牦牛HSP72基因的结构及生物信息学分析

克隆测序了牦牛HSP72基因的全序列,并分析了该基因的结构,以及HSP72蛋白的氨基酸组成、等电点、亚细胞定位、跨膜区、疏水,亲水区、结构域、特征位点、密码子偏好性、二级结构等蛋白质性质。结果表明：牦牛的HSP72基因序列全长为1926bp,无内含子,共编码641个氨基酸;牦牛的HSP72基因和HSP72蛋白与普通牛、猪、人相比存在着一定差异,这可能是导致他们之间对温度适应性差异的主要原因之一。     

牦牛Myf-5基因的克隆测序及其系统进化研究

本研究对牦牛(九龙牦牛)的生肌决定因子5(Myf-5)基因进行了T-A克隆测序和分析,并与多个物种的相应基因编码区核苷酸序列、氨基酸序列进行了比对分析,构建了物种间的系统进化树.结果 表明:①牦牛的Myf-5基因大小为3313 bp,由3个外显子和2个内含子组成,与普通牛等9个物种比较,在基因大小上有较大的差异,但外显子和内含子的组成一致.②牦牛、大额牛、瘤牛、水牛、普通牛、人、恒河猴、黑猩猩、狗、家鼠、软体贝壳、鸡、斑马鱼等物种间Myf-5基因编码区的核苷酸序列同源性较高,其中,牦牛、大额牛、瘤牛、水牛、普通牛间的同源性最高,达98.4%以上,说明Myf-5基因编码区核苷酸序列在动物物种间具有较高的保守性.③牦牛、大额牛、瘤牛、水牛、普通牛、黑猩猩、恒河猴、人、狗、家鼠、软体贝壳、鸡、斑马鱼等物种间Myf-5基因编码蛋白的氨基酸序列具有较高的同源性,保守性强,这一结果与编码区核苷酸序列的比对结果基本一致.④根据核苷酸序列,用NJ法构建的牦牛、大额牛、瘤牛、水牛、普通牛、人、恒河猴、黑猩猩、狗、家鼠、软体贝壳、鸡、斑马鱼等13个物种的分子系统进化树显示:牦牛、普通牛、瘤牛、水牛和大额牛在较近的亲缘关系下聚为一大类,人、黑猩猩和恒河猴聚为另一大类,然后这两类再和其他物种相聚.这一分类结果与各物种的动物学分类结果和血液蛋白、mtDNA水平上的聚类结果基本一致,支持牦牛、普通牛和瘤牛3个物种间不应该是属间或亚属间关系,而应是同一属下的不同种,将牦牛、普通牛和瘤牛划分在同一个属--牛属(Bos),而将水牛划分在另一个单独的属的观点.同时也显示该基因序列适合用于动物学分类.     

牦牛CAPN2基因的电子克隆及生物信息学分析

目的:对牦牛CAPN2基因进行电子克隆和序列分析,以期为进一步研究牦牛该基因与其肉质性状相关性以及基因结构和表达奠定理论基础。方法:利用人的CAPNA2基因cDNA序列为探针,在EST数据库进行同源性筛选,运用CAP3进行拼接得到完整序列并结合生物信息学进行序列分析。结果:牦牛CAPN2基因全长3 200bp,包含一个2 103bp的开放阅读框,共编码700个氨基酸;牦牛CAPN2的核苷酸序列与普通牛、羊、马、猪、小鼠、鸡、人相应序列间的一致性分别为99%、97%、90%、87%、85%、79%、90%。经聚类分析,牦牛首先与普通牛聚在一起,然后与羊、猪聚为一类;人与小鼠聚为一类;这两类相聚为一大类后,再依次与马、鸡相聚,其结果与以往的生物学分类结果一致。牦牛CAPN2基因编码蛋白的分子式为C3569H5503N255O1082S26,相对分子质量为79 935.2,理论等电点PI为4.86。结论:牦牛与普通牛、羊、马、猪、小鼠、鸡、人在CAPN2基因的编码序列具有较高有一致性。该基因编码的蛋白为位于细胞质中的水溶性蛋白。     

