牦牛PRLHR基因的克隆、序列分析及组织表达研究

试验旨在对牦牛催乳素释放激素受体（prolactin releasing hormone receptor,PRLHR）基因进行克隆、序列分析及组织表达研究。采集5头母牦牛和5头母黄牛的下丘脑、脑垂体前叶、卵巢、输卵管和子宫组织,采用RT-PCR技术扩增得到PRLHR基因cDNA全长,通过生物信息学方法分析该基因编码蛋白的生物信息学特征,利用实时荧光定量PCR技术测定PRLHR基因在牦牛及黄牛各组织中的表达量。结果显示,牦牛PRLHR基因序列长1 625 bp,其中CDS区1 113 bp、5'-UTR 22 bp和3'-UTR 490 bp,编码370个氨基酸,与黄牛、水牛、绵羊、猪和人的核苷酸序列有较高的同源性,在进化过程中十分保守;牦牛PRLHR为不稳定疏水蛋白,无信号肽,存在7个跨膜结构域;有13个丝氨酸磷酸化位点、6个苏氨酸磷酸化位点和4个酪氨酸磷酸化位点;有3个N-糖基化位点和10个O-糖基化位点;蛋白二级结构中α-螺旋、无规则卷曲、延伸链和β-转角分别为49.19%、31.89%、15.68%和3.24%;蛋白质三级结构预测显示,牦牛PRLHR蛋白具有GPCRs超级家族中PrRP家族的典型结构域。实时荧光定量PCR结果表明,PRLHR基因在牦牛输卵管组织中的表达量显著高于其他组织（P<0.05）;在牦牛下丘脑、脑垂体前叶、子宫和输卵管组织中的表达量极显著高于黄牛（P<0.01）。试验成功克隆得到牦牛PRLHR基因序列,并对其进行了生物信息学和组织表达特性分析,为进一步研究PRLHR基因在牦牛繁殖活动中的调控作用奠定了基础。     

牦牛FSHR基因克隆及其在生殖轴中的表达研究

本研究旨在阐明牦牛促卵泡素受体(follicle stimulating hormone receptor,FSHR)基因CDS序列及其在牦牛生殖轴中表达的特点,为探讨其在牦牛繁殖活动中的调控作用奠定基础。试验采集卵泡期的牦牛与黄牛下丘脑、脑垂体前叶、卵巢、输卵管及子宫组织,通过RT-PCR技术对牦牛FSHR基因cDNA进行扩增、克隆与序列分析;采用实时荧光定量PCR法检测FSHR基因在牦牛与黄牛中的组织表达差异。结果显示,牦牛FSHR基因编码区全长2 088bp,编码695个氨基酸,蛋白质分子式为C6378H10670N2088O2637S576,分子质量为177 263.85u,理论等电点(pI)为4.88,与黄牛、绵羊、山羊和猪氨基酸序列同源性较高(91.50%～99.38%)。FSHR蛋白为酸性不稳定疏水蛋白,存在信号肽与8个跨膜结构;二级结构由延伸链(15.54%)、α-螺旋(42.30%)、β-转角(1.44%)和无规则卷曲(40.72%)组成;系统进化树表明,牦牛与黄牛亲缘关系最近;实时荧光定量PCR结果显示,FSHR基因在黄牛、牦牛检测组织中均有表达,牦牛子宫中表达量显著或极显著高于除卵巢外的其他组织(P<0.05;P<0.01),而黄牛卵巢中表达量显著或极显著高于其他组织(P<0.05;P<0.01);黄牛卵巢中表达量极显著高于牦牛(P<0.01)。说明FSHR基因在动物进化中较为保守,其在牦牛卵巢中表达量低可能影响到牦牛繁殖机能。     

Whole‐genome analysis identifying candidate genes of altitude adaptive ecological thresholds in yak populations

The domestic yak (Bos grunniens) is an iconic symbol of animal husbandry on the Qinghai–Tibet Plateau. Long‐term domestication and natural selection have led to a wide distribution of yak, forming many ecological populations to adapt to the local ecological environment. High altitude is closely related to oxygen density, and it is an important environmental ecological factor for biological survival and livestock production. The aim of the present study was to perform a preliminary analysis to identify the candidate genes of altitude distribution adapted ecological thresholds in yak using next‐generation sequence technology. A total of 15,762,829 SNPs were obtained from 29 yaks with high‐ and low‐altitude distribution by genome‐wide sequencing. According to the results of the selective sweep analysis with FST and ZHp, 21 candidate genes were identified. 14 genes (serine/threonine protein kinase TNNI3K, TEN1, DYM, ITPR1, ZC4H2, KNTC1, ADGRB3, CLYBL, TANGO6, ASCC3, KLHL3, PDE4D, DEPDC1B and AGBL4) were grouped into 32 Gene Ontology terms, and four genes (RPS6KA6, ITPR1, GNAO1 and PDE4D) annotated in 35 pathways, including seven environmental information processing and one environmental adaptation. Therefore, the novel candidate genes found in the current study do not only support new theories about high‐altitude adaptation, but also further explain the molecular mechanisms of altitude adaptation threshold in yaks.     

