Background Carcass traits are crucial indicators of meat production efficiency.However,the molecular regulatory mechanisms associated with these traits remain unclear.Results In this study,we conducted comprehensive t...Background Carcass traits are crucial indicators of meat production efficiency.However,the molecular regulatory mechanisms associated with these traits remain unclear.Results In this study,we conducted comprehensive transcriptomic and genomic analyses on 399 Tiannong partridge chickens to identify key genes and variants associated with carcass traits and to elucidate the underlying regulatory mechanisms.Based on association analyses with the elastic net(EN)model,we identified 12 candidate genes(AMY1A,AP3B2,CEBPG,EEF2,EIF4EBP1,FGFR1,FOXD3,GOLM1,LOC107052698,PABPC1,SERPINB6 and TBC1D16)for 4 carcass-related traits,namely live weight,dressed weight,eviscerated weight,and breast muscle weight.SERPINB6 was identified as the only overlapping gene by 3 analyses,EN model analysis,weighted gene co-expression network analysis and differential expression analysis.Cell-level experiments confirmed that SERPINB6 promotes the proliferation of chicken DF1 cells and primary myoblasts.Further expression genome-wide association study and association analysis indicated that rs317934171 is the critical site that enhances SERPINB6 expression.Furthermore,a dual-luciferase reporter assay proved that gga-miR-1615 targets the 3′UTR of SERPINB6.Conclusions Collectively,our findings reveal that SERPINB6 serves as a novel gene for chicken carcass traits by promoting fibroblast and myoblast proliferation.Additionally,the downstream variant rs317934171 regulates SERPINB6 expression.These results identify a new target gene and molecular marker for the molecular mechanisms of chicken carcass traits.展开更多
Background:N6-methyladenosine(m^(6)A)is an abundant post-transcriptional RNA modification that affects various biological processes.The fat mass and obesity-associated(FTO)protein,a demethylase encoded by the FTO gene...Background:N6-methyladenosine(m^(6)A)is an abundant post-transcriptional RNA modification that affects various biological processes.The fat mass and obesity-associated(FTO)protein,a demethylase encoded by the FTO gene,has been found to regulate adipocyte development in an m^(6)A-dependent manner in multiple species.However,the effects of the m^(6)A methylation and FTO demethylation functions on chicken adipogenesis remain unclear.This study aims to explore the association between m^(6)A modification and chicken adipogenesis and the underlying mechanism by which FTO affects chicken preadipocyte development.Results:The association between m^(6)A modification and chicken lipogenesis was assessed by treating chicken pread-ipocytes with different doses of methyl donor betaine and methylation inhibitor cycloleucine.The results showed that betaine significantly increased methylation levels and inhibited lipogenesis,and the inverse effect was found in preadipocytes after cycloleucine treatment.Overexpression of FTO significantly inhibited m^(6)A levels and promoted proliferation and differentiation of chicken preadipocytes.Silencing FTO showed opposite results.Mechanistically,FTO overexpression increased the expression of catenin beta 1(CTNNB1)by improving RNA stability in an m^(6)A-dependent manner,and we proved that FTO could directly target CTNNB1.Furthermore,CTNNB1 may be a positive regulator of adipogenesis in chicken preadipocytes.Conclusions:m^(6)A methylation of RNA was negatively associated with adipogenesis of chicken preadipocytes.FTO could regulate CTNNB1 expression in a demethylation manner to promote lipogenesis.展开更多
Background:As a ubiquitous reversible epigenetic RNA modification,N6-methyladenosine(m6A)plays crucial regulatory roles in multiple biological pathways.However,its functional mechanisms in sex determination and differ...Background:As a ubiquitous reversible epigenetic RNA modification,N6-methyladenosine(m6A)plays crucial regulatory roles in multiple biological pathways.However,its functional mechanisms in sex determination and differentiation during gonadal development of chicken embryos are not clear.Therefore,we established a transcriptome-wide m6A map in the female and male chicken left gonads of embryonic day 7(E7)by methylated RNA immunoprecipitation sequencing(MeRIP-seq)to offer insight into the landscape of m6A methylation and investigate the