水牛脑源性神经营养因子基因克隆、序列分析及其在不同组织中的表达研究

doi:10.16431/j.cnki.1671-7236.2015.04.009

›› 2015, Vol. 42 ›› Issue (4): 830-837.doi: 10.16431/j.cnki.1671-7236.2015.04.009

水牛脑源性神经营养因子基因克隆、序列分析及其在不同组织中的表达研究

杜凤娇, 刘晓淋, 吴柱连, 李小楷, 屈春凤, 赵鑫, 雷钏, 阮秋燕, 陆凤花, 石德顺

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, 南宁 530004

收稿日期:2014-08-11 出版日期:2015-04-20 发布日期:2015-05-05
通讯作者: 陆凤花, 石德顺 E-mail:lfhgggg@163.com;ardsshi@gxu.edu.cn
作者简介:杜凤娇(1990-),女,陕西宝鸡人,硕士,研究方向:动物胚胎工程,E-mail:511201138@qq.com
基金资助:
国家农业部转基因重大专项(2011ZX08007-003);国家高技术研究发展计划(863)项目(2011AA100607);国家自然科学基金项目(31160457);广西科学基金项目(2011GXNSFA018084、2012GXNSFFA060004)

Cloning and Sequence Analysis of Buffalo BDNF Gene and Investigation of its Expression Pattern in Different Tissues

DU Feng-jiao, LIU Xiao-lin, WU Zhu-lian, LI Xiao-kai, QU Chun-feng, ZHAO Xin, LEI Chuan, RUAN Qiu-yan, LU Feng-hua, SHI De-shun

State Key Laboratory of Conservation and Utilization of Subtropical Agro-bioresources, Guangxi University, Nanning 530004, China

Received:2014-08-11 Online:2015-04-20 Published:2015-05-05

摘要/Abstract

摘要： 本研究克隆了水牛脑源性神经营养因子(brain-derived neurotrophic factor,BDNF)基因序列,并运用生物信息学方法对序列的同源性、生物进化树、蛋白的理化性质及二、三级结构等进行了分析预测,同时利用QRT-PCR方法研究了BDNF mRNA在胎儿水牛及成年水牛不同组织中的表达情况.结果表明,应用RT-PCR技术克隆获得了长800 bp水牛BDNF基因序列,其中编码区全长753 bp,编码250个氨基酸.多重序列比对分析显示,水牛BDNF核苷酸序列与黄牛、野猪、犬、人、马、小鼠同源性分别为99%、94%、93%、90%、90%和89%;生物进化树分析显示,BDNF基因在不同物种进化过程中具有较高的保守性;BDNF蛋白理论分子质量28 173.36 u,等电点9.12;蛋白二级结构由多个α-螺旋、β-折叠、T-转角及无规则卷曲组成,三级结构由多个α-螺旋、3对反向平行的β-折叠结构等构成活性中心(即NGF功能结构域).QRT-PCR结果显示,BDNF mRNA在胎儿水牛及成年水牛的心脏、肺脏、肾脏、大脑、肌肉、卵巢、睾丸组织中都有表达,且成年水牛的表达量高于胎儿水牛.

关键词: 水牛; BDNF; 克隆分析; 表达模式

Abstract: In this study, the CDS sequence of buffalo BDNF gene was cloned, and then the homology, evolutive tree of BDNF amino acid sequence and the physicochemical characteristics, secondary and tertiary structures of BDNF protein were analyzed and predicted by bioinformatics techniques, also, the expression pattern of BDNF mRNA in different tissues of fetal and adult buffalo was investigated by QRT-PCR. The results showed that with RT-PCR a 800 bp buffalo BDNF gene fragment was cloned and sequenced, including the whole CDS of 753 bp (coded 250 amino acid). The multiple sequence alignment result showed that, the BDNF gene in buffalo shared 99%, 94%, 93%, 90%, 90% and 89% similar nucleotide sequence with that of Bos taurus, Sus scrofa, Canis lupus familiaris, Homo sapiens, Equus caballus and Mus musculus, respectively. The evolutive tree analysis showed that BDNF gene was highly conservative in evolution. The molecular weight and isoelectric point of BDNF protein were predicted as 28 173.36 u and 9.12, respectively. The secondary structure of BDNF protein was composed of several α-helix, β-sheet, T-turn and randon coil structures, and the tertiary structure of BDNF protein was composed of several α-helix and 3 pairs of antiparallel β-sheet structures and so on, which configuring an active center aslo called the NGF functional domain. The QRT-PCR result showed that BDNF mRNA completely expressed in heart, lung, kidney, brain, muscle, ovary and testis in both of fetal and adult buffalo, but the mRNA expression in adult buffalo was higher than that of fetal buffalo.

Key words: buffalo; BDNF; cloning and analysis; expression pattern

中图分类号:

S823.8⁺3

杜凤娇, 刘晓淋, 吴柱连, 李小楷, 屈春凤, 赵鑫, 雷钏, 阮秋燕, 陆凤花, 石德顺. 水牛脑源性神经营养因子基因克隆、序列分析及其在不同组织中的表达研究[J]. , 2015, 42(4): 830-837.

DU Feng-jiao, LIU Xiao-lin, WU Zhu-lian, LI Xiao-kai, QU Chun-feng, ZHAO Xin, LEI Chuan, RUAN Qiu-yan, LU Feng-hua, SHI De-shun. Cloning and Sequence Analysis of Buffalo BDNF Gene and Investigation of its Expression Pattern in Different Tissues[J]. , 2015, 42(4): 830-837.

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水牛脑源性神经营养因子基因克隆、序列分析及其在不同组织中的表达研究

Cloning and Sequence Analysis of Buffalo BDNF Gene and Investigation of its Expression Pattern in Different Tissues

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