The genomic structures of Oryza sativa (A genome) and O. meyeriana (G genome) were comparatively studied using bicolor genomic in situ hybridization (GISH). GISH was clearly able to discriminate between the chro...The genomic structures of Oryza sativa (A genome) and O. meyeriana (G genome) were comparatively studied using bicolor genomic in situ hybridization (GISH). GISH was clearly able to discriminate between the chromosomes of O. sativa and O. meyeriana in the interspecific F1 hybrids without blocking DNA, and co-hybridization was hardly detected. The average mitotic chromosome length of O. meyeriana was found to be 1.69 times that of O. sativa. A comparison of 4,6-diamidino-2-phenylindole staining showed that the chromosomes of O. meyeriana were more extensively labelled, suggesting that the G genome is amplified with more repetitive sequences than the A genome. In interphase nuclei, 9-12 chromocenters were normally detected and nearly all the chromocenters constituted the G genome-specific DNA. More and larger chromocenters formed by chromatin compaction corresponding to the G genome were detected in the hybrid compared with its parents. During pachytene of the F1 hybrid, most chromosomes of A and G did not synapse each other except for 1-2 chromosomes paired at the end of their arms. At meiotic metaphase I, three types of chromosomal associations, i.e.O, sativa-O, sativa (A-A), O. sativa-O, meyeriana (A-G) and O. meyeriana-O, meyeriana (G-G), were observed in the F1 hybrid. The A-G chromosome pairing configurations included bivalents and trivalents. The results provided a foundation toward studying genome organization and evolution of O. meyeriana.展开更多
Genes encoding Wnt ligands, which have important roles in cell communication and organ development, are restricted to multicellular animals. We systematically studied W nt genes from eumetazoan genomes, with emphasis ...Genes encoding Wnt ligands, which have important roles in cell communication and organ development, are restricted to multicellular animals. We systematically studied W nt genes from eumetazoan genomes, with emphasis on the poorly studied superphylum Lophotrochozoa(four annelids, seven mollusks, eight platyhelminths, one bdelloid rotifer, and one brachiopod species). Between 3 and 39 W nt loci were identified in each genome, and the protostome-specific loss of Wnt3 genes was confirmed. We identified gastropod-specific loss of Wnt8, refining the previously proposed mollusk-specific loss. Some duplicated Wnt genes belonging to a same subfamily or closely related subfamilies showed tandem distribution in the lophotrochozoan genomes, indicating tandem duplication events during Wnt family evolution. Members of the conserved Wnt10-Wnt6-Wnt1-Wnt9 cluster showed highly correlated expression patterns over time in two assayed lophotrochozoans, the oyster C rassostrea gigas and the brachiopod L ingula anatina, reflecting the possible similar function of the clustered W nt genes.展开更多
Transgenic animal technology has been one of the fastest growing biotechnology areas. The exogenous genes have been introduced into the animal genome by genetic engineering, so that these genes can be inherited and ex...Transgenic animal technology has been one of the fastest growing biotechnology areas. The exogenous genes have been introduced into the animal genome by genetic engineering, so that these genes can be inherited and expressed by offspring to produce desired traits or evaluate function in elite livestock breeds. There are several methodologies for the production of transgenic animals, i.e., (1) microinjection of genes into pronuclei of fertilized ova; (2) DNA transfer by retroviruses; (3) injection of embryonic germ (EG)/embryonic stem (ES) cells previously treated with foreign DNA; (4) DNA transfer into cells and embryos with using liposomes; (5) exogenous DNA transfer while in vitro fertilization by using sperm; (6) electroporation of DNA into sperm, embryos or ova; (7) biolistics; (8) nuclear transfer (NT) with somatic cells, EG or ES cells; (9) germ line stem cell-mediated; (10) gene targeting; (! 1) gene silencing technology with RNA interference; (12) induced pluripotent stem cell; (13) zinc-finger nuclease gene targeting technology. Gene farming is one of the newest and most promising areas in modern biotechnology. Cattle, goats, sheep, pigs and rabbits are the main farm livestock species and fish is also used in transgenic technology. The question of "why make transgenic animals?" is very important. Some of the answers to this question are: (1) to obtain new knowledge; (2) to solve the genetic code; (3) to create genetic disease models; (4) to study the genetic control of physiological systems; (5) to improve animal production traits; (6) to produce new animal products. Transgenic technology is one of the main and important tools in the finding solutions to problems of growing population with their applications to different organisms, and takes more attention and interest every day. Transgenic technology creates opportunities and areas to play with organisms to fulfill the demands of people. Because of this, this paper based on mainly transgenic applications to take people's attention and exhibit its importance.展开更多
