The retinoic acid inducible gene-1-like receptors (RLRs)play an important role in innate immune system.RIG-1 ,as a member of RLRs,belongs to intracellular protein and is closely related to cell proliferation, differentiation and innate antiviral immunity.It is widely accepted that the RIG-1 is absent in chicken genome, whereas exists in the duck's,which may account for the stronger abilities of waterfowl than chicken in antivirus. Promoter,as the central element of gene expression regulation,can affect gene function by regulating mRNA transcription.However,there is still no report focusing on avian RIG-1 promoter.The purpose of this study was to investigate the characteristics of duck RIG-1 promoter region and its differential expression profiles in embryo stages.5′flanking promoter sequence of duck RIG-1 was obtained by PCR amplification and was analyzed by bioinformatics.The expression profiles of RIG-1 were detected by qRT-PCR in immune organs during duck embryonic development,as well as the predicted transcription factors regulating RIG-1 transcription.4372 bp of duck RIG-1 promoter region was finally obtained.Bioinformatics analysis showed that typical elements,including TATA-box,CAAT-box,and binding sites of transcription factors,such as IRF-1 ,RXR, RAR,AP1, NF-κB, SP1, IL6 and Pax-2, were distributed in duck RIG-1 promoter region.Studies had demonstrated that IRF-1 can promote the expression of human RIG-1 and Pax-2 can inhibit the expression of mouse RIG-1.Besides,a CpG island with 65.8% GC content was predicted in duck RIG-1 promoter region,which has been found in mouse too.These data indicated that a similar transcription regulation manner may exist in duck with human and mouse.The results of qRT-PCR demonstrated that RIG-1 expression levels were dynamic in immune organs during duck embryo stages,and the expression of RIG-1 in bursa of Fabricius is higher than in spleen and thymus.Clustering of gene expression pattern showed that RIG-1 had similar expression patterns with IRF-1 , RXR,AP1 ,NF-κB and IL6 in bursa of Fabricius,which may be due to that the bursa of Fabricius,compared with thymus and spleen,had relatively complete structure and function in duck embryo stages.This result indicated that they might regulate the transcription of RIG-1 .Moreover,RIG-1 always had similar expression pattern with IRF-1 and NF-κB in bursa of Fabricius,spleen and thymus,which suggested that IRF-1 and NF-κB could regulate the expression of RIG-1 .It was the first report about the promoter sequence of duck RIG-1 .The findings in characteristics of duck RIG-1 promoter region,and the relationships between RIG-1 and its transcription factors reflected by mRNA expression profiles in immune organs of embryonic stages may provide a basis and direction to explore transcriptional regulation and expression of duck RIG-1 .

Promoter sequence of gene MyoG in duck was amplified and cloned using RT–PCR, and its bioinformatic info was analyzed as well; the methylation level in CpG island (from –2536 to –1997 bp) of promoter MyoG in muscle tissues was detected using the Sequenom MassArray technique, meanwhile, its expression level was detected using qRT–PCR. The results showed that the amplified MyoG promoter sequences was 2730 bp which contained 2 CpG islands in promoter region. The CpG island (from –2536 to –1997 bp) included 5 binding sites of transcription factor and several eukaryotes structure components. The methylation data showed that methylation status from different individuals and tissues did not clustered together. Methylation frequency in CpG loci varied with individuals, and the methylation frequency on 22% of locus were negatively correlated withMyoG expression levels (P>0.05), whereas, on 78% of locus were positively correlated (P>0.05) with that, among of them, they reached significant difference level on locus of CpG 1, CpG 26.27.28.29 from leg muscles (P<0.05). The mechanism of regulating transcription for MyoG was different from mammals. Among the potential locus influencing MyoG transcription, CpG_1 and CpG_26.27.28.29 might affect MyoG transcription in skeletal muscle tissues of duck leg through DNA methylation. This study might list foundation data for studying the regulatory mechanisms of MyoG transcription in duck.

