Previous studies on mammals showed that peroxisome proliferator-activated receptor gamma coactivator-1α(PGC-1α)played a prominent role in regulating muscle fiber type transition and composition.However,the role of P...Previous studies on mammals showed that peroxisome proliferator-activated receptor gamma coactivator-1α(PGC-1α)played a prominent role in regulating muscle fiber type transition and composition.However,the role of PGC-1αin chicken muscle has seldom been explored.To investigate the effect of PGC-1αon chicken skeletal muscles in this study,the PGC-1αgene was overexpressed or silenced in chicken primary myoblasts by using lentivirus,and then the effects of the PGC-1αgene overexpression and knockdown on the mRNA expression profile of genes related to myofiber type specificity were examined during fiber formation.The results showed that overexpression of PGC-1αfrom proliferation to differentiation was accompanied by the up-regulated expression of Pax7,MyoD,and CnAα,which was significantly(P<0.01)increased after one day of transfection(1 I).The enhancement of MyoG,MEF2 c,and MyHC SM expression lagged,which was improved significantly(P<0.01)after four days of transfection(1 I3 D).Overexpression of PGC-1αdecreased(P<0.01)the MyHC FWM expression after four days of transfection(1 I3 D),and it had no significant impact(P>0.05)on the expression of CnB1,NFATc3,and MyHC FRM during myofiber formation.The effective silence(P<0.01)of PGC-1αby lentivirus mediating short hairpin RNA(shRNA)was detected after four days of transfection(1 I3 D)in cultures,and the lack of its function in chicken primary myoblasts significantly(P<0.01)down-regulated the expression of Pax7,MyoD,CnAα,MyoG,MEF2 c,and MyHC SM,significantly(P<0.01)up-regulated the expression of MyHC FWM,and had no significant impact(P>0.05)on the expression of CnB1,NFATc3,and MyHC FRM.These results indicated that the role of PGC-1αin regulating the fiber type specificity of chicken skeletal muscles might be similar to that in mammals,which interplayed with key genes related to myocyte differentiation and calcineurin signaling pathway.展开更多
The B cells translocation gene 1 （BTG1） is a member of the BTG/TOB family of anti-proliferative genes, which have recently emerged as important regulators of cell growth and differentiation among vertebrates. Here, ...The B cells translocation gene 1 （BTG1） is a member of the BTG/TOB family of anti-proliferative genes, which have recently emerged as important regulators of cell growth and differentiation among vertebrates. Here, for the first time we cloned the full-length eDNA sequence of Hyriopsis schlegelii （Hs-BTG1）, an economically important freshwater shellfish and potential indicator of environmental heavy metal pollution, for the first time. Using rapid amplification of eDNA ends （RACE） together with splicing the EST sequence from a haemocyte eDNA library, we found that Hs-BTG1 contains a 525 bp open reading frame （ORF） encoding a 174 amino-acid polypeptide, a 306 bp 5＇ untranslated region （5＇ UTR）, and a 571 bp 3＇ UTR with a Poly（A） tail as well as a transcription termination signal （AATAAA）. Homologne searching against GenBank revealed that Hs-BTG1 was closest to Crassostrea gigas BTG1, sharing 50.57% of protein identities. Hs-BTG1 also shares some typical features of the BTG/TOB family, possessing two well-conserved A and B boxes. Clustering analysis of Hs-BTG1 and other known BTGs showed that Hs-BTG1 was also closely related to BTG1 of C. gigas from the invertebrate BTG1 clade. Function prediction via homology modeling showed that both Hs-BTG1 and C. gigas BTG1 share a similar three-dimensional structure with Homo sapiens BTG1. Tissue-specific expression analysis of the Hs-BTG1 via real-time PCR showed that the transcripts were constitutively expressed, with the highest levels in the hepatopancreas and gills, and the lowest in both haemocyte and muscle tissue. Expression levels of Hs-BTG1 in hepatopancreas （2.03-fold）, mantle （2.07-fold）, kidney （2.2-fold） and haemocyte （2.5-fold） were enhanced by cadmium （Cd2＋） stress, suggesting that Hs-BTG 1 may have played a significant role in H, schlegelii adaptation to adverse environmental conditions.展开更多