post-transcriptional modification underlying gonadal differentiation.Results:The chicken embryonic gonadal transcriptome was extensively methylated.We found 15,191 and 16,111 m6A peaks in the female and male left gonads,respectively,which were mainly enriched in the coding sequence(CDS)and stop codon.Among these m6A peaks,we identified that 1013 and 751 were hypermethylated in females and males,respectively.These differential peaks covered 281 and 327 genes,such as BMP2,SMAD2,SOX9 and CYP19A1,which were primarily associated with development,morphogenesis and sex differentiation by functional enrichment.Further analysis revealed that the m6A methylation level was positively correlated with gene expression abundance.Furthermore,we found that YTHDC2 could regulate the expression of sex-related genes,especially HEMGN and SOX9,in male mesonephros/gonad mingle cells,which was verified by in vitro experiments,suggesting a regulatory role of m6A methylation in chicken gonad differentiation.Conclusions:This work provided a comprehensive m6A methylation profile of chicken embryonic gonads and revealed YTHDC2 as a key regulator responsible for sex differentiation.Our results contribute to a better understanding of epigenetic factors involved in chicken sex determination and differentiation and to promoting the future development of sex manipulation in poultry industry.展开更多
Background:B-cell CLL/lymphoma 6(BCL6)is a transcriptional master regulator that represses more than 1200 potential target genes.Our previous study showed that a decline in blood production in runting and stunting syn...Background:B-cell CLL/lymphoma 6(BCL6)is a transcriptional master regulator that represses more than 1200 potential target genes.Our previous study showed that a decline in blood production in runting and stunting syndrome(RSS)affected sex-linked dwarf(SLD)chickens compared to SLD chickens.However,the association between BCL6 gene and hematopoietic function remains unknown in chickens.Methods:In this study,we used RSS affected SLD(RSS-SLD)chickens,SLD chickens and normal chickens as research object and overexpression of BCL6 in hematopoietic stem cells(HSCs),to investigate the effect of the BCL6 on differentiation and development of HSCs.Results:The results showed that comparison of RSS-SLD chickens with SLD chickens,the BCL6 was highly expressed in RSS-SLD chickens bone marrow.The bone marrow of RSS-SLD chickens was exhausted and red bone marrow was largely replaced by yellow bone marrow,bone density was reduced,and the levels of immature erythrocytes in peripheral blood were increased.At the same time,the hematopoietic function of HSCs decreased in RSS-SLD chickens,which was manifested by a decrease in the hematopoietic growth factors(HGFs)EPO,SCF,TPO,and IL-3,as well as hemoglobinα1 and hemoglobinβexpression.Moreover,mitochondrial function in the HSCs of RSS-SLD chickens was damaged,including an increase in ROS production,decrease in ATP concentration,and decrease in mitochondrial membrane potential(ΔΨm).The same results were also observed in SLD chickens compared with normal chickens;however,the symptoms were more serious in RSS-SLD chickens.Additionally,after overexpression of the BCL6 in primary HSCs,the secretion of HGFs(EPO,SCF,TPO and IL-3)was inhibited and the expression of hemoglobinα1 and hemoglobinβwas decreased.However,cell proliferation was accelerated,apoptosis was inhibited,and the HSCs entered a cancerous state.The function of mitochondria was also abnormal,ROS production was decreased,and ATP concentration andΔΨm were increased,which was related to the inhibition of apoptosis of stem cells.Conclusions:Taken together,we conclude that the high expression of BCL6 inhibits the differentiation and development of HSCs by affecting mitochondrial function,resulting in impaired growth and development of chickens.Moreover,the abnormal expression of BCL6 might be a cause of the clinical manifestations of chicken comb,pale skin,stunted growth and development,and the tendency to appear RSS in SLD chickens.展开更多
