Bad基因在黄体期绵羊生殖器官中表达的比较研究

以黄体期小尾寒羊和萨福克羊为研究对象,采用免疫组织化学技术,针对Bad基因在母羊生殖器官卵巢,输卵管和子宫中的表达与定位进行初步研究,在卵巢中Bad基因均在多数黄体细胞中表达;在输卵管宫管结合部、峡部和壶腹部的分泌细胞Bad基因均有不同程度的表达,未发现纤毛细胞Bad阳性细胞;子宫内膜腺体小泡表达Bad阳性,子宫内膜子叶、基质的阳性细胞数极少,阳性细胞呈轻度着色,显弱阳性.利用计算机图像分析技术测量小尾寒羊和萨福克羊Bad基因表达的阳性细胞率和平均光密度,Bad基因在这两种绵羊组织中的表达规律大体相似,也存在一些明显的差异:萨福克羊的黄体细胞较小尾寒羊中的细胞凋亡更明显,萨福克羊输卵管伞部和壶腹部上皮Bad阳性细胞率均高于小尾寒羊,小尾寒羊子宫内膜子叶和基质Bad阳性细胞率和平均先密度均显著高于萨福克(P＜0.01).结果表明,小尾寒羊和萨福克羊生殖器官中细胞凋亡的差异与Bad基因表达的差异,可能是造成两者繁殖力高低差异的基础.     

Bad基因在小尾寒羊输卵管上皮组织的表达

采用免疫组织化学方法检测小尾寒羊输卵管上皮组织中Bad基因的表达水平,利用计算机图像分析技术测量小尾寒羊生殖周期的不同阶段输卵管上皮组织Bad基因表达的平均光密度和平均阳性面积。结果显示,小尾寒羊输卵管伞部、壶腹部和峡部上皮组织中的部分纤毛细胞在卵泡期和黄体期呈阳性,黄体期纤毛细胞较卵泡期表达强。同时期各部位表达强度的差异不明显。黄体期和卵泡期的分泌细胞均呈阳性,同时期不同部位差异不大,但同部位的分泌细胞黄体期比卵泡期表达强。壶腹部和峡部上皮组织阳性细胞的平均光密度值与阳性面积率差异不显著(P>0.05)。输卵管伞部在黄体期和卵泡期与同时期壶腹部和峡部比较,平均光密度和阳性面积率差异显著(P<0.05)。同一部位不同时期平均光密度值和阳性面积率差异显著(P<0.05)。表明Bad基因参与了输卵管上皮组织周期性变化的调控。     

DAPI检测黄体期绵羊生殖器官中细胞凋亡的比较研究

以黄体期小尾寒羊和萨福克羊为研究对象,采用DAPI原位荧光标记技术,对高繁殖力品种小尾寒羊和低繁殖力品种萨福克羊成年母羊主要生殖器官(卵巢、输卵管、子宫)进行细胞凋亡的确认与定位。结果表明:小尾寒羊和萨福克羊卵巢中黄体细胞有凋亡现象,黄体后期萨福克羊的细胞凋亡较小尾寒羊明显;在萨福克羊黄体期输卵管各部位上皮中发现有极少数分泌细胞具有凋亡特征,而在小尾寒羊未检测到凋亡细胞,两品种羊输卵管上皮下结缔组织有少数凋亡细胞,小尾寒羊和萨福克羊子宫内膜的基质与腺体内皮中均检测到大量凋亡细胞。细胞凋亡是母羊生殖器官变化的重要调节机制,可能与造成两者繁殖力高低差异有关。     

Caspase-9在肉用绵羊主要生殖器官中的分布研究

通过试验研究凋亡因子Caspase-9在肉用绵羊主要生殖器官中的表达,并探讨其生理意义.利用免疫组织化学的方法对Caspase-9在肉用绵羊主要生殖器官中的表达进行研究.研究结果显示,Caspase-9只在细胞质中表达,其在卵巢中主要分布于原始卵泡、初级卵泡及颗粒性黄体细胞,而卵泡膜性黄体细胞中未见表达;黄体期,主要表达于子宫的浅层腺体腺上皮细胞、子宫内膜上皮细胞、子宫颈黏膜固有层的淋巴小结、输卵管黏膜上皮分泌细胞和纤毛细胞、峡部的浆液性腺的腺上皮细胞;卵泡期,子宫和输卵管各个部位阳性反应不明显.在正常生理情况下,Caspase-9参与了肉用绵羊主要生殖器官周期性变化的调控和子宫黏膜免疫,对生殖器官功能的稳定发挥起着重要作用.     