Pichia pastoris expression system is similar to that of the mammal cell in modification of expressed protein, including refolding and glycosylation. A human aFGF gene was cloned into the intracellular expression vecto...Pichia pastoris expression system is similar to that of the mammal cell in modification of expressed protein, including refolding and glycosylation. A human aFGF gene was cloned into the intracellular expression vector pPIC9K. The Pichia pastoris KM71 strain was transformed with the recombined expression plasmid. Transgenic expression was observed after screening the transformants with G418. The expression and secretion of recombinant human aFGF (rhaFGF) into the culture medium were testified by ELISA assay. The yield peaked after two days of induction and was approximately 10 mgL-1 in shake-flask fermentation medium. The recombinant proteins were purified by the combination of heparin-Sepharose affinity chromatography and gel filtration chromatography. Two proteins with relative molecular masses (Mr) of 17 000 and 35 000 were purified as a single band in SDS-PAGE, whose biological activities were determined by MTT assay. It is found that the protein with Mr of 17 000 is nonglycosylated haFGF, and that with Mr of 35 000 is glycosylated haFGF; and the latter has a lower biological activity than the former.展开更多
To obtain the recombinant soluble protein of the extracellular fragment of human TRAIL gene and to identify its function preliminarily, this gene fragment was amplified from peripheral blood mononuclear cells (PBMC)...To obtain the recombinant soluble protein of the extracellular fragment of human TRAIL gene and to identify its function preliminarily, this gene fragment was amplified from peripheral blood mononuclear cells (PBMC) by RT-PCR and cloned into vector pGEM-T-Easy for sequence analysis. The expression vector pET-30a/TRAIL was then constructed by DNA recombination method with a His-tag gene at the front of the TRAIL fragment, and the recombinant protein was expressed in E. coli BL21 (DE3). Meanwhile, the expressed target protein was purified with Ni-NTA chromatography column and identified by SDS-PAGE and Western blotting. The proliferation inhibition activity of TRAIL-His was detected by MTF assay. PI staining and Wright-Giemsa staining were used to detect the presence of the TRAIL-induced cell apoptosis. It was demonstrated that the target protein expressed in E. coli BL21 showed the same relative molecular mass as that the protein expected and could be recognized by both the anti-TRAIL polyclonal antibody and anti-His monoclonal antibody. In addition, this protein could also inhibit proliferation of human lymphoma cell line Jurkat cells or induce apoptosis of this cell line. It is apparent that a recombinant soluble TRAIL protein with biological activity is obtained and this prospective study can lay solid foundation for further research on the biological activity and application in the anti-tumor therapy.展开更多
Objective To investigate the significance of interleukin-13 (IL-13) in patients with active lupus nephritis (LN).Methods Ten healthy volunteers and 16 patients with active LN were included in this study. The protein l...Objective To investigate the significance of interleukin-13 (IL-13) in patients with active lupus nephritis (LN).Methods Ten healthy volunteers and 16 patients with active LN were included in this study. The protein level of IL-13 in plasma was examined by enzyme linked immunosorbent assay (ELISA), and gene expression of IL-13 in peripheral blood mononuclear cells (PBMCs) by reverse transcription polymerase chain reaction (RT-PCR). Expression of IL-13 mRNA in renal tissue was studied by in situ hybridization (ISH) techniques.Results The level of IL-13 in plasma and the expression of IL-13 mRNA in PBMCs were significantly higher in LN patients than those in the controls ( P < 0.001 ). Increased expression of IL-13 mRNA was detected in renal tissue of active LN patients compared to those in the controls ( P < 0.001 ). Analysis of the linear correlation indicated that the level of IL-13 mRNA in the tubulointerstitial area in patients with active LN correlated with the concentration of serum creatinine (Scr), the glomerular activity index (GAl),the activity index of tubulointerstitium, and the level of serum C3 ( P < 0.05 for each).Conclusion The elevation of IL-13 may play an important role inthe molecular pathogenesis of active LN.展开更多
The progression of next generation sequencing is continuously changing the landscape of genomic, tran- scriptomic, and epigenomic studies. Particularly, advances in single cell manipulation and amplification technique...The progression of next generation sequencing is continuously changing the landscape of genomic, tran- scriptomic, and epigenomic studies. Particularly, advances in single cell manipulation and amplification techniques bring sequencing technology to the single-cell level. Single cell genome sequencing allows us to study tumor evolu- tion, gamete genesis, somatic mosaicism at genome-wide level; single cell transcriptome sequencing unveils the dynamic gene expression during early embryonic devel- opment, differentiation and reprogramming; single cell methylome sequencing is just taking off and shows great potential in cancer and stem cell studies. Lots of attempts are still being made in other dimensions of sequencing. The increasing need for single cell sequencing requires the future techniques with the following features: (1) high accuracy and fidelity; (2) able to perform multiple omics analyses in one cell; (3) high degree of automation and standardized pipeline. These progresses and improvements will lower the barrier for single cell sequencing to enter ordinary laboratories. The wide application of single cell sequencing techniques will substantially change biomedi- cal research in future.展开更多
