fibroin scaffold with human umbilical cord mesenchymal stem cells (hUCMSCs) after transfected with recombinant human insulin gene lentivirus. Our aim was to observe the effects of the insulin gene transfection on the adipogenesis of hUCMSCs when cultured with silk fibroin scaffolds. The hUCMSCs infected with recombinant lentiviral pLenti6.3-insulin-IRES-EGFP were seeded on silk fibroin scaffolds and cultured in adipogenic differentiation medium for 5 - 7 days. The expression of adipogenic gene PPARγ-2 was tested by RT-PCR after 7 days culture of adipogenic induction. The accumulation of cytoplasmic droplets of neutral lipids was assessed by Oil Red O staining. The RNA and protein expression of transfected insulin gene in hUCMSCs were detected by QPCR and western blot. The effect of recombinant lentivirus transfection on the growth and proliferation of hUCMSCs was observed by MTT test. We observed that the 2-ΔΔCt value of insulin gene expression of hUCMSCs in the transfected group was 300.25 times higher than that in the untransfected group. The western blot showed that a positive band was discerned at the site of a relative molecular mass of 8 × 103 Dalton in transfected group. After adipogenic culture for 7 days, under the fluorescent inverted phase-contrast microscope, after Oil Red O staining, a lot of adipocytes appeared in silk fibroin scaffold;round adipose droplets showed intracellularly;the size of the adipocytes was not homogenous, and the density of adipocytes in transfected group was significantly higher than that in untransfected group (P = 0.007, P < 0.01). RT-PCR results showed that the expression of adipogenic gene PPARγ-2 in transfected group was much stronger than t展开更多
Antheraea yamamai (Japanese oak silkworm) is a kind of silkworm of great economic value. and the process of the expression of its silkprotem genes is a perfect model for the study of molecular regulation during the de...Antheraea yamamai (Japanese oak silkworm) is a kind of silkworm of great economic value. and the process of the expression of its silkprotem genes is a perfect model for the study of molecular regulation during the development and differentiation. So studying its fibroin and allied genes is of both theoretic and practical magnitude A YAC library with an average size of 570kb is constructed from the posterior silk gland, using pYAC4 as a vector. The library was screened by means of polymerase chain reaction, and clones representing fibroin gene were isolated and characterized.展开更多
Lepidopteran insects produce cocoons with unique properties.The cocoons are made of silk produced in the larval tissue silk gland and our understanding of the silk genes is still very limited.Here,we investigated silk...Lepidopteran insects produce cocoons with unique properties.The cocoons are made of silk produced in the larval tissue silk gland and our understanding of the silk genes is still very limited.Here,we investigated silk genes in the bagworm moth Eumeta variegata,a species that has recently been found to produce extraordinarily strong and tough silk.Using short-read transcriptomic analysis,we identified a partial sequence of the fibroin heavy chain gene and its product was found to have a C-terminal structure that is conserved within nonsaturniid species.This is in accordance with the presence of fibroin light chain/fibrohexamerin genes and it is suggested that the bagworm moth is producing silk composed of fibroin ternary complex.This indicates that the fibroin structure has been evolutionarily conserved longer than previously thought.Other than fibroins we identified candidates for sericin genes,expressed strongly in the middle region of the silk gland and encoding serine-rich proteins,and other silk genes,that are structurally conserved with other lepidopteran homologues.The bagworm moth is thus considered to be producing conventional lepidopteran type of silk.We further found a number of genes expressed in a specific region of the silk gland and some genes showed conserved expression with Bombyx mori counterparts.This is the first study allowing comprehensive silk gene identification and expression analysis in the lepidopteran Psychidae family and should contribute to the understanding of silk gene evolution as well as to the development of novel types of silk.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
基金This work was supported by the Key Project from National Natural Science Foundation of China (Grant No. 39730730) General Program from National Natural Science Foundation of China (Grant No. 39770574) Foundation for University Key Teacher by the
文摘The sericulture industry plays a very important role in our national economy. Silkworm (Bombyx mori) is always regarded as a model animal and biological reactor. There have been detailed studies on the structure, expression and control and molecular evolution of silk genes. However, few, if any, reports are available on the localization of structural genes in silkworm by molecular cytogenetics. The present experiment has tentatively localized the Fib-H gene at the distal end of the 25th linkage group, namely at the 25-0.0 position, and verified that Fib-H has only one locus, thus providing a temporary solution to the problem about its localization.