A number of eukaryotic expression vectors have been developed for use as DNA vaccines. They showed varying abilities to initiate immune responses;however, there is little data to indicate which of these vectors will b...A number of eukaryotic expression vectors have been developed for use as DNA vaccines. They showed varying abilities to initiate immune responses;however, there is little data to indicate which of these vectors will be the most useful and practical for DNA vaccines in different species. This report examines the use of five expression vectors with different promoters and Kozak sequence to express the same hemagglutinin (HA) protein of an H6N2 avian influenza virus for DNA vaccination in chickens. Although intramuscular vaccination with seven DNA constructs elicited no or limited measurable H6 HA antibody responses in Hy-Line chickens, variable reduction in virus shedding for either oropharyngeal or cloacal swabs post-virus challenge were observed. This indicated that all DNA constructs generated some levels of protective immunity against homologous virus challenge. Interestingly, lower dose (50 or 100 μg) of plasmid DNAs consistently induced better immune response than higher dose (300 or 500 μg). In the transfection experiments there appeared to be a hierarchy in the in vitro expression efficiency in the order of pCAG-optiHAk/ pCAG-HAk > pCI-HAk > VR-HA > pCI-HA > pCI-neo-HA > pVAX-HA. Since the level of in vitro expression correlates with the level of immune response in vivo, in vitro expression levels of the DNA constructs can be used as an indicator for pre-selection of plasmid vaccines prior to in vivo assessment. Moreover, our results suggested that the Kozak sequence could be used as an effective tool for DNA vaccine design.展开更多
【目的】建立针对鸡白介素6(IL-6)、白介素17(IL-17)和γ干扰素(IFN-γ)的实时荧光定量PCR检测方法,为细胞因子的定量检测及病毒致病机制的研究奠定基础。【方法】根据GenBank已发表的鸡IL-6、IL-17、IFN-γ和3-磷酸甘油醛脱氢酶(GAPDH...【目的】建立针对鸡白介素6(IL-6)、白介素17(IL-17)和γ干扰素(IFN-γ)的实时荧光定量PCR检测方法,为细胞因子的定量检测及病毒致病机制的研究奠定基础。【方法】根据GenBank已发表的鸡IL-6、IL-17、IFN-γ和3-磷酸甘油醛脱氢酶(GAPDH)基因保守序列,设计并合成4对相应的特异性引物,以鸡胚成纤维细胞的cDNA为模板构建重组质粒,对退火温度、引物浓度等SYBR Green I实时荧光定量PCR反应条件进行优化,建立各基因的标准曲线,并进行特异性、敏感性、重复性试验。【结果】GAPDH、IL-6、IL-17和IFN-γ的SYBR Green I实时荧光定量PCR扩增效率分别是98.2%、99.2%、102.0%和100.8%,相应的标准误差为0.00666、0.00813、0.00365和0.00458。特异性试验结果显示各基因的溶解曲线均呈单一溶解峰,敏感性试验结果表明各基因的检测下限均为100拷贝,重复性试验结果表明组内变异系数均小于2.00%。【结论】建立的SYBR Green I实时荧光定量PCR具有敏感性高、重复性好、特异性强等特点,为快速检测和定量鸡源IL-6、IL-17和IFN-γ的表达水平提供了技术平台。展开更多
基金supported by grants from the Key Projects of National Natural Science Foundation of China (No. 32230101)the Project of Qingyuan Science and Technology (2020A01, 2021SJXM011)+1 种基金the Agriculture Research System (CARS-41)the Core Breed Source Research Project JBGS (2021) 107
文摘Background Carcass traits are crucial indicators of meat production efficiency.However,the molecular regulatory mechanisms associated with these traits remain unclear.Results In this study,we conducted comprehensive transcriptomic and genomic analyses on 399 Tiannong partridge chickens to identify key genes and variants associated with carcass traits and to elucidate the underlying regulatory mechanisms.Based on association analyses with the elastic net(EN)model,we identified 12 candidate genes(AMY1A,AP3B2,CEBPG,EEF2,EIF4EBP1,FGFR1,FOXD3,GOLM1,LOC107052698,PABPC1,SERPINB6 and TBC1D16)for 4 carcass-related traits,namely live weight,dressed weight,eviscerated weight,and breast muscle weight.SERPINB6 was identified as the only overlapping gene by 3 analyses,EN model analysis,weighted gene co-expression network analysis and differential expression analysis.Cell-level experiments confirmed that SERPINB6 promotes the proliferation of chicken DF1 cells and primary myoblasts.Further expression genome-wide association study and association analysis indicated that rs317934171 is the critical site that enhances SERPINB6 expression.Furthermore,a dual-luciferase reporter assay proved that gga-miR-1615 targets the 3′UTR of SERPINB6.Conclusions Collectively,our findings reveal that SERPINB6 serves as a novel gene for chicken carcass traits by promoting fibroblast and myoblast proliferation.Additionally,the downstream variant rs317934171 regulates SERPINB6 expression.These results identify a new target gene and molecular marker for the molecular mechanisms of chicken carcass traits.
基金funded by the Natural Scientific Foundation of China(U1901206)Local Innovative and Research Teams Project of Guangdong Province(2019BT02N630)+4 种基金the Science and Technology Program of Guangdong province,China(2020B1212060060)the Science and Technology Program of Guangzhou,China(202103000084)the Construction Project of Modern Agricultural Science and Technology Innovation Alliance in Guangdong Province(2021KJ128)National Key R&D Program of China(2021YFD1300100)China Agriculture Research System(CARS-41-G03).
