| 贝学军.柑橘钙离子结合蛋白基因克隆及植物表达载体构建[J].中国南方果树,2012,41(3): |
| 柑橘钙离子结合蛋白基因克隆及植物表达载体构建 |
| Cloning of a calcium binding protein gene from Citrus sinensis and construction of sense and antisense expression vectors |
| 投稿时间：2012-03-12 修订日期：2012-04-25 |
| DOI： |
| 中文关键词: 柑橘 钙离子结合蛋白基因 基因克隆 遗传转化 |
| 英文关键词:Citrus calcium binding protein gene cloning Genetic transformation |
| 基金项目:重庆市科技攻关计划项目（CSTTC, 2007AA1018）; 现代农业（柑橘）产业技术体系建设专项 |
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| 中文摘要: |
| 以伏令夏橙叶片中分离的总RNA为模板，经RT-PCR扩增到一条约600bp的富含柑橘钙离子结合蛋白（CsCaBP）的基因片段，将此片段克隆到pMD-19T中，经测序分析该片段与甜橙基因组中的对应序列完全吻合。设计2对带有限制性内切酶位点的特异性引物，以cDNA为模板扩增到2个CsCaBP片段，经双酶切消化后，分别以正反2个方向插入到植物表达载体pFGC5941的查耳酮合成酶(CHSA)内含子两侧，构建成功CsCaBP基因的RNA干扰载体，但未得获转基因植株。将钙离子结合蛋白基因的正向片段定向克隆到具有CaMV35S启动子的pFGC5941表达质粒上，构建成功CsCaBP过量表达载体。将构建好的表达载体导入根癌农杆菌LBA4404菌株，转化酸橙下胚轴，经PCR检测，获得10株过量表达转基因植株。 |
| 英文摘要: |
| A calcium binding protein gene (CsCaBP) was amplified by Reverse Transcription Polymerse Chain Reaction from Citrus sinensis cv ‘Valencia’, and a 600bp-long PCR product was obtained and cloned into pMD-19T plasmid. Sequence analysis showed that the nucleotide sequence of the cDNA was exactly the same as the corresponding genomic sequence. Two PCR products were re-amplified from cDNA and inserted in inverted orientations into the RNAi vector at the two sides of the intron of chalcone synthase gene. The over expression construct was obtained by inserting the full-length CsCaBP cDNA into pFGC5941 under the control of 35S promoter. Both constructs were verified by PCR and sequencing. The constructs were transformed into Agrobacterium tumefaciens, and the transformants were tested positive by PCR. Transformation of Citrus auantium hypocotyledon segments by co-culturing the segments with the bacteria followed by regeneration of transgenic plants yielded only the overexpression transgenic plants but not the RNAi plant. |
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