柑桔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 sinensisand Construction of Sense and Antisense Expression Vectors

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、含钙离子结合蛋白基因(CsCaBP)的片段,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,并连接到TA克隆载体pMD-19T上;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的RNA干扰载体,但未获得转基因植株。将CsCaBP的正向片段定向克隆到具有CaMV35S启动子的pF-GC5941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9株过量表达转基因植株,荧光定量PCR验证发现目的基因在转基因植株中有不同程度的表达。

A calcium binding protein gene （CsCaBP） was amplified by Reverse Transcription Polymerse Chain Reaction （RT-PCR）from Citrus sinensis cv. ＇Valencia＇, and a 600 bp-long PCR product was obtained and cloned into pMD-19T plasmid. Sequence analysis showed that the nucleotide sequence of the eDNA 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 ove^expression construct was obtained by inserting the full-length CsCaBP eDNA 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.