为研究Ccna1基因在鸭骨骼肌发育中的作用,本研究应用RT-PCR扩增和克隆了鸭Ccna1基因启动子区并作生物信息学分析,应用荧光定量PCR技术检测Ccna1基因以及参与该基因表达调控的多个肌源性转录因子在鸭胚骨骼肌组织发育过程中的表达模式.结果表明,扩增得到鸭Ccna1基因启动子2 213 bp.序列分析表明,鸭Ccna1启动子存在典型的TATA-box、CAAT-box调控元件,有MyoD、MRF4、MyoG、Sp1、PITX1及MEF2等多个转录因子结合位点.定量结果发现,MyoD、MRF4、MyoG和Ccna1在鸭胚胎骨骼肌发育过程中均有表达.在鸭胸肌中,MRF4在E23时期表达量最高,显著高于D8的表达量(P＜0.05);在腿肌中,MRF4和MyoG表达量在E17时期达到最高,且显著高于D2、D8(P＜0.05).聚类分析表明,在鸭胸肌组织中Ccna1表达模式与MyoD一致,在腿肌组织中Ccna1表达模式与MRF4、MyoG一致.初步推测,Ccna1参与到了鸭骨骼肌组织的发育,且在鸭腿肌的生长发育中,MRF4、MyoG可能与Ccna1相互作用进而调控骨骼肌发育,而在鸭胸肌组织中,Ccna1的表达可能与MyoD的转录调控有关.

禽类绿壳蛋性状不仅有躲避捕食的作用,还是禽类的一个重要经济性状,由于进化和遗传背景差异,不同禽类的绿壳蛋性状形成机制不同.本研究以绿壳蛋鸭(Anas platyrhynchos)和白壳蛋鸭输卵管壳腺部组织为研究对象,基于高通量测序技术,富集和分析参与绿壳蛋性状形成的小RNA(microRNAs,miRNAs)及其靶基因.结果显示,在绿壳蛋鸭和白壳蛋鸭壳腺部共预测到269个miRNAs,其中已知的miRNAs 245个,新预测的miRNAs 24个;相对于白壳蛋鸭,绿壳蛋鸭壳腺部存在169个差异miRNAs,包括85个上调的miRNAs,84个下调的miRNAs,共预测获得4 872个靶基因.样本间miRNAs表达量分析发现,各样品之间相关性均较高.富集基因本体(Top Gene Ontology,TopGO)分析结果显示,存在参与血红素结合的分子功能(富集基因23个,P=O.008),并且大部分均伴有电子和Fe2+的转运功能.另外,ggamiR-133b、gga-miR-193b-3p和gga-miR-449a(log2(差异倍数(fold change,FC)＞2)＞1,P＞0.05)预测到了参与胆绿素合成的血红素加氧酶1基因(heme oxygenase 1,HMOX1)和大量的有机物转运代谢基因.以上结果提示,部分miRNAs可能参与了鸭绿壳蛋性状的形成调控,但可能不是影响鸭绿壳蛋形成的主要因素.研究结果为揭示家禽绿壳蛋的形成分子机制提供了理论依据,为基于基因操作技术培育绿壳蛋禽类提供了基础资料.

The objective of this study was to obtain the complete mitochondrial DNA (mtDNA) sequence of Sichuan Sheldrake duck,and to provide reference data for protection and utilization of this genetic resources of duck.Sixteen pairs of primers were designed by Primer 6.0 according to the mitochondrial genome sequences of duck homologous species.The genomic sequence of Sichuan Sheldrake duck's mtDNA were then amplified by PCR and sequencing technology.The results showed that the length of Sichuan Sheldrake duck's mtDNA genome was 16 604 bp,which composed of A (29.19％),C (32.82％),G (15.79％) and T (22.20％).The whole genome contained 22 tRNA genes,2 rRNA genes,13 protein-encoding genes and a non-encoding control area (D-loop area).The analysis by online software of tRNAscan-SE 1.21 and RNA structure 5.6 found that 22 tRNA genes were the standard secondary structures with 4 constant arms which like a clover.The D-loop region of Sichuan Sheldrake duck's mtDNA genome contained Poly (c),TAS,E-box,F-box,D-box,Bird-similarity box and CXB1 conserved box.The neighbor joint (NJ) phylogenetic tree was established by MEGA6.0 basing on the D-loop region and the complete mtDNA sequence,respectively,and the results showed that the Longsheng duck was more closer to Sichuan Sheldrake duck.