牦牛MC4R基因及其蛋白结构预测分析

根据普通牛MC4R基因序列设计同源引物,扩增克隆牦牛MC4R基因,对牦牛与普通牛MC4R基因及其蛋白结构进行生物信息学分析。与普通牛MC4R基因相比,牦牛在该基因存在5个多态位点,其中3个在密码子第3位,2个位点在密码子第1位;4个牦牛个体中有1个与其他个体存在2个变异位点且在密码子第3位;牦牛与普通牛在MC4R蛋白2个氨基酸的差异只引起两物种MC4R蛋白二级结构组分的细微差异。MC4R蛋白在牦牛和普通牛之间保守性较强,MC4R基因可能在哺乳类数百万年的进化历程中受到较强的进化抑制或净化进化。为研究MC4R基因在牦牛食欲控制、体重调节、脂肪沉积和能量平衡调节等方面提供基础资料。     

牦牛MC1R基因的克隆测序及其分析研究

以麦洼牦牛、斯布牦牛、天祝牦牛和九龙牦牛为研究对象,对黑色素皮质素受体1(Melanocortin receptor I,MCIR)基因编码区进行了克隆测序及分析.结果表明,牦牛的MC1R基因编码区全长954 bp,编码317个氨基酸:4个牦牛品种间及与普通牛间在MC1R基因的编码区内共有13个碱基差异,无碱基的插入和缺失现象,编码蛋白共有9个氨基酸差异.MC1R蛋白为亲水性蛋白,无信号肽,有糖基化位点和7个跨膜区.系统进化分析显示,麦洼牦牛与斯布牦牛的MC1R基因相似性最近.本研究结果时今后开展MC1R基因与牦牛毛色性状的相关性分析以及牦牛的毛色遗传机理、基因定位、基因表达调控等研究具有重要的意义.     

牦牛ZP3基因的克隆及蛋白质结构的预测

本研究对牦牛ZP3基因的编码区进行了克隆,在此基础上对ZP3蛋白的分子结构预测,为研究牦牛受精生物学提供基础。根据GenBank中普通牛的ZP3核苷酸序列设计特异性引物,以牦牛卵巢组织总RNA为模板,通过RT-PCR技术扩增牦牛ZP3基因cDNA序列(GenBank登录号为GQ856646),利用DNAMAN生物软件进行核苷酸和氨基酸序列分析、蛋白质专家系统ExPASy进行ZP3蛋白质分子结构预测。结果表明,扩增出的牦牛ZP3基因编码序列长1 266 bp,编码421个氨基酸。牦牛与牛、猪、狗、人、鼠和鸡ZP3基因核苷酸相应序列的同源性分别为98.42%、96.73%、79.67%、78.71%、69.15%和56.61%,氨基酸同源性分别为98.10%、83.85%、74.24%、70.26%、62.62%和46.12%,符合物种进化规律。预测的ZP3蛋白二级和三级结构显示它是一个具有22个氨基酸信号肽的亲水性β-桶状跨膜蛋白。牦牛ZP3基因编码区的成功克隆,为进一步研究该基因的结构与功能及其在受精过程中的作用提供了基础。     

牛科动物HSL基因序列分析及其分子进化研究

在对牛科中4种动物即牦牛、瘤牛、普通牛和水牛HSL基因外显子Ⅰ部分核苷酸序列进行测定的基础上,与Gen-Bank中其他物种相应基因核苷酸序列、氨基酸序列进行了比对分析,并构建了牦牛与其他物种间分子系统进化树。结果表明:牦牛与普通牛、瘤牛、水牛、猪、人、小鼠、大鼠7个物种HSL基因外显子Ⅰ部分核苷酸序列间保守性较高,同源性大小依次为99.8%、99.6%、97.4%、90.6%、88.4%、83.5%、82.3%。相应氨基酸序列间保守性更高,同源性分别为100%、100%、98.2%、94.0%、92.2%、89.8%、89.8%。牦牛与各物种该基因部分核苷酸序列间碱基变异类型主要表现为碱基转换和颠换,无碱基插入和缺失发生,碱基转换的频率高于颠换的频率;在核苷酸水平上的多数碱基替换都是同义替换;序列间单碱基变异位点大多出现在同一位点,多发生在密码子第3位,其次是第1位,最少发生在第2位,符合分子进化的中性学说。HSL基因外显子Ⅰ部分核苷酸序列进行多序列对位排列构建的各物种间分子系统进化树结果表明,普通牛和瘤牛首先聚为一类,再分别与牦牛、水牛、猪、人聚类,最后与大鼠、小鼠聚为一类。该聚类结果与动物学上的分类结果一致,表明HSL基因外显子Ⅰ部分核苷酸序列适合于构建物种间分子系统进化树。研究表明,牦牛、普通牛和瘤牛3个物种间的遗传距离大小相近,牦牛和水牛间的遗传距离与普通牛、瘤牛和水牛间的遗传距离大小相当。牦牛、普通牛和瘤牛3个物种间的遗传距离远小于它们各自与水牛这一物种的遗传距离,它们三者之间的亲缘关系也相对于它们各自与水牛间的亲缘关系都较近,故将牦牛、普通牛和瘤牛划分在同一个属——牛属(Bos)更为合理。     

