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园艺学报 ›› 2010, Vol. 37 ›› Issue (7): 1139-1146.

• 研究报告 • 上一篇    下一篇

柿果实扩张蛋白基因cDNA克隆及原核表达

常晓晓,饶景萍*,刘 乐   

  1. (西北农林科技大学园艺学院,陕西杨凌 712100)
  • 收稿日期:2010-03-12 修回日期:2010-05-19 出版日期:2010-07-25 发布日期:2010-07-25
  • 通讯作者: 饶景萍

Cloning and Prokaryotic Expression of Expansin cDNA in Persimmon Fruits

CHANG Xiao-xiao,RAO Jing-ping*,and LIU Le   

  1. (College of Horticulture,Northwest A & F University,Yangling,Shaanxi 712100,China)
  • Received:2010-03-12 Revised:2010-05-19 Online:2010-07-25 Published:2010-07-25
  • Contact: RAO Jing-ping

摘要: 以‘富平尖柿’(Diospyros kaki L.‘Fuping Jianshi’)果实不同发育时期提取的总RNA为模板,利用RT-PCR结合RACE技术扩增得到5个扩张蛋白基因:成熟期CDK-Exp3(1 168 bp),转色期CDK-Exp4(1 120 bp)和CDK-Exp5(1 018 bp),膨大期CDK-Exp6(1 129 bp)和CDK-Exp7(1 121 bp);5个基因的开放阅读框(ORF)均为765 bp,编码254个氨基酸;构建了CDK-Exp3原核表达载体pET-CDK-Exp3,并转化于E.coli BL21(DE3),SDS-PAGE检测结果显示表达了一个约43 kD的蛋白。

关键词: 柿, 果实, 扩张蛋白, 基因, cDNA克隆, 原核表达

Abstract: Using the total RNA from different stages of persimmon(Diospyros kaki L.‘Fuping Jianshi’)fruit as the template,five expansin cDNA:CDK-Exp3(1 168 bp)from mature stage,CDK-Exp4(1 120 bp)and CDK-Exp5(1 018 bp)from colour-changed stage,CDK-Exp6(1 129 bp)and CDK-Exp7(1 121 bp)from fruit expanding stage were isolated by RT-PCR and RACE. The open reading frame of the five genes was 765 bp and encoded a protein of 254 amino acid residues. The CDK-Exp3 was cloned into pET-32a(+)vector to construct recombination prokaryotic expression vector pET-CDK-Exp3,and which was transformed to E.coli BL21. Recombinant protein about 43 kD was expressed in pET-CDK-
Exp3 system and separated by SDS-PAGE electrophoresis.

Key words: persimmon, fruit, expansin, gene, cDNA clone, prokaryotic expression

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