【目的】研究鸡gga-mir-1658前体区基因遗传变异/单倍型及其在品种间的分布,分析其对micro RNA二级茎环结构和靶基因选择的影响,旨在筛选其中具有潜在生物学功能的变异位点,为进一步揭示其对gga-mir-1658基因表达调控的影响及表型效应...【目的】研究鸡gga-mir-1658前体区基因遗传变异/单倍型及其在品种间的分布,分析其对micro RNA二级茎环结构和靶基因选择的影响,旨在筛选其中具有潜在生物学功能的变异位点,为进一步揭示其对gga-mir-1658基因表达调控的影响及表型效应奠定基础。【方法】根据鸡gga-mir-1658基因组序列(Gen Bank登录号:NR_035151.1)设计一对特异性引物,采用PCR产物直接测序的方法,对太行鸡(95只)、北京油鸡(83只)和来航鸡(42只)3个鸡种220只个体的gga-mir-1658基因前体区进行多态性检测。使用DNAman、MEGA和mfold软件进行pre-gga-mir-1658基因组序列的比对分析和二级结构模拟。通过SHEsis软件和Haploview软件进行配对连锁不平衡分析和单倍型分析。使用mi Randa软件对gga-mir-1658的靶基因及其复合物自由能变化进行预测分析。【结果】在pre-gga-mir-1658基因共发现6个变异位点,其中次要等位基因频率≥5%位点有g.28 C>G、g.31C>T、g.70 G>A和g.71 G>–,4个变异位点均定位于gga-mir-1658基因成熟体的种子区。遗传变异特征分析显示,g.70 G>A位点表现为低度多态(PIC<0.25),其余3个位点均表现为中度多态(0.25<PIC<0.50)。适合性检验表明,除了来航鸡的g.31 C>T、g.71 G>–位点、太行鸡的g.71 G>–位点以及北京油鸡的g.70 G>A位点之外,其他变异位点在各鸡种均处于哈代-温伯格平衡状态(P>0.05)。连锁不平衡和单倍型分析表明,各突变位点之间存在弱连锁平衡;从3个品种鸡中共检测到11种单倍型,其中H1(C C G–)和H11(G T G G)是群体的优势单倍型,频率均大于25%。生物信息学分析表明,种子区的突变可影响gga-mir-1658基因前体二级结构的空间构型和自由能,其中H6单倍型突变体的自由能最高(41.00 kcal·mol-1),H2和H5单倍型突变体的自由能最低(35.70 kcal·mol-1);群体的优势单倍型H1和H11突变体的自由能分别为-36.10和40.04 kcal·mol-1。gga-mir-1658基因不同单倍型成熟体种子区序列存在差异,在gga-mir-1658-5p存在"AUACCAU"、"AUACCAC"2种种子区序列,gga-mir-1658-3p共有"AACUCUG"、"AGCUGUG"、"AACUGUG"和"AGCUCUG"4种种子区序列。针对gga-mir-1658的预测靶基因的生物信息学分析显示,它们主要在基因表达调控、细胞凋亡、免疫系统的发育和B细胞激活等基本生物学过程中显著富集;此外,种子区变化可以影响gga-mir-1658成熟体对靶基因的选择。【结论】(1)gga-mir-1658基因种子区存在4个具有潜在生物学功能和表型效应的突变位点,可组成11种单倍型,其中H1(C C G–)和H11(G T G G)在北京油鸡、太行鸡和来航鸡为优势单倍型。(2)种子区突变影响gga-mir-1658基因前体二级结构的稳定性和靶基因的选择,可能是具有潜在表型效应的重要功能位点。展开更多
Background:Excessive abdominal fat deposition in commercial broilers presents an obstacle to profitable meat quality,feed utilization,and reproduction.Abdominal fat deposition depends on the proliferation of preadipoc...Background:Excessive abdominal fat deposition in commercial broilers presents an obstacle to profitable meat quality,feed utilization,and reproduction.Abdominal fat deposition depends on the proliferation of preadipocytes and their maturation into adipocytes,which involves a cascade of regulatory molecules.Accumulating evidence has shown that microRNAs(miRNAs)serve as post-transcriptional regulators of adipogenic differentiation in mammals.However,the miRNA-mediated molecular mechanisms underlying abdominal fat deposition in chickens are still poorly understood.This study aimed to investigate the biological functions and regulatory mechanism of miRNAs in chicken abdominal adipogenesis.Results:We established a chicken model of abdominal adipocyte differentiation and analyzed miRNA and mRNA expression in abdominal adipocytes at different stages of differentiation(0,12,48,72,and 120 h).A total of 217 differentially expressed miRNAs(DE-miRNAs)and 3520 differentially expressed genes were identified.Target prediction of DE-miRNAs and functional enrichment analysis revealed that the differentially expressed targets were significantly enriched in lipid metabolism-related signaling pathways,including the PPAR signaling and MAPK signaling pathways.A candidate miRNA,gga-miR-106-5p,exhibited decreased expression during the proliferation and differentiation of abdominal preadipocytes and was downregulated in the abdominal adipose tissues of fat chickens compared to that of lean chickens.gga-miR-106-5p was found to inhibit the proliferation and adipogenic differentiation of chicken abdominal preadipocytes.A dual-luciferase reporter assay suggested that the KLF15 gene,which encodes a transcriptional factor,is a direct target of gga-miR-106-5p.gga-miR-106-5p suppressed the posttranscriptional activity of KLF15,which is an activator of abdominal preadipocyte proliferation and differentiation,as determined with gain-and loss-of-function experiments.Conclusions:gga-miR-106-5p functions as an inhibitor of abdominal adipogenesis by targeting the KLF15 gene in chickens.These findings not only improve our understanding of the specific functions of miRNAs in avian adipogenesis but also provide potential targets for the genetic improvement of excessive abdominal fat deposition in poultry.展开更多
