牦牛StAR基因克隆及其生物信息学与组织表达特性的研究

旨在克隆获得牦牛StAR基因编码序列（CDS）并进行生物信息学分析,探究其mRNA组织表达特性。本研究以屠宰场采集的成年母牦牛心、肝、脾、肺、肾、卵巢、输卵管、子宫组织（n=5）,不同年龄（胎牛、1岁、2岁）牦牛的卵巢（n=3）,不同发情周期（卵泡期、黄体期）的牦牛卵巢（n=3）,黄体期黄牛的卵巢（n=3）及实验室冻存的牦牛颗粒细胞为研究材料。以牦牛黄体期卵巢cDNA为模板,用逆转录PCR克隆StAR基因,并使用MEGA7.0和ExPASy-ProtParam等软件分析其生物信息学特性;采用实时荧光定量PCR技术分析牦牛StAR基因组织表达特性。结果发现,StAR基因CDS区长858 bp,编码285个氨基酸,StAR蛋白总体带正电荷,属于碱性亲水稳定蛋白,无跨膜结构及信号肽,主要存在于细胞质和线粒体; StAR基因具有较高的保守性,符合物种进化规律。牦牛StAR基因在卵巢表达水平最高（P<0.01）,且2岁时卵巢表达水平极显著高于胎牛和1岁龄（P<0.01）,黄体期卵巢表达水平极显著高于卵泡期（P<0.01）;黄体期黄牛卵巢中StAR基因的表达量极显著高于牦牛（P<0.01）;在颗粒细胞的体外培养过程中StAR基因表达量逐渐上升,在培养24 h时达到高峰（P<0.01）,随后显著降低。综上所述,StAR基因序列较为保守,在牦牛卵巢组织中表达最高,且表达水平随年龄与卵巢周期而变化,提示StAR基因可能参与牦牛卵巢及黄体功能相关的繁殖调控。     

鸡FKBP5基因的克隆、生物信息学及组织表达谱分析

本研究旨在对鸡FK506结合蛋白5(FK506 binding protein 5,FKBP5)基因进行克隆和生物信息学分析,并检测其在鸡不同组织中的表达量。以鸡脾脏cDNA为模板,通过PCR方法扩增和克隆鸡FKBP5基因完整CDS区序列,并进行同源性比对及系统进化树构建;使用在线软件分析FKBP5蛋白的理化性质、疏水性、跨膜结构、信号肽、二级结构和三级结构;利用实时荧光定量PCR检测其在肢体内外翻畸形(valgus-varus deformity,VVD)组肉鸡和正常组肉鸡组织中的表达量,并绘制组织表达谱。结果显示,鸡FKBP5基因CDS区序列全长1350 bp,编码449个氨基酸;同源性比对和进化树分析表明,鸡FKBP5基因氨基酸序列与鹌鹑、鸭、壁虎、中华鳖、小鼠、人、猪、斑马鱼的同源性分别为98.4%、96.2%、85.8%、87.4%、81.1%、85.2%、86.1%和61.2%,与鹌鹑、鸭的亲缘关系最近,爬行类和哺乳类动物次之,与斑马鱼(鱼类)亲缘关系最远。鸡FKBP5蛋白分子质量为50.43 ku,理论等电点(pI)为5.94,半衰期为30 h,肽链N端为蛋氨酸(Met),不稳定系数为23.80,属于稳定蛋白;跨膜区和信号肽预测结果显示,FKBP5蛋白不属于跨膜蛋白和分泌型蛋白。结构域分析结果表明,该蛋白包括2个FKBP型肽基脯氨酰异构酶(FKBP-type peptidylprolyl isomerases)、3个四肽重复(TPR)序列,主要包含α-螺旋(46.77%)、延伸链(12.47%)和无规则卷曲(40.76%),且该蛋白与HSPA2、PPID、STIP1、HSP90AA1和HSP90AB1等互作蛋白具有很强的相关性。实时荧光定量PCR结果显示,FKBP5基因在鸡各组织中广泛表达,其中在软骨和法氏囊中表达量较高,在发病组肉鸡组织中的表达量均高于正常组肉鸡,且在心脏、腿肌、法氏囊、胸腺、软骨中表达量差异显著(P<0.05),在脾脏中表达量差异极显著(P<0.01)。本试验结果可为肉鸡骨骼疾病及腿部健康选育提供参考。     

Characterization of tuberculous granulomas in different stages of progression and associated tertiary lymphoid tissue in goats experimentally infected with Mycobacterium avium subsp. hominissuis