The technology of homology cloning and anchored PCR was used to clone the IL-1β gene from the Japanese sea perch （Lateolabrax iaponicus）. The full-length cDNA of sea perch IL-1β was 1 310 bp, including a 5＇ untra...The technology of homology cloning and anchored PCR was used to clone the IL-1β gene from the Japanese sea perch （Lateolabrax iaponicus）. The full-length cDNA of sea perch IL-1β was 1 310 bp, including a 5＇ untranslated regiop （UTR） of 136 bp, a 3＇ UTR ot 430 bp, and an ORF of 774 bp encoding a polypeptide of 258 amino acids with an estimated molecular mass of 29.31 kDa. The searches for nucleotides and protein sequence similarities with the BLAST analysis indicated that the deduced amino acid sequence of sea perch IL-1β was homological to the IL-1β in other fish species and even the mammalian. Conserved signature sequences of the IL-1β gene family were found in the sea perch IL-1β deduced amino acid sequence. Temporal expressions of the IL-1β gene in LPS or iridovirus challenged group and in control group were measured by the semi-quantitative RT-PCR. The mRNA transcripts of IL-1β could be detected in head-kidney, spleen, liver, gill and heart of the healthy individuals, and the expression level of IL-1β in head-kidney, spleen and gill was higher than that in liver and heart, but it was hard to be detected in the brain. After being stimulated by the LPS or iridovirus, the IL-1β expression in most of examined tissues was up-regulated, and also could be detected in the brain. These results indicated that the expression of sea perch IL-1β was constitutive and could be up-regulated by immune effector stimulation. Therefore the sea perch IL-1β could play a critical role in the host-pathogen interaction.展开更多
目的建立实时SYBR Green I定量RT-PCR检测人TIM-1和TIM-3mRNA的方法。方法从人外周血单个核细胞提取的总RNA中逆转录扩增人TIM-1和TIM-3的cDNA,将将纯化的人TIM-1和TIM-3的扩增产物分别与pMD18-T Simple载体进行连接,转化宿主菌DH5α,...目的建立实时SYBR Green I定量RT-PCR检测人TIM-1和TIM-3mRNA的方法。方法从人外周血单个核细胞提取的总RNA中逆转录扩增人TIM-1和TIM-3的cDNA,将将纯化的人TIM-1和TIM-3的扩增产物分别与pMD18-T Simple载体进行连接,转化宿主菌DH5α,提取重组质粒DNA,PCR鉴定并测序分析。纯化质粒并检测260nm吸光值,确定重组质粒原液的拷贝浓度并以此制备荧光定量PCR梯度浓度标准品,进行实时荧光定量PCR实验。结果建立了TIM-1和TIM-3基因基因mRNA表达实时荧光定量PCR检测方法,检测灵敏度达103拷贝,线性范围为103-107拷贝。阈值循环数(Ct)与PCR体系中起始模板量的对数值之间有着良好的线性关系,线性相关系数分别为1.00和1.00,扩增效率分别为1.070和1.023,批内及批间变异系数<5%。熔解曲线分析表明,产物为特异的单峰。结论我们成功建立检测人TIM-1和TIM-3的实时荧光定量PCR方法,为进一步研究人TIM-1和TIM-3功能奠定基础。展开更多
基金supported by the National Natural Science Foundation of China(31301967)the Natural Science Foundation of Jiangsu Province,China(BK20161322)+4 种基金the projects of Key Laboratory for Poultry Genetics and Breeding of Jiangsu Province(JQLAB-ZZ-201703)the Major Breeding Programs in Jiangsu Province,China(PZCZ201728)the earmarked fund for China Agriculture Research System(CARS-41)the Independent Scientific Foundation of Public Welfare Scientific Institutes in Jiangsu Province,China(BM2018026)the Open Projects of Key Laboratory of Chicken Genetics and Breeding,Ministry of Agriculture and Rural Affairs of China(CGB-201704)。
文摘Previous studies on mammals showed that peroxisome proliferator-activated receptor gamma coactivator-1α(PGC-1α)played a prominent role in regulating muscle fiber type transition and composition.However,the role of PGC-1αin chicken muscle has seldom been explored.To investigate the effect of PGC-1αon chicken skeletal muscles in this study,the PGC-1αgene was overexpressed or silenced in chicken primary myoblasts by using lentivirus,and then the effects of the PGC-1αgene overexpression and knockdown on the mRNA expression profile of genes related to myofiber type specificity were examined during fiber formation.The results showed that overexpression of PGC-1αfrom proliferation to differentiation was accompanied by the up-regulated expression of Pax7,MyoD,and CnAα,which was significantly(P<0.01)increased after one day of transfection(1 I).The enhancement of MyoG,MEF2 c,and MyHC SM expression lagged,which was improved significantly(P<0.01)after four days of transfection(1 I3 D).Overexpression of PGC-1αdecreased(P<0.01)the MyHC FWM expression after four days of transfection(1 I3 D),and it had no significant impact(P>0.05)on the expression of CnB1,NFATc3,and MyHC FRM during myofiber formation.The effective silence(P<0.01)of PGC-1αby lentivirus mediating short hairpin RNA(shRNA)was detected after four days of transfection(1 I3 D)in cultures,and the lack of its function in chicken primary myoblasts significantly(P<0.01)down-regulated the expression of Pax7,MyoD,CnAα,MyoG,MEF2 c,and MyHC SM,significantly(P<0.01)up-regulated the expression of MyHC FWM,and had no significant impact(P>0.05)on the expression of CnB1,NFATc3,and MyHC FRM.These results indicated that the role of PGC-1αin regulating the fiber type specificity of chicken skeletal muscles might be similar to that in mammals,which interplayed with key genes related to myocyte differentiation and calcineurin signaling pathway.
