越橘低温响应因子VcICE1的克隆和功能鉴定

Cloning and Functional Analysis of Cold Response Factor VcICE1 in Blueberry

为挖掘越橘低温胁迫响应基因在低温逆境胁迫应答中的作用,探讨越橘低温逆境胁迫响应机制,以北陆越橘为试材,克隆低温响应基因 VcICE1 (Inducer of CBF3 expression 1),分析其表达模式及对低温的响应,探讨 VcICE1 在抵御低温胁迫中的生物学功能。利用农杆菌介导法获得转基因拟南芥,比较转基因和野生型拟南芥对低温胁迫响应的差异。利用瞬时转化烟草叶片试验,分析VcICE1对 AtCBF3 的转录调控。结果表明,克隆获得越橘低温响应因子 VcICE1 ,该基因ORF为1 566 bp,编码含有522个氨基酸的蛋白质,含有1个保守的bHLH结构域。系统进化分析表明,VcICE1与AtICE1同源性最高。表达分析显示, VcICE1 在根、枝条、幼叶、花和果实中均表达,在幼叶中表达量最高,在果实中最低,并响应低温处理。在拟南芥中异位表达 VcICE1 ,其低温抗性较野生型显著升高。瞬时表达试验结果表明,VcICE1可激活 AtCBF3 的表达。 VcICE1 能够对低温处理有明显响应,推测其在响应低温胁迫过程中发挥重要的调控作用。

In order to excavate the role of blueberry cold response genes in cold stress and explore the regulation mechanism of cold stress response,a cold response factor gene VcICE1 (Inducer of CBF3 expression 1)from blueberry was isolated by PCR,and its expression pattern and response to cold stress were analyzed. The transgenic Arabidopsis thaliana were generated via Agrobacterium -mediated transformation,and the expression differences in response to cold stress were compared between the transgenic line and wild-type of Arabidopsis . The transient expression assays in tobacco leaves were carried out to test the transcriptional regulation of AtCBF3 by VcICE1. Sequence analysis showed that the open reading frame(ORF)of VcICE1 was 1 566 bp in length, encoding 522 amino acids. Protein structure analysis showed that VcICE1 contained a basic helix-loop-helix domain. The phylogenetic tree indicated that the blueberry VcICE1 exhibited the highest sequence similarity with the Arabidopsis thaliana AtICE1. Expression analysis showed that VcICE1 could express in roots, stems,young leaves,flowers and fruits,but the expression levels varied in different organs,the highest in young leaves and relatively low in fruits. The expression analysis result indicated that VcICE1 could be induced by cold stress treatment. The VcICE1 protein could activate the expression of AtCBF3 and favorably contributed to cold tolerance in transgenic Arabidopsis . VcICE1 could respond significantly to cold treatment. It was speculated that VcICE1 played a regulatory role in response to cold stress.