Background: Increasing evidence indicates that micro RNAs(mi RNAs) are involved in inflammatory response and immune regulation following pathogen invasion. The purpose of this study was to elucidate the roles played b...Background: Increasing evidence indicates that micro RNAs(mi RNAs) are involved in inflammatory response and immune regulation following pathogen invasion. The purpose of this study was to elucidate the roles played by Gallus gallus micro RNA-1306-5 p(gga-mi R-1306-5 p) in host responses against potential invasion by Salmonella enteritidis(SE) in chickens and the underlying mechanisms.Results: In present study, the expression levels of gga-mi R-1306-5 p were determined in both tissues and HD11 cells. The results showed that gga-mi R-1306-5 p was significantly increased following SE infection or lipopolysaccharide(LPS) stimulation. The dual luciferase reporter assay further validated that gga-mi R-1306-5 p targeted the Toll-interacting protein(Tollip), and thereby participated in the regulation of immune response against SE or LPS stimulation through binding with the 3′-untranslated region(3’UTR) of Tollip. Additionally, the expression of Tollip was significantly blocked by over-expressed gga-mi R-1306-5 p. The underlying mechanisms by which ggami R-1306-5 p modulated the production of pro-inflammatory cytokines were also investigated. Molecular biological assays demonstrated that overexpression of gga-mi R-1306-5 p promoted the production of pro-inflammatory mediators, including NF-κB, TNF-α, IL-6, and IL-1β, which produced effects similar to those of Tollip knockdown.Conclusions: Taken together, gga-mi R-1306-5 p induced by SE or LPS, regulates the immune response by inhibiting Tollip, which activates the production of inflammatory cytokines. This study has provided the first direct evidence that gga-mi R-1306-5 p targets Tollip, and is involved in the host response against SE.展开更多
文摘【目的】研究鸡gga-mir-1658前体区基因遗传变异/单倍型及其在品种间的分布,分析其对micro RNA二级茎环结构和靶基因选择的影响,旨在筛选其中具有潜在生物学功能的变异位点,为进一步揭示其对gga-mir-1658基因表达调控的影响及表型效应奠定基础。【方法】根据鸡gga-mir-1658基因组序列(Gen Bank登录号:NR_035151.1)设计一对特异性引物,采用PCR产物直接测序的方法,对太行鸡(95只)、北京油鸡(83只)和来航鸡(42只)3个鸡种220只个体的gga-mir-1658基因前体区进行多态性检测。使用DNAman、MEGA和mfold软件进行pre-gga-mir-1658基因组序列的比对分析和二级结构模拟。通过SHEsis软件和Haploview软件进行配对连锁不平衡分析和单倍型分析。使用mi Randa软件对gga-mir-1658的靶基因及其复合物自由能变化进行预测分析。【结果】在pre-gga-mir-1658基因共发现6个变异位点,其中次要等位基因频率≥5%位点有g.28 C>G、g.31C>T、g.70 G>A和g.71 G>–,4个变异位点均定位于gga-mir-1658基因成熟体的种子区。遗传变异特征分析显示,g.70 G>A位点表现为低度多态(PIC<0.25),其余3个位点均表现为中度多态(0.25<PIC<0.50)。适合性检验表明,除了来航鸡的g.31 C>T、g.71 G>–位点、太行鸡的g.71 G>–位点以及北京油鸡的g.70 G>A位点之外,其他变异位点在各鸡种均处于哈代-温伯格平衡状态(P>0.05)。连锁不平衡和单倍型分析表明,各突变位点之间存在弱连锁平衡;从3个品种鸡中共检测到11种单倍型,其中H1(C C G–)和H11(G T G G)是群体的优势单倍型,频率均大于25%。生物信息学分析表明,种子区的突变可影响gga-mir-1658基因前体二级结构的空间构型和自由能,其中H6单倍型突变体的自由能最高(41.00 kcal·mol-1),H2和H5单倍型突变体的自由能最低(35.70 kcal·mol-1);群体的优势单倍型H1和H11突变体的自由能分别为-36.10和40.04 kcal·mol-1。gga-mir-1658基因不同单倍型成熟体种子区序列存在差异,在gga-mir-1658-5p存在"AUACCAU"、"AUACCAC"2种种子区序列,gga-mir-1658-3p共有"AACUCUG"、"AGCUGUG"、"AACUGUG"和"AGCUCUG"4种种子区序列。针对gga-mir-1658的预测靶基因的生物信息学分析显示,它们主要在基因表达调控、细胞凋亡、免疫系统的发育和B细胞激活等基本生物学过程中显著富集;此外,种子区变化可以影响gga-mir-1658成熟体对靶基因的选择。【结论】(1)gga-mir-1658基因种子区存在4个具有潜在生物学功能和表型效应的突变位点,可组成11种单倍型,其中H1(C C G–)和H11(G T G G)在北京油鸡、太行鸡和来航鸡为优势单倍型。(2)种子区突变影响gga-mir-1658基因前体二级结构的稳定性和靶基因的选择,可能是具有潜在表型效应的重要功能位点。
基金supported by the National Key Research and Development Program of China(2021YFD1200803)Sanya Yazhou Bay Science and Technology City Administration(SYND-2022-28)China Agriculture Research System of MOF and MARA(CARS-40).