文摘Background:N6-methyladenosine(m^(6)A)is an abundant post-transcriptional RNA modification that affects various biological processes.The fat mass and obesity-associated(FTO)protein,a demethylase encoded by the FTO gene,has been found to regulate adipocyte development in an m^(6)A-dependent manner in multiple species.However,the effects of the m^(6)A methylation and FTO demethylation functions on chicken adipogenesis remain unclear.This study aims to explore the association between m^(6)A modification and chicken adipogenesis and the underlying mechanism by which FTO affects chicken preadipocyte development.Results:The association between m^(6)A modification and chicken lipogenesis was assessed by treating chicken pread-ipocytes with different doses of methyl donor betaine and methylation inhibitor cycloleucine.The results showed that betaine significantly increased methylation levels and inhibited lipogenesis,and the inverse effect was found in preadipocytes after cycloleucine treatment.Overexpression of FTO significantly inhibited m^(6)A levels and promoted proliferation and differentiation of chicken preadipocytes.Silencing FTO showed opposite results.Mechanistically,FTO overexpression increased the expression of catenin beta 1(CTNNB1)by improving RNA stability in an m^(6)A-dependent manner,and we proved that FTO could directly target CTNNB1.Furthermore,CTNNB1 may be a positive regulator of adipogenesis in chicken preadipocytes.Conclusions:m^(6)A methylation of RNA was negatively associated with adipogenesis of chicken preadipocytes.FTO could regulate CTNNB1 expression in a demethylation manner to promote lipogenesis.
基金funded in part by grants from China Agricultural Research System(CARS-40).
文摘Background:As a ubiquitous reversible epigenetic RNA modification,N6-methyladenosine(m6A)plays crucial regulatory roles in multiple biological pathways.However,its functional mechanisms in sex determination and differentiation during gonadal development of chicken embryos are not clear.Therefore,we established a transcriptome-wide m6A map in the female and male chicken left gonads of embryonic day 7(E7)by methylated RNA immunoprecipitation sequencing(MeRIP-seq)to offer insight into the landscape of m6A methylation and investigate the post-transcriptional modification underlying gonadal differentiation.Results:The chicken embryonic gonadal transcriptome was extensively methylated.We found 15,191 and 16,111 m6A peaks in the female and male left gonads,respectively,which were mainly enriched in the coding sequence(CDS)and stop codon.Among these m6A peaks,we identified that 1013 and 751 were hypermethylated in females and males,respectively.These differential peaks covered 281 and 327 genes,such as BMP2,SMAD2,SOX9 and CYP19A1,which were primarily associated with development,morphogenesis and sex differentiation by functional enrichment.Further analysis revealed that the m6A methylation level was positively correlated with gene expression abundance.Furthermore,we found that YTHDC2 could regulate the expression of sex-related genes,especially HEMGN and SOX9,in male mesonephros/gonad mingle cells,which was verified by in vitro experiments,suggesting a regulatory role of m6A methylation in chicken gonad differentiation.Conclusions:This work provided a comprehensive m6A methylation profile of chicken embryonic gonads and revealed YTHDC2 as a key regulator responsible for sex differentiation.Our results contribute to a better understanding of epigenetic factors involved in chicken sex determination and differentiation and to promoting the future development of sex manipulation in poultry industry.
基金This work was supported by grants from the Key-Area Research and Development Program of Guangdong Province(Grant No.2020B020222002)the Guangdong Provincial Promotion Project on Preservation and Utilization of Local Breed of Livestock and Poultry,National Natural Science Foundation of China(Grant No.31401046)+1 种基金the China Agriculture Research System(CARS-41-G03)Guangdong Youth Talent Project.