基金National Natural Science Foundation of China(No. 51173125)Natural Science Foundations of Jiangsu Province of China(No. BK2010253,No. BK2012633)+2 种基金College Natural Science Research Project of Jiangsu Province of China(No. 12KJA43004)Science and Technology Plan Foundation of Suzhou of China(No. ZXS2012002)Priority Academic Program Development of Jiangsu Higher Education Institutions,China
文摘To provide materials used in investigating the relationship between amino acid compositions of silk-like protein, structure, and functions, especially the biological functions, the motif genes encoding the silk fibroin amorphous domain, SGFGPVANGGSGEASSESDFGSSGFGPVANASSGEASSESDFAG(F) were designed and extended using a "head-to-tail" construction strategy. The designed genes were cloned into PSLFA1180FA and multimerized to form structures containing a two-timer, a four-timer, an eight-timer, and a twelve-timer. All the resulting plasmids were digested using the restriction enzyme BamHI and the double-enzymes BglII/HindIII. Restriction enzyme analysis and DNA sequencing revealed the motif was successfully cloned into PSLFA1180FA and multimerized to form a twelve-timer without gene deletion or mutation.
文摘The resorption of the transplanted fat over time limited the use of autologous fat for the reconstruction of soft tissue defect. Tissue engineering (TE) adipose with silk fibroin scaffold could be a promising substitute for soft tissue filling. In this study, we try to develop a tissue engineering adipose in vitro by seeding silk fibroin scaffold with human umbilical cord mesenchymal stem cells (hUCMSCs) after transfected with recombinant human insulin gene lentivirus. Our aim was to observe the effects of the insulin gene transfection on the adipogenesis of hUCMSCs when cultured with silk fibroin scaffolds. The hUCMSCs infected with recombinant lentiviral pLenti6.3-insulin-IRES-EGFP were seeded on silk fibroin scaffolds and cultured in adipogenic differentiation medium for 5 - 7 days. The expression of adipogenic gene PPARγ-2 was tested by RT-PCR after 7 days culture of adipogenic induction. The accumulation of cytoplasmic droplets of neutral lipids was assessed by Oil Red O staining. The RNA and protein expression of transfected insulin gene in hUCMSCs were detected by QPCR and western blot. The effect of recombinant lentivirus transfection on the growth and proliferation of hUCMSCs was observed by MTT test. We observed that the 2-ΔΔCt value of insulin gene expression of hUCMSCs in the transfected group was 300.25 times higher than that in the untransfected group. The western blot showed that a positive band was discerned at the site of a relative molecular mass of 8 × 103 Dalton in transfected group. After adipogenic culture for 7 days, under the fluorescent inverted phase-contrast microscope, after Oil Red O staining, a lot of adipocytes appeared in silk fibroin scaffold;round adipose droplets showed intracellularly;the size of the adipocytes was not homogenous, and the density of adipocytes in transfected group was significantly higher than that in untransfected group (P = 0.007, P < 0.01). RT-PCR results showed that the expression of adipogenic gene PPARγ-2 in transfected group was much stronger than t
文摘Antheraea yamamai (Japanese oak silkworm) is a kind of silkworm of great economic value. and the process of the expression of its silkprotem genes is a perfect model for the study of molecular regulation during the development and differentiation. So studying its fibroin and allied genes is of both theoretic and practical magnitude A YAC library with an average size of 570kb is constructed from the posterior silk gland, using pYAC4 as a vector. The library was screened by means of polymerase chain reaction, and clones representing fibroin gene were isolated and characterized.
基金This work was supported by grants-in-aid from the JST/JICA,SATREPS(Science and Technology Research Partnership for Sustainable Devel Devel opment)and Ministry of Agriculture,Forestry,and Fisheries,Japan.
文摘Lepidopteran insects produce cocoons with unique properties.The cocoons are made of silk produced in the larval tissue silk gland and our understanding of the silk genes is still very limited.Here,we investigated silk genes in the bagworm moth Eumeta variegata,a species that has recently been found to produce extraordinarily strong and tough silk.Using short-read transcriptomic analysis,we identified a partial sequence of the fibroin heavy chain gene and its product was found to have a C-terminal structure that is conserved within nonsaturniid species.This is in accordance with the presence of fibroin light chain/fibrohexamerin genes and it is suggested that the bagworm moth is producing silk composed of fibroin ternary complex.This indicates that the fibroin structure has been evolutionarily conserved longer than previously thought.Other than fibroins we identified candidates for sericin genes,expressed strongly in the middle region of the silk gland and encoding serine-rich proteins,and other silk genes,that are structurally conserved with other lepidopteran homologues.The bagworm moth is thus considered to be producing conventional lepidopteran type of silk.We further found a number of genes expressed in a specific region of the silk gland and some genes showed conserved expression with Bombyx mori counterparts.This is the first study allowing comprehensive silk gene identification and expression analysis in the lepidopteran Psychidae family and should contribute to the understanding of silk gene evolution as well as to the development of novel types of silk.
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