Oral infection of goats with Mycobacterium avium subsp. hominissuis (MAH) resulted in a large variety of granulomas in organized gut-associated lymphatic tissues and intestinal lymph nodes. To characterize the cellular composition of granulomas, CD4⁺, CD8⁺, γδ, B lymphocytes and plasma, CD25⁺, CD68⁺, MHC-II⁺, Ki67⁺ and endothelial cells were labeled in consecutive frozen sections by immunohistochemistry and acid fast bacilli (AFB) by Kinyoun stain. Granulomas with extensive necrosis, little mineralization and variable numbers of AFB surrounded by many CD4⁺ T cells, but only few epitheloid macrophages were observed in severely sick goats at 2–3 mpi. They were interpreted as exuberant immune reaction. Organized granulomas with very few AFB were seen in clinically healthy goats at 13 mpi. The necrotic cores were surrounded by a zone of granulomatous infiltrate with many epitheloid macrophages and few lymphocytes. This zone was initially wide and highly vascularized and became progressively smaller. It was enclosed by an increasing layer of connective tissue. All organized granulomas were surrounded by compartimentalized tertiary lymphoid tissue. The granulomas in experimental infection of goats with MAH reflect the heterogeneity of lesions seen in mycobacterial infections of humans and ruminants and are therefore valuable for comparative research.     

Effect of urea supplementation on performance and safety in diets of Dorper crossbred sheep

This study was conducted to evaluate the efficacy and safety of dietary urea in sheep rations having a 50:50 concentrate:roughage ratio. Sixty‐four Dorper × thin‐tailed Han crossbred ram lambs with an average body weight of 30.8 (±0.02) kg were randomly divided into four groups of 16 sheep each, and each group was fed one of the following diets: a basal diet (CON), or CON supplemented with 0.5% (0.5UTM), 1.5% (1.5UTM) or 2.5% (2.5UTM) urea. Growth performance, carcass characteristics, non‐carcass offals, meat quality and peptic tissue lesions were assayed. The average daily weight gains for CON, 0.5UTM, 1.5UTM and 2.5UTM were 216, 218, 200 and 170 g, respectively, with the CON and 0.5UTM groups higher than 2.5UTM group (p < 0.05). Sheep from the 2.5UTM treatment had a significantly lower dry matter intake (1.29 kg/day) than those from the CON and 0.5UTM treatments (1.42 and 1.43 kg/day, p < 0.05), and the feed conversion ratio in the 2.5UTM group was the highest (p < 0.05). Carcass characteristics, including shrunk body weight, empty body weight, hot carcass weight, dress percentage, and the absolute or relative weight (% body weight) of heart, liver, spleen, lung and kidney, were not altered by the treatments (p > 0.05). The muscular pH of 2.5UTM was higher than that of CON (5.68 vs. 5.52, p < 0.05), and shear force in 0.5UTM was lower compared with CON and 2.5UTM (p < 0.05). The anatomical structure lesions in kidneys became more serious with the increasing dietary urea concentrations, with the 2.5UTM animals showing the most severe lesions compared with CON animals. Therefore, supplementary urea as a non‐protein nitrogen source for sheep should not exceed 1.5% of ration having a 50:50 concentrate:roughage ratio to ensure efficacy and safety.     

TeMV与PWV侵染对西番莲代谢生理及组织微结构的影响

以西番莲品种‘台农1号’为实验材料,通过电镜扫描技术比较观测TeMV和PWV侵染与健康西番莲的叶片、幼嫩果实外果皮组织形态结构,分析测定其叶片生理生化变化,研究西番莲病毒侵染对西番莲叶片和幼嫩果实外果皮组织结构和生理生化的影响。结果表明：TeMV与PWV侵染后,西番莲植株叶片的海绵组织细胞间隙缩小,发生成团堆积状排列,栅栏组织细胞萎缩,呈不规则长条形;幼嫩果实外表皮组织中石细胞层发生严重断裂,排列松散,海绵层细胞变得松散,且发生较严重的木质化现象;叶片与幼嫩果实外表皮表面纹理粗糙、褶皱,气孔保卫细胞萎缩,气孔周围组织皱缩、粗燥;感病果实外果皮表面上的腺毛凸起无序。病毒侵染后,感病植株第1～3叶的蔗糖含量较第4～6叶的高23.11%;健康叶片则相对平衡,降低了淀粉积累、PG酶、纤维素酶与PPO酶活性,提高了感病植株第1～3叶的α-淀粉酶、GS酶与POD酶活性。西番莲受TeMV与PWV侵染后,植株叶片第1～6叶营养物质分配不平衡,相关物质的合成分解与抗氧化防御酶系统受伤害,叶片与果实组织形态结构受到不同程度的损坏,植株正常吸收功能遭到破坏。     

超高效液相色谱-串联质谱法测定猪牛组织中甲基盐霉素残留量

建立超高效液相色谱-串联质谱法测定猪牛组织中甲基盐霉素残留量。猪、牛组织中残留的甲基盐霉素,经乙腈溶液提取,硅胶固相萃取柱净化,超高效液相色谱-串联质谱法测定,外标法定量。甲基盐霉素在5 ng/mL～250 ng/mL的浓度范围内呈现良好的线性关系,甲基盐霉素在猪、牛肌肉、肝脏、肾脏、脂肪中的检测限均为2.5μg/kg,定量限均为5.0μg/kg,猪牛肌肉、肝脏、肾脏、脂肪中5.0 ng/g～100 ng/g添加浓度范围内的回收率均值为71.9%～88.4%,批内批间RSD值均<15%。