文摘Background:B-cell CLL/lymphoma 6(BCL6)is a transcriptional master regulator that represses more than 1200 potential target genes.Our previous study showed that a decline in blood production in runting and stunting syndrome(RSS)affected sex-linked dwarf(SLD)chickens compared to SLD chickens.However,the association between BCL6 gene and hematopoietic function remains unknown in chickens.Methods:In this study,we used RSS affected SLD(RSS-SLD)chickens,SLD chickens and normal chickens as research object and overexpression of BCL6 in hematopoietic stem cells(HSCs),to investigate the effect of the BCL6 on differentiation and development of HSCs.Results:The results showed that comparison of RSS-SLD chickens with SLD chickens,the BCL6 was highly expressed in RSS-SLD chickens bone marrow.The bone marrow of RSS-SLD chickens was exhausted and red bone marrow was largely replaced by yellow bone marrow,bone density was reduced,and the levels of immature erythrocytes in peripheral blood were increased.At the same time,the hematopoietic function of HSCs decreased in RSS-SLD chickens,which was manifested by a decrease in the hematopoietic growth factors(HGFs)EPO,SCF,TPO,and IL-3,as well as hemoglobinα1 and hemoglobinβexpression.Moreover,mitochondrial function in the HSCs of RSS-SLD chickens was damaged,including an increase in ROS production,decrease in ATP concentration,and decrease in mitochondrial membrane potential(ΔΨm).The same results were also observed in SLD chickens compared with normal chickens;however,the symptoms were more serious in RSS-SLD chickens.Additionally,after overexpression of the BCL6 in primary HSCs,the secretion of HGFs(EPO,SCF,TPO and IL-3)was inhibited and the expression of hemoglobinα1 and hemoglobinβwas decreased.However,cell proliferation was accelerated,apoptosis was inhibited,and the HSCs entered a cancerous state.The function of mitochondria was also abnormal,ROS production was decreased,and ATP concentration andΔΨm were increased,which was related to the inhibition of apoptosis of stem cells.Conclusions:Taken together,we conclude that the high expression of BCL6 inhibits the differentiation and development of HSCs by affecting mitochondrial function,resulting in impaired growth and development of chickens.Moreover,the abnormal expression of BCL6 might be a cause of the clinical manifestations of chicken comb,pale skin,stunted growth and development,and the tendency to appear RSS in SLD chickens.
文摘A number of eukaryotic expression vectors have been developed for use as DNA vaccines. They showed varying abilities to initiate immune responses;however, there is little data to indicate which of these vectors will be the most useful and practical for DNA vaccines in different species. This report examines the use of five expression vectors with different promoters and Kozak sequence to express the same hemagglutinin (HA) protein of an H6N2 avian influenza virus for DNA vaccination in chickens. Although intramuscular vaccination with seven DNA constructs elicited no or limited measurable H6 HA antibody responses in Hy-Line chickens, variable reduction in virus shedding for either oropharyngeal or cloacal swabs post-virus challenge were observed. This indicated that all DNA constructs generated some levels of protective immunity against homologous virus challenge. Interestingly, lower dose (50 or 100 μg) of plasmid DNAs consistently induced better immune response than higher dose (300 or 500 μg). In the transfection experiments there appeared to be a hierarchy in the in vitro expression efficiency in the order of pCAG-optiHAk/ pCAG-HAk > pCI-HAk > VR-HA > pCI-HA > pCI-neo-HA > pVAX-HA. Since the level of in vitro expression correlates with the level of immune response in vivo, in vitro expression levels of the DNA constructs can be used as an indicator for pre-selection of plasmid vaccines prior to in vivo assessment. Moreover, our results suggested that the Kozak sequence could be used as an effective tool for DNA vaccine design.
文摘【目的】建立针对鸡白介素6(IL-6)、白介素17(IL-17)和γ干扰素(IFN-γ)的实时荧光定量PCR检测方法,为细胞因子的定量检测及病毒致病机制的研究奠定基础。【方法】根据GenBank已发表的鸡IL-6、IL-17、IFN-γ和3-磷酸甘油醛脱氢酶(GAPDH)基因保守序列,设计并合成4对相应的特异性引物,以鸡胚成纤维细胞的cDNA为模板构建重组质粒,对退火温度、引物浓度等SYBR Green I实时荧光定量PCR反应条件进行优化,建立各基因的标准曲线,并进行特异性、敏感性、重复性试验。【结果】GAPDH、IL-6、IL-17和IFN-γ的SYBR Green I实时荧光定量PCR扩增效率分别是98.2%、99.2%、102.0%和100.8%,相应的标准误差为0.00666、0.00813、0.00365和0.00458。特异性试验结果显示各基因的溶解曲线均呈单一溶解峰,敏感性试验结果表明各基因的检测下限均为100拷贝,重复性试验结果表明组内变异系数均小于2.00%。【结论】建立的SYBR Green I实时荧光定量PCR具有敏感性高、重复性好、特异性强等特点,为快速检测和定量鸡源IL-6、IL-17和IFN-γ的表达水平提供了技术平台。
