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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Sc CBF1。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 k D,理论等电点为10.31。氨基酸序列比对结果表明,Sc CBF1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Sc CBF1与高粱的亲缘关系最近。q RT-PCR表达分析结果表明,Sc CBF1在甘蔗根、茎、叶中均有表达,在根中表达量最高。Sc CBF1在低温、干旱、脱落酸(ABA)胁迫下诱导表达,而高盐抑制Sc CBF1的表达。成功构建了原核表达载体p GEX-6P-1-Sc CBF1,通过IPTG诱导,实现了Sc CBF1蛋白在大肠杆菌中的表达。
C-repeat / dehydration-responsive element binding factor (CBF) induces the expression of a series of abiotic stress response genes in plants under the abiotic stresses of low temperature, high salt, and drought. In improving plant stress resistance Has an important role. In this study, a new CBF gene was cloned from sugarcane by using the method of bioinformatics and RT-PCR (reverse transcription-polymerase chain reaction) and named Sc CBF1. The open reading frame (ORF) of this gene is 603 bp in length and encodes a protein of 200 amino acids with a relative molecular mass of 22.80 kD and a theoretical isoelectric point of 10.31. Amino acid sequence alignment revealed that the similarity of Sc CBF1 to Sorghum bicolor and Zea mays was 96% and 94%, respectively. Phylogenetic analysis showed that Sc CBF1 has the closest relationship with sorghum. q RT-PCR analysis showed that Sc CBF1 was expressed in roots, stems and leaves of sugarcane, and highest in roots. Sc CBF1 induced expression under low temperature, drought and abscisic acid (ABA) stress, while high salt inhibited the expression of Sc CBF1. The prokaryotic expression vector pGEX-6P-1-Sc CBF1 was successfully constructed, and the expression of Sc CBF1 protein in E.coli was induced by IPTG.