SPLUNC1基因在2种绵羊不同组织器官中差异表达分析

SPLUNC1是抗病原微生物感染的关键蛋白.为研究SPLUNC1基因在2种绵羊不同组织器官中的差异表达情况,试验选取健康新疆阿勒泰羊5只,多胎萨福克羊5只,放血处死后分别采集气管、支气管、肺脏、胃、食管、结肠、心脏、脾脏、皮肤,提取RNA,反转录合成cDNA.以cDNA为模板,扩增SPLUNC1基因748bp特征片段,...     

BMPR-IB基因对绵羊外周血生殖激素水平的影响

为研究不同BMPR-IB基因型多胎萨福克羊外周血中生殖激素水平的差异,以培育的发情期多胎萨福克母羊为实验对象,应用PCR-RFLP技术检测个体的BMPR-IB基因型,ELISA方法检测绵羊外周血中E2、FSH、LH、PRL和P4的含量,分析BMPR-IB基因型与5种生殖激素水平的相关性.结果表明：多胎萨福克羊群体中存在BMPR-IB基因的BB、B＋和＋＋三种基因型.＋＋基因型个体的平均E2含量和P4含量显著低于B＋基因型和BB基因型个体（P＜0.05）,3种基因型个体间平均FSH含量、LH含量和PRL含量差异均不显著（P＞0.05）.BMPR-IB基因突变导致发情期母羊的雌二醇和孕酮的分泌量增加.研究结果可为合理利用BMPR-IB基因培育多胎肉羊提供依据.     

USP26基因在热应激前后绵羊睾丸中的差异表达及生物信息学分析

试验旨在探究USP26基因在热应激前后的吐鲁番黑羊及萨福克羊睾丸中的差异表达,以期为研究USP26的生物学功能及作用机制提供依据。通过生物信息学软件分析绵羊USP26基因序列、编码蛋白的理化性质、疏水性、跨膜区域、信号肽、二级结构和三级结构,然后选取热应激前后的吐鲁番黑羊及萨福克羊活体去势后提取睾丸总RNA,反转录后进行实时荧光定量PCR,分析USP26基因在热应激前后不同羊之间mRNA的差异表达。结果表明,绵羊USP26基因序列与山羊的亲缘关系最近,与弯角大羚羊次之,与野猪最远。绵羊USP26蛋白分子量100 617.20,不稳定系数46.91,总平均亲水系数-0.580,属于不稳定的亲水性蛋白;通过对蛋白亚细胞定位发现,绵羊USP26蛋白主要存在于细胞核（69.6%）和细胞质（17.4%）中;绵羊USP26蛋白不属于跨膜蛋白和分泌蛋白;通过对二级结构和三级结构预测发现,其主要包含α-螺旋、β-转角、延伸链和无规则卷曲4种结构,其中α-螺旋（39.27%）及无规则卷曲（44.75%）占绝大部分。吐鲁番黑羊的USP26基因在热应激前后的睾丸中mRNA的表达无差异（P>0.05）,...     

家蚕凋亡蛋白酶(caspase-1)基因在大肠杆菌中的表达

为了研究家蚕凋亡蛋白酶(caspase-1)的原核表达及剪切方式,用PCR法扩增出家蚕caspase-1基因的cD-NA,然后插入pET28a载体,得到阳性质粒pET28a-caspase-1,并转化感受态细胞BL21,用异丙基硫代-β-D-半乳糖苷(IPTG)诱导了caspase-1蛋白的表达。应用SDS-PAGE和Western blot分析对其性质作检测,诱导3 h后caspase-1蛋白得到剪切,5 h开始降解。研究结果表明,caspase-1基因可在大肠杆菌BL21中诱导表达,并且能够进行自我剪切。     

家禽免疫器官内细胞凋亡及其凋亡相关基因调控研究进展

细胞凋亡是生物界广泛存在的一种重要的生命过程,是多细胞动物为调节机体发育、维护内环境稳定、有基因调控的细胞主动性死亡过程。在细胞凋亡的分子生物学研究过程中,发现已有多种基因参与细胞凋亡的调控,其中包括Bcl-2家族和Bax、p53、Fas和FasL等。家禽免疫器官胸腺、脾脏和法氏囊内存在着自然凋亡现象,许多学者用物理性、化学性和生物性因素诱导了家禽免疫器官细胞凋亡,证实了细胞凋亡相关基因参与了家禽免疫器官内细胞凋亡的调控。     