文摘The genomic structures of Oryza sativa (A genome) and O. meyeriana (G genome) were comparatively studied using bicolor genomic in situ hybridization (GISH). GISH was clearly able to discriminate between the chromosomes of O. sativa and O. meyeriana in the interspecific F1 hybrids without blocking DNA, and co-hybridization was hardly detected. The average mitotic chromosome length of O. meyeriana was found to be 1.69 times that of O. sativa. A comparison of 4,6-diamidino-2-phenylindole staining showed that the chromosomes of O. meyeriana were more extensively labelled, suggesting that the G genome is amplified with more repetitive sequences than the A genome. In interphase nuclei, 9-12 chromocenters were normally detected and nearly all the chromocenters constituted the G genome-specific DNA. More and larger chromocenters formed by chromatin compaction corresponding to the G genome were detected in the hybrid compared with its parents. During pachytene of the F1 hybrid, most chromosomes of A and G did not synapse each other except for 1-2 chromosomes paired at the end of their arms. At meiotic metaphase I, three types of chromosomal associations, i.e.O, sativa-O, sativa (A-A), O. sativa-O, meyeriana (A-G) and O. meyeriana-O, meyeriana (G-G), were observed in the F1 hybrid. The A-G chromosome pairing configurations included bivalents and trivalents. The results provided a foundation toward studying genome organization and evolution of O. meyeriana.
基金Supported by the National Natural Science Foundation of China(Nos.31402285,31530079)the Scientific and Technological Innovation Project financially supported by the Qingdao National Laboratory for Marine Science and Technology(No.2015ASKJ02)the earmarked fund for Modern Agro-Industry Technology Research System(No.CARS-48)
文摘Genes encoding Wnt ligands, which have important roles in cell communication and organ development, are restricted to multicellular animals. We systematically studied W nt genes from eumetazoan genomes, with emphasis on the poorly studied superphylum Lophotrochozoa(four annelids, seven mollusks, eight platyhelminths, one bdelloid rotifer, and one brachiopod species). Between 3 and 39 W nt loci were identified in each genome, and the protostome-specific loss of Wnt3 genes was confirmed. We identified gastropod-specific loss of Wnt8, refining the previously proposed mollusk-specific loss. Some duplicated Wnt genes belonging to a same subfamily or closely related subfamilies showed tandem distribution in the lophotrochozoan genomes, indicating tandem duplication events during Wnt family evolution. Members of the conserved Wnt10-Wnt6-Wnt1-Wnt9 cluster showed highly correlated expression patterns over time in two assayed lophotrochozoans, the oyster C rassostrea gigas and the brachiopod L ingula anatina, reflecting the possible similar function of the clustered W nt genes.
文摘Transgenic animal technology has been one of the fastest growing biotechnology areas. The exogenous genes have been introduced into the animal genome by genetic engineering, so that these genes can be inherited and expressed by offspring to produce desired traits or evaluate function in elite livestock breeds. There are several methodologies for the production of transgenic animals, i.e., (1) microinjection of genes into pronuclei of fertilized ova; (2) DNA transfer by retroviruses; (3) injection of embryonic germ (EG)/embryonic stem (ES) cells previously treated with foreign DNA; (4) DNA transfer into cells and embryos with using liposomes; (5) exogenous DNA transfer while in vitro fertilization by using sperm; (6) electroporation of DNA into sperm, embryos or ova; (7) biolistics; (8) nuclear transfer (NT) with somatic cells, EG or ES cells; (9) germ line stem cell-mediated; (10) gene targeting; (! 1) gene silencing technology with RNA interference; (12) induced pluripotent stem cell; (13) zinc-finger nuclease gene targeting technology. Gene farming is one of the newest and most promising areas in modern biotechnology. Cattle, goats, sheep, pigs and rabbits are the main farm livestock species and fish is also used in transgenic technology. The question of "why make transgenic animals?" is very important. Some of the answers to this question are: (1) to obtain new knowledge; (2) to solve the genetic code; (3) to create genetic disease models; (4) to study the genetic control of physiological systems; (5) to improve animal production traits; (6) to produce new animal products. Transgenic technology is one of the main and important tools in the finding solutions to problems of growing population with their applications to different organisms, and takes more attention and interest every day. Transgenic technology creates opportunities and areas to play with organisms to fulfill the demands of people. Because of this, this paper based on mainly transgenic applications to take people's attention and exhibit its importance.
