扁桃CBF1转录因子的克隆及原核表达分析

Cloning and Prokaryotic Expression of Almond CBF1 Transcription Factor

为了探明CBF转录因子在扁桃中的抗寒分子机理,以新疆栽培扁桃‘矮丰’叶片为材料,通过PCR技术从扁桃基因组DNA中获得CBF1转录因子,命名为AF-Pd CBF1,Gen Bank登录号为KM245570。序列分析表明,该基因开放阅读框为729bp,编码242个氨基酸,推测蛋白质分子量为27.405 k D,并且该蛋白没有信号肽。进化树分析表明,AF-Pd CBF1与甜樱桃和中国梅的亲缘关系最近。将该基因片段连接到原核表达载体p ET-32a(+)中,构建融合表达质粒p ET-Pd CBF1,转化到E.coli Rosetta(DE3)中进行表达。SDS-PAGE电泳检测结果表明,表达蛋白与预期大小一致,分子量大小约为47.8 k D。对重组蛋白的诱导条件进行优化后结果表明,重组蛋白p ET-Pd CBF1在IPTG浓度为0.2 mmol/L、诱导4 h时,其表达量最佳。试验结果可为进一步纯化和鉴定目的蛋白及研究其功能奠定基础。

In order to identify the role of CBF transcription factors in plant resistance of almond. The CBF1 homologue gene named AF-PdCBF1 （ Genbank Accession No. KM245570） was isolated from the leaf of almond ＇ Aifeng＇ （ Prunus dulcis） in Xinjiang with the technology and method of PCR. Sequence analysis revealed that the open reading frame was 729 bp in full-length and encoded a protein of 242 amino acids, and its relative molecular mass was approximately 27. 405 kD, and AF-PdCBF1 had no signal peptide. The homology tree showed that AF- PdCBF1 with Prunus avium and Prunus mume had recently genetic relationship. The recombinant prokaryotic ex- pression vector pET-PdCBF1 was constructed by inserting the DNA fragment into the prokaryotie expression vector pET-32a（ ＋ ）, and then transformed into E. coli Rosetta （ DE3 ）. The SDS-PAGE displayed that the expressed proteins consistented with the size of expected protein and then 47.8 kD protein was obtained. The optimal expression condition was set O. 2 mmol/L IPTG, induced temperature at 28 -C, and induced time for 4 hours. The results provided a foundation for further purifying and identifying target protein and studying the function of AF- PdCBF1.