甘蔗转录激活因子ScCBF1基因的克隆与表达分析

doi:10.3724/SP.J.1006.2015.00717

摘要/Abstract

摘要：

植物在受到低温、高盐、干旱等非生物逆境胁迫后，CBF结合因子(C-repeat/dehydration-responsive element binding factor)会诱导一系列非生物胁迫应答基因的表达，在提高植物抗逆性方面具有重要作用。本研究利用生物信息学和RT-PCR (reverse transcription-polymerase chain reaction)方法从甘蔗中克隆到1个新的CBF类基因，命名为ScCBF1。该基因的开放读码框(open reading frame, ORF)长度为603 bp，编码200个氨基酸，编码蛋白相对分子质量为22.80 kD，理论等电点为10.31。氨基酸序列比对结果表明，ScCBF1与高粱(Sorghum bicolor)和玉米(Zea mays)中该蛋白相似度分别是96%和94%。进化分析表明ScCBF1与高粱的亲缘关系最近。qRT-PCR表达分析结果表明，ScCBF1在甘蔗根、茎、叶中均有表达，但在根中表达量最高。ScCBF1在低温、干旱、脱落酸(ABA)胁迫下诱导表达，而高盐抑制ScCBF1的表达。成功构建了原核表达载体pGEX-6P-1-ScCBF1，通过IPTG诱导，实现了ScCBF1蛋白在大肠杆菌中的表达。

关键词: 甘蔗, CBF结合因子, 荧光定量PCR, 原核表达

Abstract:

C-repeat/dehydration-responsive element binding factor (CBF) plays an important role in improving plant stress resistance. The CBF can induce a series of abiotic stress responsive gene expression when the plants are subjected to low temperature, high salt, drought or other abiotic stresses. In this paper, a new CBF-like gene, designed as ScCBF1, was cloned by bioinformatics and RT-PCR method from sugarcane. Sequence analysis showed that ScCBF1 contained a 603 bp open reading frame (ORF) and encoded a deduced protein of 200 amino acids. Its molecular weight and isoelectric point were predicted as 22.80 kD and 10.31, respectively. The amino acid sequence alignment results showed that ScCBF1 shared the similarity of 96% and 94% with the CBF1 protein from Sorghum bicolor and Zea mays, respectively. Phylogenetic tree analysis revealed that ScCBF1 had closer relationships with Sorghum bicolor. qRT-PCR showed that ScCBF1 expressed in root, stem and leaf in sugarcane with the highest expression level in roots. ScCBF1 gene was induced by low temperature, drought or abscisic acid (ABA), but was downregulated under high salinity. The protokaryotic expression vector pGEX-6P-1-ScCBF1 was successfully constructed and transformed into E. coli. Under the induction of IPTG, ScCBF1was successfully expressed in E. coli. This study sheds light on the understanding of the function of ScCBF1.

Key words: Sugarcane, C-repeat/Dehydration-responsive element binding factor, Quantitative Real-Time PCR, Prokaryotic expression

成伟,郑艳茹,葛丹凤,程光远,翟玉山,邓宇晴,彭磊,谭向尧,徐景升*. 甘蔗转录激活因子ScCBF1基因的克隆与表达分析[J]. 作物学报, 2015, 41(05): 717-724.

CHENG Wei,ZHENG Yan-Ru,GE Dan-Feng,CHENG Guang-Yuan,ZHAI Yu-Shan,DENG Yu-Qing,PENG Lei,TAN Xiang-Yao,XU Jing-Sheng. Molecular Cloning and Expression Analysis of Transcriptional Activators ScCBF1 Gene from Sugarcane[J]. Acta Agron Sin, 2015, 41(05): 717-724.

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