CK1 inhibitor affects in vitro maturation and developmental competence of bovine oocytes

The objectives of present study were to evaluate the effect of casein kinase 1 (CK1) inhibition D4476 on in vitro maturation (IVM) and developmental competence of bovine oocytes. The cumulus oocyte complexes (COCs) were cultured in maturation medium with D4476 (0, 2, 5, 10, 20 μM) for 24 hr. After IVM and in vitro fertilization, through expansion average scores of cumulus cells (CCs), oocyte maturation efficiency, cleavage rate and blastocyst rate of zygote, we found 5 μM D4476 could increase the development potential of oocytes. After the COCs were treated with 5 μM D4476, the results of quantitative real‐time PCR analysis, Lichen red staining and PI staining showed that under without affecting germinal vesicle breakdown and nuclear morphology, D4476 could significantly decrease CK1 and upregulate TCF‐4 in oocytes. Furthermore, without influencing the level of Bad and CTSB, D4476 could significantly increase the expression of β‐catenin, TCF‐4, Cx43, MAPK, PTGS‐2, PTX‐3, TGS‐6, Bax and Bcl‐2 in CCs. Western blot analysis revealed that the addition of 5 μM D4476 during the maturation of COCs resulted in a lower level of Cx43 protein at 12 hr and a higher expression of Cx43 protein at 24 hr compared to the group without D4476. These results indicate that adding optimum D4476 (5 μM) to maturation medium is beneficial to maturity efficiency and development competence of bovine oocytes.     

低温贮藏对调理牦牛肉品质的影响

本试验以经过一定加工工艺处理后的调理牦牛肉为试验原料,将调理牦牛肉在4℃和-4℃两种条件下进行贮藏,测定调理牦牛肉pH值、蒸煮损失、肉色、质构等品质特性,并进行相关性分析。结果表明:在4℃贮藏条件下,随着贮藏时间的增加,调理牦牛肉的pH值、蒸煮损失、亮度值L^*值呈增大的趋势;红度值a^*、硬度呈减小的趋势。在-4℃贮藏条件下,随着贮藏时间的增加,调理牦牛肉的pH值、亮度值L^*、黄度值b^*、咀嚼性呈增大的趋势;红度值a^*、硬度、弹性呈减小的趋势。     

青稞替代玉米对育肥牦牛生产性能和肉品质的影响

本试验旨在研究青稞替代玉米对育肥牦牛生产性能、屠宰性能、养分表观消化率及肉品质的影响。采用2×3交叉设计,以日粮类型(玉米、60%青稞+40%玉米)和不同精粗比(30∶70、40∶60和50∶50)为两因素,选取36头年龄4周岁左右、体重为(144±21.0)kg的麦洼公牦牛,随机分为6组,每组6个重复,每个重复1头牛。预饲期15d,正试期180d。结果表明:1)日粮类型及精粗比对牦牛生产性能无显著影响(P>0.05)。2)青稞-玉米组的净肉率显著高于玉米组(P<0.05),但屠宰率和眼肌面积无显著差异(P>0.05);精粗比对屠宰性能无显著影响(P>0.05);日粮类型和精粗比二因素对眼肌面积的互作影响显著(P<0.05),以青稞-玉米(40∶60)组效果最优。3)日粮类型对养分表观消化率无显著影响(P>0.05);与30∶70相比,40∶60和50∶50极显著提高了有机物(OM)和总能(GE)的表观消化率(P<0.01)、显著提高了粗蛋白(CP)的表观消化率(P<0.05);日粮类型和精粗比二因素对GE、OM和粗脂肪(EE)的表观消化率有显著的互作影响(P<0.05),以玉米(40∶60)组效果最佳。4)青稞-玉米组和玉米组背最长肌亮度值(L*)、红度值(a*)、pH值均无显著差异(P>0.05),但青稞-玉米组的黄度值(b*)和滴水损失显著低于玉米组(P<0.05)、蒸煮损失极显著低于玉米组(P<0.01);随着精粗比的升高,蒸煮损失有逐渐降低的趋势(P=0.075)。5)日粮类型对背最长肌的嫩度无显著影响(P>0.05);随着精粗比的升高,剪切力逐渐减小(P>0.05),肌纤维直径有减小的趋势(P=0.059),肌纤维密度显著增加(P<0.05),肌纤维面积显著减小(P<0.05)。综上所述,以青稞替代日粮中60%的玉米能够提高育肥牦牛的屠宰性能,改善牦牛肉的品质;日粮精粗比为40∶60时饲喂效果最佳。     

牦牛EPF的原核表达及多克隆抗体制备

本试验旨在制备牦牛早孕因子(early pregnancy factor,EPF)多克隆抗体,为牦牛妊娠早期高效快速诊断技术研究奠定基础.采用RT-PCR方法,从牦牛胎盘和卵巢组织中提取RNA,反转录为模板后进行EPF基因扩增,并将其克隆到改造后的载体pET28-His10-Sumo上,构建pET28-His10-Su...     

酪蛋白源阿片物质的分离、纯化及活性鉴定

酪蛋白的胃蛋白酶水解物通过RP-HPLC进行分离,并用NG108-15 细胞对其进行阿片样活性鉴定。实验结果表明:酪蛋白经胃蛋白酶处理2 h 后, 其酶解产物中有阿片样活性物质产生, 而酪蛋白本身无阿片活性。     

云南主要地方牛种肉质特性研究

 对大额牛、云南瘤牛、中甸耗牛、迪庆黄牛和中甸犏牛的肉质特性作了系统测试和分析.结果表明,它们在自然放牧条件下,均表现出优良的肉质特性.肌肉的蛋白质含量均高于19%,脂肪含量均低于1.40%.