基金supported by the Key Scientific and Technological Programme of Jiangxi Province,China(20121BBF60036)the Special Fund for Agro-scientific Research in the Public Interest,State Agriculture Ministry of China(200903028)+2 种基金the Science and Technology Landing Project of Jiangxi Province,China(KJLD12001)the Youth Fund of the Education Department of Jiangxi Province,China(GJJ14219)the National Natural Science Foundation of China(31160534)
文摘The B cells translocation gene 1 （BTG1） is a member of the BTG/TOB family of anti-proliferative genes, which have recently emerged as important regulators of cell growth and differentiation among vertebrates. Here, for the first time we cloned the full-length eDNA sequence of Hyriopsis schlegelii （Hs-BTG1）, an economically important freshwater shellfish and potential indicator of environmental heavy metal pollution, for the first time. Using rapid amplification of eDNA ends （RACE） together with splicing the EST sequence from a haemocyte eDNA library, we found that Hs-BTG1 contains a 525 bp open reading frame （ORF） encoding a 174 amino-acid polypeptide, a 306 bp 5＇ untranslated region （5＇ UTR）, and a 571 bp 3＇ UTR with a Poly（A） tail as well as a transcription termination signal （AATAAA）. Homologne searching against GenBank revealed that Hs-BTG1 was closest to Crassostrea gigas BTG1, sharing 50.57% of protein identities. Hs-BTG1 also shares some typical features of the BTG/TOB family, possessing two well-conserved A and B boxes. Clustering analysis of Hs-BTG1 and other known BTGs showed that Hs-BTG1 was also closely related to BTG1 of C. gigas from the invertebrate BTG1 clade. Function prediction via homology modeling showed that both Hs-BTG1 and C. gigas BTG1 share a similar three-dimensional structure with Homo sapiens BTG1. Tissue-specific expression analysis of the Hs-BTG1 via real-time PCR showed that the transcripts were constitutively expressed, with the highest levels in the hepatopancreas and gills, and the lowest in both haemocyte and muscle tissue. Expression levels of Hs-BTG1 in hepatopancreas （2.03-fold）, mantle （2.07-fold）, kidney （2.2-fold） and haemocyte （2.5-fold） were enhanced by cadmium （Cd2＋） stress, suggesting that Hs-BTG 1 may have played a significant role in H, schlegelii adaptation to adverse environmental conditions.
文摘The technology of homology cloning and anchored PCR was used to clone the IL-1β gene from the Japanese sea perch （Lateolabrax iaponicus）. The full-length cDNA of sea perch IL-1β was 1 310 bp, including a 5＇ untranslated regiop （UTR） of 136 bp, a 3＇ UTR ot 430 bp, and an ORF of 774 bp encoding a polypeptide of 258 amino acids with an estimated molecular mass of 29.31 kDa. The searches for nucleotides and protein sequence similarities with the BLAST analysis indicated that the deduced amino acid sequence of sea perch IL-1β was homological to the IL-1β in other fish species and even the mammalian. Conserved signature sequences of the IL-1β gene family were found in the sea perch IL-1β deduced amino acid sequence. Temporal expressions of the IL-1β gene in LPS or iridovirus challenged group and in control group were measured by the semi-quantitative RT-PCR. The mRNA transcripts of IL-1β could be detected in head-kidney, spleen, liver, gill and heart of the healthy individuals, and the expression level of IL-1β in head-kidney, spleen and gill was higher than that in liver and heart, but it was hard to be detected in the brain. After being stimulated by the LPS or iridovirus, the IL-1β expression in most of examined tissues was up-regulated, and also could be detected in the brain. These results indicated that the expression of sea perch IL-1β was constitutive and could be up-regulated by immune effector stimulation. Therefore the sea perch IL-1β could play a critical role in the host-pathogen interaction.
文摘目的建立实时SYBR Green I定量RT-PCR检测人TIM-1和TIM-3mRNA的方法。方法从人外周血单个核细胞提取的总RNA中逆转录扩增人TIM-1和TIM-3的cDNA,将将纯化的人TIM-1和TIM-3的扩增产物分别与pMD18-T Simple载体进行连接,转化宿主菌DH5α,提取重组质粒DNA,PCR鉴定并测序分析。纯化质粒并检测260nm吸光值,确定重组质粒原液的拷贝浓度并以此制备荧光定量PCR梯度浓度标准品,进行实时荧光定量PCR实验。结果建立了TIM-1和TIM-3基因基因mRNA表达实时荧光定量PCR检测方法,检测灵敏度达103拷贝,线性范围为103-107拷贝。阈值循环数(Ct)与PCR体系中起始模板量的对数值之间有着良好的线性关系,线性相关系数分别为1.00和1.00,扩增效率分别为1.070和1.023,批内及批间变异系数<5%。熔解曲线分析表明,产物为特异的单峰。结论我们成功建立检测人TIM-1和TIM-3的实时荧光定量PCR方法,为进一步研究人TIM-1和TIM-3功能奠定基础。
