慈竹CBF1全长基因的克隆及其分析

Molecular Cloning and Expression Analysis of CBF1 Gene in Neosinocalamus affinis

分离和克隆慈竹CBF1基因,探明低温下转基因烟草诱导的表达情况,以慈竹叶片为材料,通过RT-PCR和RACE技术克隆慈竹CBF1基因的全长cDNA序列,并进行生物信息学分析;同时选用转基因烟草T2代株系为材料,进行半定量RT-PCR,分析NaCBF1基因在烟草中的表达情况。结果表明:克隆得到慈竹NaCBF1基因(Gen Bank登陆号为JN896707),全长序列为1 051 bp,其中包括5'-UTR 54 bp,3'-UTR 334 bp,编码区663 bp,编码223个氨基酸,分子量为23.48 kDa,等电点为5.27,为不稳定疏水性蛋白。氨基酸序列比对分析表明,NaCBF1与CtCBF1、HvCBF1和TaCBF1一致性分别为93%、86%和85%,且NaCBF1与CtCBF1、PeDREB1聚为一类。不同低温胁迫下,转NaCBF1基因烟草植株均能正常转录,表明具有耐寒性。NaCBF1参与植物对低温胁迫的调控,对其环境胁迫响应还有待探讨,为进一步分析其在不同信号途径相互作用中的功能和调控机制提供有力证据。

The aim of this study was to isolate of CBF1 gene from Neosinocalamus affinis and analyze its expression patterns in the transgenic tobacco under low temperature stresses. In this experiment,the leaves of Neosinocalamus affinis was used as material, the full length cDNA sequence of Neosinocalamus affinis CBF1 transcription factor gene was cloned by using RT-PCR and RACE techniques. The expression of NaCBF1 gene in the T2 lines of trangentic tobacco was analyzed by using semi-quantitative RT-PCR. The results showed that the gene was designated as NaCBF1 that GenBank accession number was JN896707. This gene was 1051 bp DNA fragment in full length ,including a 5＇-UTR （untranslated region） of 54 bp ,a 3＇-UTR of 334 bp and opening reading-frame （ORF） was 663 bp, encoding 221 amino acids with a conservative DNA binding domain. Its relative molecular mass was approximately 23.48kDa and isoelectric point was 5.27. After alignment of amino acid, the sequence of NaCBF1 gene was respectively 93% ,86% and 85% identical to CBF1 gene of Chimonobambusa tumidissinoda, Hordeum vulgare and Triticum aestivum. NaCBF1, Ct CBF1 and PeDREB1 were clustered into one group. The transcript level was increased in response to cold stress. It confirmed that NaCBFlwas related to the stress response, and NaCBF1 was related to the environmental stress. It was the basis for the research of gene structure and biological function, and for the analysis of the function of interaction in different signal pathway and regulating mechanism.