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  • 张亮,李疆,代培红,李鹏,罗淑萍,彭弯弯.扁桃CBF1转录因子的克隆及原核表达分析[J].植物遗传资源学报,2015,16(3):612-617.    [点击复制]
  • zhangliang,lijiang,daipeihong,lipeng,luoshuping,彭弯弯.Cloning and Prokaryotic Expression of Almond CBF1 Transcription Factor[J].植物遗传资源学报,2015,16(3):612-617.   [点击复制]
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扁桃CBF1转录因子的克隆及原核表达分析
张亮,李疆,代培红,李鹏,罗淑萍,彭弯弯
0
(新疆农业大学农学院;新疆农业大学林学与园艺学院)
摘要:
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-PdCBF1,GenBank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 kD,并且该蛋白没有信号肽。进化树分析表明,AF-PdCBF1与甜樱桃与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pET-32a(+)中,构建融合表达质粒pET-PdCBF1,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 kD。对重组蛋白的诱导条件进行优化后结果表明,重组蛋白pET-PdCBF1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。
关键词:  扁桃  CBF1转录因子  原核表达
DOI:
投稿时间:2014-10-23修订日期:2015-01-03
基金项目:国家自然科学基金项目(面上项目,重点项目,重大项目)
Cloning and Prokaryotic Expression of Almond CBF1 Transcription Factor
zhangliang,lijiang,daipeihong,lipeng,luoshuping,彭弯弯
()
Abstract:
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 encodes a protein of 242 amino acids, and its relative molecular mass was approximately 27.405 kDa, and AF-PdCBF1 has no signal peptide. The homology tree showed that AF-PdCBF1 with Prunus avium and Prunus mume has recently genetic relationship. The recombinant prokaryotic expression vector pET-PdCBF1 was constructed by inserting the DNA fragment into the prokaryotic 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 kDa protein was obtained. The optimal expression condition was set 0.2 mmol/L IPTG,induced temperature 28℃, and induced time 4 hours. The results have provided a foundation for further purifying and identifying target protein and studying the function of AF-PdCBF1.
Key words:  almond  CBF1 transcription factor  prokaryotic expression

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