雷琼牛GH基因第五外显子遗传多样性及其分化

对16头雷琼牛GH基因第5外显子序列进行分析,发现了1个变异位点,定义了2种单倍型。引用巴州牦牛2个个体GH基因同源区序列并结合GenBank中牛属普通牛、其它瘤牛和牦牛3个种群与水牛1个远缘种GH基因同源区序列,分别采用邻接（NJ）法和最大简约（MP）法构建分子系统发育树,得到基本一致的拓扑结构,结果显示GH基因的分化早于雷琼牛（瘤牛）、其它瘤牛、普通牛、牦牛和水牛的分化,瘤牛物种内存在多型,同时证实了GH基因第5外显子区有着较高的突变率。     

牦牛分类地位研究概述

牦牛的分类地位一直存在着争议,牦牛究竟是属于牛亚科牛属还是属于牛亚科牦牛属,到目前为止还没有形成一个明确的定论.本文通过对牦牛与牛亚科其他属在古生物学证据、形态学特征、血液蛋白多态性、微卫星多态性、mtDNA序列变异、rDNA的RFLP数据和功能基因序列信息等各方面研究资料的比较分析,发现牦牛无论在古生物学证据、形态学特征,还是在分子生物学特征上均表现出与牛属中的普通牛Bos taurus、瘤牛Bos indicus不同,而与美洲野牛Bison bison的亲缘关系更近一些,因此将牦牛划分为牛亚科中1个独立属(即牦牛属),似乎比将牦牛作为牛属中的1个亚属或1个种更合适.     

牦牛MEF2C基因克隆、生物信息学及表达谱分析

本试验旨在分析牦牛(Bos grunniens)肌细胞增强因子2C(myocyte enhancer factor 2C,MEF2C)基因的分子特征和表达规律,探索其影响牦牛肌肉发育的作用机制.试验以大通牦牛肌肉组织cDNA为模板,采用PCR扩增技术扩增牦牛MEF2C基因,用DNAStar,ExPASy,ABCpred等生物信息学软件分析MEF2C基因序列和其编码的蛋白质结构,利用实时荧光定量PCR技术(RT-qPCR)检测了 MEF2C基因的表达情况.试验克隆获得的牦牛MEF2C基因编码区全长1 302 bp,编码433个氨基酸;蛋白质结构预测结果显示,MEF2C蛋白具有30h的半衰期,为亲水性碱性蛋白,没有信号肽但拥有跨膜结构.系统关系中牦牛与普通牛的亲缘关系最为接近,与小鼠亲缘关系较远.组织表达谱结果显示,MEF2C基因在牦牛7个组织中都有表达且在臀二头肌组织中表达量最高;不同时期MEF2C基因表达情况为胎牛＞成年牛＞6月龄牛.研究结果将为进一步探讨MEF2C基因在牦牛肌肉发育中的作用提供科学依据,同时也为解析牦牛肌肉发育的分子机制提供数据支撑.     

Cerebral Blood Flow Threshold and Regional Heterogeneity of Heat Shock Protein 72 Induction Following Transient Forebrain Ischemia in Rats

Heat shock proteins (HSPs) induced by brain ischemia may play an important role in neuroprotection from neuronal degeneration. In this study, we examined the cerebral blood flow (CBF) threshold to produce regional differences in HSP72 induction after transient forebrain ischemia in spontaneously hypertensive rats (SHRs). Female SHRs were subjected to 20 min of cerebral ischemia induced by bilateral carotid artery occlusion. The CBF was measured by laser Doppler flowmetry. At forty-eight hours after cerebral ischemia and reperfusion, the rats were decapitated and the brains were removed. Specific areas (hippocampal CA1, CA2-3, dentate gyrus, dorsolateral and ventromedial striatum, and parietal cortex) were thereafter dissected from the brain. The amounts of HSP72 in these samples were determined using Western blot analysis. In the hippocampus, HSP72 was induced when the CBF decreased to less than 18–25% of the resting level. The mean values of HSP72 produced in the CA1 area, CA2-3 area, and the dentate gyrus following ischemia and reperfusion treatment were 4.44 ± 1.43 (±SD) ng/g prtein, 3.51 ± 0.72 ng/g protein and 3.77 ± 1.05 ng/g protein, respectively. In the parietal cortex, the amount of HSP72 induction was less pronounced (2.55 ± 0.40 ng/g protein), while HSP72 was hardly detected at all in the striatum, even under conditions of very severe CBF reduction and reperfusion. We demonstrated the existence of both a CBF threshold (i.e., approximately 20% of the resting level) for HSP72 induction and regional heterogeneity for the induction of HSP72 protein.     