基金the National Natural Science Foundation of China (No.19732003)
文摘Pichia pastoris expression system is similar to that of the mammal cell in modification of expressed protein, including refolding and glycosylation. A human aFGF gene was cloned into the intracellular expression vector pPIC9K. The Pichia pastoris KM71 strain was transformed with the recombined expression plasmid. Transgenic expression was observed after screening the transformants with G418. The expression and secretion of recombinant human aFGF (rhaFGF) into the culture medium were testified by ELISA assay. The yield peaked after two days of induction and was approximately 10 mgL-1 in shake-flask fermentation medium. The recombinant proteins were purified by the combination of heparin-Sepharose affinity chromatography and gel filtration chromatography. Two proteins with relative molecular masses (Mr) of 17 000 and 35 000 were purified as a single band in SDS-PAGE, whose biological activities were determined by MTT assay. It is found that the protein with Mr of 17 000 is nonglycosylated haFGF, and that with Mr of 35 000 is glycosylated haFGF; and the latter has a lower biological activity than the former.
文摘To obtain the recombinant soluble protein of the extracellular fragment of human TRAIL gene and to identify its function preliminarily, this gene fragment was amplified from peripheral blood mononuclear cells (PBMC) by RT-PCR and cloned into vector pGEM-T-Easy for sequence analysis. The expression vector pET-30a/TRAIL was then constructed by DNA recombination method with a His-tag gene at the front of the TRAIL fragment, and the recombinant protein was expressed in E. coli BL21 (DE3). Meanwhile, the expressed target protein was purified with Ni-NTA chromatography column and identified by SDS-PAGE and Western blotting. The proliferation inhibition activity of TRAIL-His was detected by MTF assay. PI staining and Wright-Giemsa staining were used to detect the presence of the TRAIL-induced cell apoptosis. It was demonstrated that the target protein expressed in E. coli BL21 showed the same relative molecular mass as that the protein expected and could be recognized by both the anti-TRAIL polyclonal antibody and anti-His monoclonal antibody. In addition, this protein could also inhibit proliferation of human lymphoma cell line Jurkat cells or induce apoptosis of this cell line. It is apparent that a recombinant soluble TRAIL protein with biological activity is obtained and this prospective study can lay solid foundation for further research on the biological activity and application in the anti-tumor therapy.
基金ThisstudywassupportedbytheFoundationofScientificResearchfromtheScienceCommitteeofGuangdong (No 1995 5 6 )
文摘Objective To investigate the significance of interleukin-13 (IL-13) in patients with active lupus nephritis (LN).Methods Ten healthy volunteers and 16 patients with active LN were included in this study. The protein level of IL-13 in plasma was examined by enzyme linked immunosorbent assay (ELISA), and gene expression of IL-13 in peripheral blood mononuclear cells (PBMCs) by reverse transcription polymerase chain reaction (RT-PCR). Expression of IL-13 mRNA in renal tissue was studied by in situ hybridization (ISH) techniques.Results The level of IL-13 in plasma and the expression of IL-13 mRNA in PBMCs were significantly higher in LN patients than those in the controls ( P < 0.001 ). Increased expression of IL-13 mRNA was detected in renal tissue of active LN patients compared to those in the controls ( P < 0.001 ). Analysis of the linear correlation indicated that the level of IL-13 mRNA in the tubulointerstitial area in patients with active LN correlated with the concentration of serum creatinine (Scr), the glomerular activity index (GAl),the activity index of tubulointerstitium, and the level of serum C3 ( P < 0.05 for each).Conclusion The elevation of IL-13 may play an important role inthe molecular pathogenesis of active LN.
基金supported by the Recruitment Program of Global Youth Experts to Fan Bai
文摘The progression of next generation sequencing is continuously changing the landscape of genomic, tran- scriptomic, and epigenomic studies. Particularly, advances in single cell manipulation and amplification techniques bring sequencing technology to the single-cell level. Single cell genome sequencing allows us to study tumor evolu- tion, gamete genesis, somatic mosaicism at genome-wide level; single cell transcriptome sequencing unveils the dynamic gene expression during early embryonic devel- opment, differentiation and reprogramming; single cell methylome sequencing is just taking off and shows great potential in cancer and stem cell studies. Lots of attempts are still being made in other dimensions of sequencing. The increasing need for single cell sequencing requires the future techniques with the following features: (1) high accuracy and fidelity; (2) able to perform multiple omics analyses in one cell; (3) high degree of automation and standardized pipeline. These progresses and improvements will lower the barrier for single cell sequencing to enter ordinary laboratories. The wide application of single cell sequencing techniques will substantially change biomedi- cal research in future.