BMPR-IB基因作为影响蒙古羊双羔性状候选基因的研究

为了探索多胎候选基因BMPR-IB与蒙古羊双羔群体繁殖性状之间的关系,试验采用聚合酶链式反应-限制性片段长度多态性分子标记技术(PCR-RFLP),以蒙古羊单羔群体(18只)、多胎类型小尾寒羊(30只)为对照组对蒙古羊双羔群体(50只)进行了BMPR-IB基因的遗传多态性分析。结果表明:蒙古羊双羔群体和蒙古羊单羔群体中都不存在BMPR-IB基因的FecB(A746G)突变,说明BMPR-IB基因不是影响蒙古羊双羔性状的主效基因;而在多胎品种小尾寒羊中检测到了相应的突变,发现了3种基因型,其突变纯合子(BB)、杂合子(B+)和野生型(++)的基因型频率分别是0.100,0.733,0.167,将其与产羔数进行关联分析,结果BB基因型母羊和B+基因型母羊平均产羔数差异极显著(P0.01);经x2适合性检验,小尾寒羊该基因位点显著偏离Hardy-Weinberg平衡(P0.05),由此推断BMPR-IB基因是控制小尾寒羊多胎性能的主效基因。     

Combined microRNAome and transcriptome analysis of follicular phase and luteal phase in porcine ovaries

The aim of this study was to explore the expression difference of miRNAs and mRNAs between the follicular phase (FP) and luteal phase (LP) in porcine ovaries and provide a theoretical basis for the research on mammalian reproductive regulation. RNA‐Seq and miRNA‐Seq were used to identify differentially expressed genes (DEGs) and miRNAs (DEMs) between the FP and LP in ovaries of six sows (3‐year‐old Yorkshire pigs with similar weights and same parities). Bioinformatic analysis was used to screen potential genes and miRNAs related to porcine ovarian function. Real‐time qualitative PCR was used to validate the sequencing results. RNA‐Seq results showed that 3,078 genes were up‐regulated, and 1,444 genes were down‐regulated in the LP compared with the FP, and DEGs were significantly enriched in 242 Gene Ontology (GO) terms and 33 Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. miRNA‐Seq identified 112 DEMs, of which 25 were up‐regulated and 87 were down‐regulated in the LP compared with the FP. We obtained 186 intersection genes (IGs) between the 4,522 DEGs and 2,444 target genes predicted from the 112 DEMs. After constructing a miRNA‐gene‐pathway network, we identified key miRNAs and genes including miR‐17‐3p, miR‐214, miR‐221‐5p, miR‐125b, FGF1, YWHAG, YWHAZ, FDFT1 and DHCR24, which are enriched in Hippo and PI3K‐Akt signalling pathways, and various metabolic pathways. These results indicate that these key genes and miRNAs may play important roles in the developmental transition from FP to LP in porcine ovaries and represent candidate targets for further study.     

Intron 1 of the interferon γ gene: Its role in nematode resistance in Suffolk and Texel sheep breeds

Genetic variation at intron 1 of the interferon gamma gene has recently been associated with variation in faecal egg count in Romney, Merino and Soay breeds of sheep. The Texel breed is more resistant to gastrointestinal nematode infection than the Suffolk breed, based on faecal egg count. Hence, the objective of this experiment was to characterise the polymorphisms of intron 1 of the interferon gammagene in Suffolk and Texel sheep, and to determine if the characterised haplotype variants were associated with faecal egg count variation in these breeds. Intron 1 haplotypes and faecal egg measurements were determined for Suffolks (n=113) and Texels (n=135). Four haplotypes were identified in Suffolks (A, B, C and D), two haplotypes as previously described, and two previously unknown haplotypes. However, there was no association between interferon gamma intron 1 haplotype and faecal egg count in Suffolks. In contrast, two previously identified haplotypes (A and B) were present in the Texel breed, and the B haplotype was associated with resistance to nematode infection (P=0.02). These results suggest that intron 1 of the interferon gamma gene has an important role in resistance to nematode infection in the Texel breed.     

The expression of VEGF,myoglobin and CRP2 proteins regulating endometrial remodeling in the porcine endometrial tissues during follicular and luteal phase

Endometrial remodeling is important for successful embryo development and implantation in pigs. Therefore, this study investigated change of proteins regulating endometrial remodeling on follicular and luteal phase in porcine endometrial tissues. The endometrial tissue samples were collected from porcine uterus during follicular and luteal phase, vascular endothelial growth factor (VEGF), myoglobin and cysteine‐rich protein 2 (CRP2) proteins were expressed by immnofluorescence, immunoblotting, and determined by 2‐DE and MALDI‐TOF/MS. We found that VEGF, myoglobin and CRP2 were strongly localized in endometrial tissues during luteal phase, but not follicular phase. The protein levels of VEGF, myoglobin and CRP2 in endometrial tissues were higher than luteal phase (P < 0.05). These results may provide understanding of intrauterine environment during estrous cycle in pigs, and will be used in animal reproduction for developing specific biomarkers in the future.     