文摘Background:Excessive abdominal fat deposition in commercial broilers presents an obstacle to profitable meat quality,feed utilization,and reproduction.Abdominal fat deposition depends on the proliferation of preadipocytes and their maturation into adipocytes,which involves a cascade of regulatory molecules.Accumulating evidence has shown that microRNAs(miRNAs)serve as post-transcriptional regulators of adipogenic differentiation in mammals.However,the miRNA-mediated molecular mechanisms underlying abdominal fat deposition in chickens are still poorly understood.This study aimed to investigate the biological functions and regulatory mechanism of miRNAs in chicken abdominal adipogenesis.Results:We established a chicken model of abdominal adipocyte differentiation and analyzed miRNA and mRNA expression in abdominal adipocytes at different stages of differentiation(0,12,48,72,and 120 h).A total of 217 differentially expressed miRNAs(DE-miRNAs)and 3520 differentially expressed genes were identified.Target prediction of DE-miRNAs and functional enrichment analysis revealed that the differentially expressed targets were significantly enriched in lipid metabolism-related signaling pathways,including the PPAR signaling and MAPK signaling pathways.A candidate miRNA,gga-miR-106-5p,exhibited decreased expression during the proliferation and differentiation of abdominal preadipocytes and was downregulated in the abdominal adipose tissues of fat chickens compared to that of lean chickens.gga-miR-106-5p was found to inhibit the proliferation and adipogenic differentiation of chicken abdominal preadipocytes.A dual-luciferase reporter assay suggested that the KLF15 gene,which encodes a transcriptional factor,is a direct target of gga-miR-106-5p.gga-miR-106-5p suppressed the posttranscriptional activity of KLF15,which is an activator of abdominal preadipocyte proliferation and differentiation,as determined with gain-and loss-of-function experiments.Conclusions:gga-miR-106-5p functions as an inhibitor of abdominal adipogenesis by targeting the KLF15 gene in chickens.These findings not only improve our understanding of the specific functions of miRNAs in avian adipogenesis but also provide potential targets for the genetic improvement of excessive abdominal fat deposition in poultry.
基金supported by grants from the National Natural Science Foundation of China(No.31572393)the National Key Technology R&D Program(2015BAD03B03)+1 种基金the China Agricultural Science and Technology Innovation Project(ASTIPIAS04)the earmarked fund for Modern AgroIndustry Technology Research System(CARS-41)
文摘Background: Increasing evidence indicates that micro RNAs(mi RNAs) are involved in inflammatory response and immune regulation following pathogen invasion. The purpose of this study was to elucidate the roles played by Gallus gallus micro RNA-1306-5 p(gga-mi R-1306-5 p) in host responses against potential invasion by Salmonella enteritidis(SE) in chickens and the underlying mechanisms.Results: In present study, the expression levels of gga-mi R-1306-5 p were determined in both tissues and HD11 cells. The results showed that gga-mi R-1306-5 p was significantly increased following SE infection or lipopolysaccharide(LPS) stimulation. The dual luciferase reporter assay further validated that gga-mi R-1306-5 p targeted the Toll-interacting protein(Tollip), and thereby participated in the regulation of immune response against SE or LPS stimulation through binding with the 3′-untranslated region(3’UTR) of Tollip. Additionally, the expression of Tollip was significantly blocked by over-expressed gga-mi R-1306-5 p. The underlying mechanisms by which ggami R-1306-5 p modulated the production of pro-inflammatory cytokines were also investigated. Molecular biological assays demonstrated that overexpression of gga-mi R-1306-5 p promoted the production of pro-inflammatory mediators, including NF-κB, TNF-α, IL-6, and IL-1β, which produced effects similar to those of Tollip knockdown.Conclusions: Taken together, gga-mi R-1306-5 p induced by SE or LPS, regulates the immune response by inhibiting Tollip, which activates the production of inflammatory cytokines. This study has provided the first direct evidence that gga-mi R-1306-5 p targets Tollip, and is involved in the host response against SE.
