水牛PHB基因克隆与表达模式  

Cloning and expression pattern of buffalo PHB gene

作　　者：熊炤成 屈春凤 沈开元[1] 刘庆友[1] 王英群 石德顺[1] 李湘萍[1] 

机构地区：[1]广西大学亚热带农业生物资源保护与利用国家重点实验室,广西南宁530004 [2]广西畜禽品种改良站,广西南宁530001

出　　处：《中国兽医学报》2016年第8期1422-1428,共7页Chinese Journal of Veterinary Science

基　　金：国家“863”计划资助项目(2011AA100607);广西自然科学基金资助项目(2014GXNSFAA118099,2014GXNSFAA118084)

摘　　要：本研究克隆水牛PHB(prohibitin)基因并运用生物信息学方法对其进行分析,同时初步探索其表达模式。首先应用RT-PCR技术扩增获得水牛PHB基因片段,应用生物信息学方法分析和预测了其理化性质、蛋白质二级及三级结构。测序结果表明,水牛PHB基因编码区全长948bp,推测编码272个氨基酸,理论蛋白质相对分子质量29 830,等电点为5.58。多重序列比较分析显示,水牛PHB基因核苷酸序列与牛、野猪、羊、黑猩猩、人、小鼠等相应序列相似性分别为99.4%,94.3%,97.8%,92.8%,92.9%,89.6%;系统进化树分析结果推测,PHB基因在不同物种以及进化过程中具有高度的保守性。QRT-PCR分析结果显示,水牛PHB基因在垂体、大脑、肝脏、卵巢、肌肉、肾脏和睾丸组织具有不同程度的表达,其中在肾脏表达量最高,肝脏、睾丸、卵巢和垂体次之,肌肉表达最低。对高、低活率水牛精子样本PHB基因表达进行了定量检测,结果显示高活率精子中PHB基因表达显著高于低活率精子(P<0.05)。Western blot分析结果发现,高、低活率精子样品的β-actin蛋白条带清晰一致,PHB蛋白在高、低活率精子样品的表达差异显著,低活率精子样品的PHB蛋白表达下调,这与mRNA水平的检测结果一致。本研究为阐明PHB基因在水牛精子发生过程中的作用及其对水牛精子活率的影响奠定了基础。Buffalo PHB （prohibitin） gene was cloned in the present study,sequences were analy- sised by bioinformatics techniques, and the expression pattern of PHB in buffalo tissues and sperms were also assayed. According to the published Bos taurus PHB gene sequences in Gen Bank, specific primers were designed, the target gene fragment was obtained by RT-PCR method. The physical and chemical properties, protein secondary and tertiary structure of buffalo PHB were analyzed and forecasted using bioinformatics techniques. The sequencing result showed that, buffalo PHB gene included a 948 bp CDS （coding 279, amino acids）. The relative molecular quality and isoelectric point of buffalo PHB protein were predicted as 29 830 and 5.58 respectively. The result of multiple comparison of the sequences showed that buffalo PHB gene shared 99. 4%, 94.3%,97.8%,94. 3%,92. 9% and 89.6% of similar nucleotide sequence with Bos Taurus,Sus scrofa, Ovis aries, Pan troglodytes, Hom sapiens, Mus musculus, respectively. The results of the phylogenetic tree analysis showed that,the PHB gene was highly conservative in different species and in the process of evolution. In addition,the expression of PHB gene in buffalo tissues and the sperm samples were analyzed with high and low motility rate through QRT-PCR method. The re- sults showed that,the mRNA of PHB gene had the most abundant expression in the kidney,next in the liver,testis,ovary and pituitary gland, the minimal expression was in the muscle. The mR- NA of PHB gene in the buffalo sperm sample with high motility rate was significantly higher than that of the low motility rate（P〈0.05）. Results of Western blot analysis showed that,the band of β-actin protein in the two sperm samples were clear and almost same,the PHB protein in the buf- falo sperm sample with high motility rate was significantly different with that of low motility rate, the PHB protein was down-regulated in the buffalo sperm sample with low motility rate, which was consistent with the result of mRNA level. This s

关 键 词：水牛PHB基因 生物信息学分析 QRT-PCR Western BLOT 

分 类 号：S823.83[农业科学—畜牧学] S814[农业科学—畜牧兽医]