Amino acids profile within peripheral blood and follicular fluid based on high‐performance liquid chromatography methods may explain differences in folliculogenesis between short‐term under/over‐fed treatments during luteal phase of Hu sheep

The nutritional alteration of amino acids (AAs) profile in physiological fluid was poorly characterized in livestock. After oestrus synchronization, 24 ewes were randomly assigned to two groups based on the nutrient requirement recommended for maintenance (M): the feed‐supplemented group (S, 1.5 × M, N = 12) and feed‐restricted group (R, 0.5 × M, N = 12) on days 6–12 of their oestrous cycle, which occurred shortly before ovulation. The concentration of 30 AAs in peripheral blood (PB) and follicular fluid (FF) was quantified to calculate the PB‐to‐FF concentration gap for each AA and determine its correlation with metabolites and hormones in PB and FF. Results showed that the feed restriction enlarged the oestrous cycle length, decreased the number of follicles 2.5–3.5 mm, increased the number of follicles >3.5 mm and augmented the volume of follicles >2.5 mm. Nineteen AAs from PB were significantly different between the groups. The phosphoethanolamine (PEtN) and ration of essential AAs to nonessential AAs (EAA/NEAA) in FF significantly (p < 0.05) increased and decreased in the R group, respectively. Most AAs, except aspartate (Asp) and carnosine (Car) in the R group and alanine (aAla) in both groups, were significantly lower within FF than those within PB. The correlation of AAs with FSH and progesterone (P₄) was more significant than that of AAs with other endocrine milieu characteristics. In conclusion, our results revealed that the influence of short‐term nutritional manipulation during luteal phase on folliculogenesis might not be due to the variation of intrafollicular AAs profile but rather attribute to the peripheral blood AAs profile alteration.     

鸡褪黑激素受体基因在开产前后不同组织中的表达差异

为了从分子水平上了解鸡褪黑激素受体（MTNR）基因在开产前后不同组织中的表达差异,以β-actin基因为内标基因,运用实时荧光定量PCR方法,比较了MTNR1A、MTNR1B和MTNR1C这3个受体基因在心脏、肝脏、输卵管、卵巢、脑和视交叉各组织及在鸡开产前后表达量的差异。结果表明,MTNR1A只在开产后各组织中的表达量存在差异,其中以卵巢组织表达量最高,心脏中表达量最低。MTNR1B和MTNR1C的表达量在开产前或开产后均表现出组织间的显著差异;在开产前后的各组织中,MTNR1B的表达量均以视交叉为最高,卵巢为最低;而MTNR1C的表达量在开产前以心脏中最高,开产后在视交叉中最高,在卵巢组织中的表达量开产前后均为最低。同一组织开产后与开产前比较,MTNR1C基因在心脏、输卵管和脑组织中的表达量呈显著下调趋势。因此,3个受体基因在鸡的不同组织和生理时期存在表达差异。     

血管活性肠肽及其基因在小鼠卵巢中的定位

为探明血管活性肠肽（VIP）及其基因在青春期、妊娠期、哺乳期和泌乳后期小鼠卵巢中分布的变化规律,分别采用免疫组织化学PV-9000两步法和原位杂交法确定VIP及其mRNA在卵巢中的定位。结果显示：VIP及其基因主要定位在是黄体细胞和卵泡上,通过单因素方差分析表明在小鼠卵巢内,泌乳后期表达量相对其他3期差异极显著（P〈0.01）。VIP及其基因在不同生理时期内小鼠卵巢的表达量不同,即随着日龄的增加表达量呈上升趋势,且泌乳后期表达量最大。     

牛c-Myc/TAT/9R融合表达载体构建及其原核表达研究

原癌基因c-Myc虽然在许多肿瘤组织中特异性高表达,但它也是机体正常细胞生长与增殖所必需的因子,然而目前家畜诱导多潜能干细胞（iPS）研究中关于多功能转录因子c-Myc的报道很少.为此,研究拟利用蛋白转导区域（PTD）的跨膜作用,构建TAT-bc-Myc-9R融合表达载体,并对其进行原核表达,旨在为转录因子蛋白重编程牛体细胞奠定基础.试验以牛c-Myc基因编码区序列为参考,设计并合成含酶切位点、跨膜转导肽TAT和9R的引物,利用PCR等基因工程方法获得包括牛c-Myc、TAT和9R的重组原核表达载体（pTAT-HA-bc-Myc-9R）;酶切鉴定和DNA测序结果表明,试验成功获得bc-Myc-9R重组序列,成功构建重组质粒pTAT-bc-Myc-9R;不同IPTG浓度和诱导时间条件对该重组载体的诱导表达和SDS-PAGE分析表明,TAT-bc-Myc-9R在0.6 mmol/L IPTG和37℃诱导6h条件下表达约57KDa的目的蛋白.这些结果为PTD介导的转录因子蛋白直接重编程牛体细胞以获得iPS研究积累了科学资料.