Synthesis of 60- and 72 kDa heat shock proteins in early porcine embryogenesis

Proteins of selected embryonic stages were metabolically labeled with [(35)S]-methionine and analyzed by two-dimensional SDS-polyacrylamide gel electrophoresis (2-D PAGE) to study protein expression from 4- to 8-cell to blastocyst stage of porcine embryos. Two proteins with molecular weights of 60 and 72kDa were de novo synthesized during the 4- to 8-cell stage were the earliest that were detected. They were identified as HSP60 and HSP72 according to their locations on 2-D autoradiography and the immunoblotting result of anti-HSP 60 and HSP 72 antibodies of 1-cell stage of porcine embryos. In protein translation in early pig embryogenesis the timing of their synthesis suggests that HSP60 and HSP72 play significant roles as chaperones.     

Heat shock protein 72 overexpression protects against hyperthermia, circulatory shock, and cerebral ischemia during heatstroke.

This study extends our earlier studies in rats by applying our heatstroke model to a new species. Additionally, transgenic mice are used to examine the role of heat shock protein (HSP) 72 in experimental heatstroke. Transgenic mice that were heterozygous for a porcine HSP70i gene ([+]HSP72), transgene-negative littermate controls ([-]HSP72), and normal Institute of Cancer Research strain mice (ICR) under pentobarbital sodium anesthesia were subjected to heat stress (40 degrees C) to induce heatstroke. In [-]HSP72 or ICR, the values for mean arterial pressure, the striatal blood flow, and the striatal PO2 after the onset of heatstroke were significantly lower than those in preheat controls. The core and brain temperatures, the extracellular concentrations of ischemic and injury markers in the striatum, and the striatal neuronal damage scores were significantly greater than those in the preheat controls. In [-]HSP72 or ICR, the body temperatures, cell ischemia content, and injury marker in the striatum were significantly higher, and the mean arterial pressure, striatal blood flow, and striatal PO2 concentration were significantly lower during heatstroke than in [+]HSP72. Accordingly, the latency and the survival times for [+]HSP72 significantly exceeded those of [-]HSP72 or ICR. These results demonstrate that the overexpression of HSP72 in multiple organs improves survival during heatstroke by reducing hyperthermia, circulatory shock, and cerebral ischemia and damage in mice.     

Specific stimulation of human apurinic/apyrimidinic endonuclease by heat shock protein 70

We previously demonstrated the stimulation of human apurinic/apyrimidinic endonuclease 1 (HAP1) by heat shock protein 70 (HSP70). In this work, we further defined the functional interaction between these proteins. Digestion of HSP70 by trypsin released 48 and 43 kDa amino terminal fragments that retained the ability to stimulate HAP1. In agreement with this result, an HSP70 N-terminal deletion mutant protein containing amino acids 1-385 was comparable to the full-length protein in its ability to enhance HAP1 activity. HSP70 mutants containing carboxy terminal amino acids 386-640 stimulated HAP1 only slightly, as did unrelated proteins. These results implicate the amino terminal portion of HSP70 in stimulating the activity of HAP1.     

Isolation and some properties of bovine brain 100 kDa heat shock protein

1. The 100 kDa protein was purified from bovine brains. 2. The antibody against the 100 kDa brain protein was prepared and was monospecific to the antigen. 3. The antibody cross-reacted with HeLa cell HSP100 (100 kDa heat shock protein). 4. The physicochemical, immunochemical properties and a partially amino acid sequence indicated that the 100 kDa protein was HSP100. 5. Peptide mapping using Staphylococcus aureus V8 protease showed a core peptide with 10 kDa molecular mass common to both HSP100 and HSP90. 6. The amino acid sequence of the 10 kDa fragment of the 100 kDa protein showed a high homology with that of human HSP90 (38-60); the difference was only two of 23 amino acid residues determined.