扁桃CBF2基因的克隆及原核表达

Cloning and Prokaryotic Expression of Almond CBF2 Gene

为进一步研究扁桃CBF2目的蛋白功能,探明CBF2转录因子在扁桃中的抗寒分子机制,以新疆栽培扁桃(Amygdalus communis L.)‘双软’叶片为材料,通过PCR技术从基因组DNA中克隆CBF2基因,命名为AcCBF2,将该基因片段连接到原核表达载体pET-32a(+)上,构建重组质粒pET-AcCBF2并转化E.coli Rosetta(DE3)感受态细胞中进行诱导表达;生物信息学推测显示该基因的开放阅读框(ORF)为717 bp,编码238个氨基酸,其分子量为26.59 kD,等电点为5.29。系统发育树分析结果显示,AcCBF2基因与扁桃CBF1、光核桃和桃的亲缘关系最近。SDS-聚丙烯酰胺凝胶电泳结果表明,该蛋白分子量约47 kD,与预期蛋白大小基本一致。AcCBF2基因在E.coli Rosetta中的最佳诱导条件为IPTG浓度0.2 mmol/L、诱导4 h。

The aims are to further study the function of CBF2 target protein, and investigate the molecular mechanism of cold resistance of CBF2 transcription factor in almond. The leaves of Xinjiang cultivated almond ‘Shuangruan＇ were used as materials. The CBF2 gene was cloned from the genomic DNA with PCR technique,and named Ac CBF2. The recombinant prokaryotic expression vector pET-AcCBF2 was constructed by inserting the DNA fragment into the prokaryotic expression vector pET-32 a（＋）, and then transformed into E.coli Rosetta（DE3）. The bioinformatics results showed that the open reading frame was 717 bp and encoded a protein of 238 amino acids, its molecular weight was 26.59 kD and isoeletctric point was 5.29. Phylogenetic tree analysis showed that AcCBF2 had closest genetic relationship with the genes of almond CBF1, Prunus mira Koehne and Peach. The SDS-PAGE displayed that the weight of expressed proteins was 47 kD, which was consistent with the expected protein size. The optional induction condition of AcCBF2 gene in E. coli Rosetta was 4 h of induction in 0.2 mmol/L IPTG.