| 水牛水通道蛋白9基因克隆及其表达分析 |
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| 引用本文： | 屈春凤,李胜,李卉,杜凤娇,雷薇,吴柱连,李湘萍,石德顺.水牛水通道蛋白9基因克隆及其表达分析[J].中国畜牧兽医,2015,42(7):1621-1629. |
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| 作者姓名： | 屈春凤 李胜 李卉 杜凤娇 雷薇 吴柱连 李湘萍 石德顺 |
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| 作者单位： | 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 |
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| 基金项目： | 高等学校博士学科点专项科研基金(20126401110001);广西自然科学基金(2012GXNSFCB053002、2014GXNSFAA118099) |
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| 摘 要： | 本研究旨在对水牛水通道蛋白9 (aquaporins 9,AQP9) 基因进行克隆,并对其在水牛不同组织中的表达规律及其在水牛卵巢和睾丸组织中的表达差异进行探索。根据GenBank上黄牛AQP9基因序列(登录号:NM_001205833.1)设计特异性引物,以水牛睾丸组织cDNA为模板,应用RT-PCR方法扩增AQP9基因编码区片段;运用生物信息学方法分析其核苷酸序列的保守性和氨基酸的理化性质;应用实时荧光定量PCR技术分析AQP9基因在水牛组织中的表达情况;免疫组织化学方法分析AQP9蛋白在不同发育阶段水牛卵泡及睾丸组织中的表达差异。结果表明,克隆获得了888 bp的水牛AQP9基因编码区序列,其编码295个氨基酸。多重序列比较显示,水牛AQP9核苷酸序列与牛、猪、绵羊和人相应序列相似性分别为99%、90%、97%、88%;氨基酸序列的同源性分别为99%、86%、97%、83%,系统进化树分析结果推测,AQP9基因在物种进化过程中具有高度保守性。实时荧光定量PCR结果显示,AQP9基因在水牛肝脏、肺脏、大脑、皮肤、睾丸和卵巢组织中有不同程度的表达,在肝脏组织中表达最高,皮肤和睾丸次之,肺脏和卵巢表达较低。免疫组化结果显示,在卵巢组织中,AQP9蛋白表达随卵泡发育时期的不同而变化,并随着卵泡发育其表达逐渐增强;在睾丸组织中,AQP9蛋白在各级精母细胞和间质细胞中均有表达。结果提示,成功克隆得到水牛AQP9基因序列;AQP9在水牛卵巢和睾丸中的表达及其功能可能与水牛卵泡发育和精子发生有重要的关联。
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| 关 键 词： | 水牛 水通道蛋白 AQP9基因 克隆 表达分析 |
| 收稿时间： | 2014-11-12 |
Cloning of Buffalo AQP9 Gene and Analysing its Expression in the Buffalo Tissues |
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| QU Chun-feng,LI Sheng,LI Hui,DU Feng-jiao,LEI Wei,WU Zhu-lian,LI Xiang-ping,SHI De-shun.Cloning of Buffalo AQP9 Gene and Analysing its Expression in the Buffalo Tissues[J].China Animal Husbandry & Veterinary Medicine,2015,42(7):1621-1629. |
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| Authors: | QU Chun-feng LI Sheng LI Hui DU Feng-jiao LEI Wei WU Zhu-lian LI Xiang-ping SHI De-shun |
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| Institution: | State Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources, Guangxi University, Nanning 530004, China |
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| Abstract: | Cloning buffalo AQP9 gene and analyzing its expression in buffalo tissues.A pair of primers was designed according to the released bovine AQP9 sequences in GenBank,which was used to clone buffalo AQP9 gene.The AQP9 gene was amplified by RT-PCR,whose nucleotide sequence and protein structure were analyzed by bioinformatics methods.The expression of AQP9 in buffalo tissues was assayed by Real-time quantitative PCR.The expression of AQP9 gene in buffalo ovary and testis tissue was detected by immunohistochemical staining method.The results showed that the cloned ORF length of buffalo AQP9 gene was 888 bp,which coded 295 amino acids.The results of multiple sequence comparison showed that the nucleotide sequence of buffalo AQP9 shared 99%,90%,97% and 88% homologeous compared with that of Bos taurus,Sus scrofa,Ovis ariessis and Homo sapiens,respectively,while shared 99%,86%,97%,83% homologeous for amino acids,respectively.Phylogenetic tree analysis indicated that AQP9 gene was highly conservative in the evolutionary process.Real-time quantitative PCR results showed that AQP9 gene expressed in buffalo liver,lung,brain,skin,testis and ovary tissues with different levels,had the most abundant expression in liver,followed by in skin and testis,less observed in lung and ovary.The results of immunohistochemical staining showed that the expression of AQP9 protein varied with the development of buffalo ovarian tissue,and gradually enhanced with follicle development.In testicular tissue,AQP9 protein expressed in spermatocyte and leydig cells of developmental stage testis.These results indicated that we had successfully cloned buffalo AQP9 gene sequences.The expression and its function of AQP9 in buffalo ovaries and testes might play an important role in follicle development and spermatogenesis. |
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| Keywords: | buffalo aquaporins AQP9 gene cloning expression analysis |
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