过氧化物酶体增殖物激活受体(peroxisome proliferators-activated receptors,PPARs)是核激素受体超家族的一员,分α、β/δ、γ3个亚型,是调节细胞生长和分化的重要因子.为了解PPARs各成员在鸭(Anas platyrhynchos)骨骼肌发育中的不同作用,本研究克隆了鸭PPARα、PPARβ和PPARγ基因启动子区,预测其共同转录因子结合位点,用qRT-PCR检测PPARs及共同转录因子在鸭胚及出生后骨骼肌发育过程中的表达,比较表达模式并进行聚类分析.结果表明,扩增出鸭PPARα、PPAEβ和PPARγ启动子序列分别为2 526、1 631和2 942 bp,GenBank登录号分别为KX845431、KX845432和KX845433.鸭PPARs启动子区存在典型的CAAT-box、TATA-box顺式作用元件,预测存在共同的特异性蛋白1(specificity protein 1,Sp1)、核因子κB(nuclear factor kappa B,NF-κB)和CCAAT增强子结合蛋白(CCAAT/enhancer binding protein alpha,C/EBP-α)转录因子结合位点.PPARs成员在鸭胸肌、腿肌组织中均有表达.在胸肌中,转录因子NF-κB与PPARβ基因的表达模式一致;在腿肌中,Sp1、NF-κB、PPARβ和PPARγ的表达模式一致.PPARs各成员均参与了骨骼肌的发育调控,PPARβ作用可能更大;PPAPβ、PPARγ在鸭骨骼肌发育过程中的功能可能相似;此外,NF-κB可能调控PPARβ在骨骼肌中的表达.研究结果为PPARs基因的表达和功能鉴定提供理论依据.

为研究鸭IL-2基因调控区单核苷酸多态性对其转录调控的影响,克隆获得了鸭IL-2基因启动子2850 bp序列,与人、小鼠和原鸡的同源性分别为35.37％,37.52％和34.74％.其中,-1 400/-1000存在集中的核心转录因子结合位点.对鸭IL-2基因启动子(-1 932/-742)进行单核苷酸多态检测和遗传多态性分析发现,该区域存在两个突变位点(C-1353A、C-1406T),且均处于Hardy-Weinberg极不平衡状态;等位基因A均为优势等位基因.单核苷酸多态与其表达水平的相关性分析发现,突变位点不同基因型与IL-2基因mRNA表达水平和IL-2蛋白水平均无显著相关关系,但基因型AA个体的mRNA表达量均高于其他基因型个体(P＞0.05),表明鸭IL-2启动子等位基因A可能有促进IL-2基因转录的趋势.研究结果为IL-2基因转录调控机制的研究提供了理论基础.

研究旨在建立快速分离鸭早期胚胎的方法,并观察鸭早期胚胎体节、神经系统、心脏、肢芽与胚胎外部轮廓发育特点,为以鸭胚胎为模型开展相关生物学研究提供资料.通过分离鸭早期不同阶段的胚胎、制作石蜡切片和显微观察,了解鸭胚早期发育各阶段的不同形态学特点.结果表明:鸭的卵裂为盘状卵裂;体节形成开始于孵化后27 h左右;心脏从原基经发育成外形呈“S”型,直至心脏随躯体翻转完全向左;胚胎外部轮廓的变化依次为:长条状、头颈部开始弯曲,整体呈“C”型,整体呈“O”型.研究初步整理了鸭早期胚胎发育形态特点,并建立了以滤纸为载体分离鸭早期胚胎的方法,该方法对胚胎的损伤小,更易清理胚胎上的残余物质.