| 摘要: |
| 【目的】克隆沼泽型水牛VASA基因5′侧翼序列，对启动子进行生物信息学分析及转录活性检测，为后续水牛繁殖性能的分子机理研究奠定基础。【方法】根据GenBank已公布的河流型水牛（Bubalus bubalis）的VASA 5′ 侧翼序列，经过同源比对，设计PCR引物。以沼泽型水牛血液基因组为模板扩增VASA基因5′ 侧翼序列并进行测序和生物信息学分析。将扩增的VASA基因5′ 侧翼片段插入pEGFP-1多克隆位点处，构建pVASA-EGFP-1载体。载体经脂质体转染HEK-293T和CHO细胞系，分析其转录活性。【结果】成功克隆沼泽型水牛VASA 5′侧翼序列及部分CDS区序列，共3 081 bp，同源性分析表明，沼泽型水牛VASA基因5′侧翼序列与河流型水牛、黄牛、绵羊、山羊、小鼠和人的相似性分别为99%，96%，93%，95%，90%和79%。对其5′侧翼序列2 000 bp进行启动子预测及转录因子结合位点预测，发现在其翻译起始位点上游－635-－586处存在TATA box，启动子区存在Nobox、Stat1、Stat3、Stat4、Stat5a、Stat5b、Stat6、Gata3、Smad2、Smad3和Smad4等反式作用因子结合位点，其中Stat家族基因在VASA启动子区存在多个结合位点，且同一位点又存在多个Stats基因结合的情况。水牛VASA启动子能启动EGFP在HEK-293T细胞中的表达，但非常微弱；能启动EGFP在CHO细胞中表达，且启动活性与CMV启动子相似。【结论】成功克隆了沼泽型水牛VASA基因启动子，分析了其启动子序列特征并成功验证其组织特异性和转录活性。 |
| 关键词: 沼泽型水牛 VASA基因 生物信息学分析 转录活性 |
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| 基金项目:农业部转基因重点项目(2014ZX08010-012B)；广西科技攻关国际交流与合作项目（桂科合14123001-5)；广西水牛研究所基本业务费项目（水牛基160206） |
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| Cloning and transcriptional activity of VASA gene promoter of swamp buffalo |
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DUAN Anqin,PANG Chunying,ZHU Peng,et al
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| Abstract: |
| 【Objective】Cloning and bioinformatics analyses of VASA 5′flanking sequence were conducted to lay foundation for investigating molecular regulatory mechanism of swamp buffalo reproduction.【Method】PCR primers of VASA 5′flanking were designed according to the homologous sequence in Bubalus bubalis published in GenBank.The VASA 5′flanking fragment was cloned by PCR and then bioinformatics analysis was conducted.This fragment was then inserted into pEGFP-1 reporter vector to construct pVASA-EGFP-1 vector.Transcription activity was analyzed by transfecting the vector into HEK-293T and CHO cell lines.【Result】The 3 081 bp VASA 5′ flanking and partial CDS region of swamp buffalo were successfully cloned and sequenced.The sequence showed high homologous with river type buffalo,cattle,sheep,goat,mouse and human with nucleotide similarities of 99%,96%,93%,95%,90% and 79%,respectively.A TATA box was predicted at the －635――586 from the translation initiation site,and other transcription factor binding sites of Nobox,Stat1,Stat3,Stat4,Stat5a,Stat5b,Stat6,Gata3 and Smad2,Smad3,Smad4.Many potential binding sites of Stat family genes were also found in the VASA promoter region.The promoter of VASA gene could weakly activate EGFP expression in HEK-293T cells,and activate it in CHO cells lines as strong as promoted by CMV promoter.【Conclusion】The promoter of VASA gene was successfully cloned,its sequence features were analyzed and its tissue specificity and transcriptional activity were verified. |
| Key words: swamp buffalo VASA gene bio-informatics analysis transcriptional activity |
