萝卜低温胁迫转录因子的克隆及遗传转化

Cloning and Genetic Transformation of Low Temperature Transcription Factor from Raphanus sativus

采用RT-PCR和电子克隆技术从抗寒植物萝卜(Raphanus sativusL.)中分离了一个低温胁迫转录因子的cDNA全长序列,命名为RsICE1(GenBank登录号为HQ891287).序列分析显示,该基因全长1 375 bp,编码区为1266 bp,编码421个氨基酸.序列比对表明,该蛋白C端含有一个典型的bHLH结构域,与其他植物的ICE1蛋白具有较高的同源性.进化树分析表明,RsICE1编码的蛋白与油菜的ICE1编码蛋白亲缘关系最近,处在同一进化分枝.半定量RT-PCR分析表明,RsICE1是冷诱导条件下差异表达的基因.构建该基因的植物表达载体,利用农杆菌介导法转化粳稻品种龙粳24,经PCR和RT-PCR分子检测,证明RsICE1基因已经整合到水稻基因组中,并正常表达.与对照相比,低温处理后转基因株系存活率和脯氨酸含量明显增加,相对电导率积累速率明显下降,提高了抗低温胁迫能力.

ICE1（inducer of CBF expression 1） is a low temperature stress transcription factor encoding MYC-like bHLH and a upstream transcription regulator of CBF/DREB cold response pathway.By binding specifically to the MYC element in the CBF/DREB promoter,ICE1 induces the expression of CBF/DREB and its downstream genes.The cDNA sequence of a low temperature stress transcription factor designated as RsICE1（GenBank Accession No.HQ891287） was successfully isolated from cold-tolerant radish（Raphanus sativus L.） by RT-PCR and in silicon cloning.Sequence analysis showed that the full-length cDNA of RsICE1 was 1 375 bp and contained a complete open reading frame（ORF） of 1 266 bp,which encoded 421 amino acid residues.Sequence multialignment demonstrated that the C-terminal of deduced RsICE1 protein exhibited a typical bHLH domain and was high identity with other ICE1 proteins from different species.The homology tree showed that RsICE1 was at the same evolutionary branch with CbICE1.The semi-quantitative RT-PCR result suggested that RsICE1 was difference expression gene under low temperature stress.With constructed plant expression vector,RsICE1 gene was successfully transferred into rice variety ＇Longjing 24＇ by Agrobacterium-mediated transgenic technique.PCR and RT-PCR detection confirmed that RsICE1 gene had been integrated into rice genome and was expressed normally.After low temperature stress treatment,compared to control,the transgenic lines had obvious higher survival rate and proline content.In the meantime,the accumulation rate of relative conductivity was much lower.The RsICE1 gene improved low temperature stress tolerant capacities in transgenic rice lines.