短枝型苹果MdRGL基因的克隆及原核表达分析

doi:10.3864/j.issn.0578-1752.2012.07.012

摘要/Abstract

摘要： 【目的】克隆短枝型苹果（Malus domestica Borkh.）的MdRGL基因，并对其进行生物信息学分析，构建该基因的原核表达载体，在大肠杆菌中诱导表达融合蛋白，为明确短枝型苹果MdRGL基因与短枝芽变特性之间的关系奠定基础。【方法】采用同源克隆法，以短枝型苹果叶片总RNA为模板，通过RT-PCR获得短枝型苹果MdRGL的cDNA片段，T/A克隆后进行序列测定。将克隆到的短枝型苹果MdRGL克隆到表达载体pGEX-4T-1上，构建融合表达载体pGEX-4T-MdRGL，转化到大肠杆菌BL21（DE3）并诱导表达。【结果】从短枝型苹果中克隆到5条MdRGL基因：MdRGL1a/b、MdRGL2a/b和MdRGL3b。序列分析表明，除MdRGL3b外，另外4条序列都具有DELLA和VHYNP结构域。利用所构建的原核表达载体，经IPTG诱导和SDS-PAGE电泳检测结果表明，表达蛋白与预期蛋白大小一致。【结论】短枝型苹果中DELLA蛋白的克隆和原核表达的成功，为进一步纯化和鉴定目的蛋白及研究其功能奠定了试验基础。

关键词: 短枝型苹果, DELLA蛋白, 基因克隆, 序列分析, 原核表达

Abstract: 【Objective】Cloning, sequence analysis of the DELLA genes from Malus domestica Borkh and its expression it in E. coli were conducted to further explore the relationship between DELLA and mutation shoot of spur type bud sport apple.【Method】The MdRGL cDNA fragments amplified from Malus domestica Borkh by reverse transcription PCR (RT-PCR) and then the cloned genes of MdRGL were inserted into vector pGEX-4T-1. The recombinant plasmids pGEX-4T-MdRGL were expressed in a prokaryotic expression system after its transformation into E. coli BL21 (DE3).【Result】 Five DELLA genes, MdRGL1a/b, MdRGL2a/b and MdRGL3b were isolated. Molecular structure analysis revealed that these five genes exhibited the typical structures of the DELLA and VHYNP domain except MdRGL3b. The results of SDS-PAGE demonstrated that the expressed proteins were consistent with the size of expected protein in the prokaryotic expression system.【Conclusion】The five DELLA genes of spur-type apple were cloned and successfully expressed in E. coli. This study will provide a foundation for studying the function of the target protein.

Key words: spur-type apple, DELLA protein, gene clone, sequence analysis, prokaryotic expression

宋杨, 张艳敏, 刘美艳, 王传增, 刘金, 冯守千, 王延玲, 陈学森. 短枝型苹果MdRGL基因的克隆及原核表达分析[J]. 中国农业科学, 2012, 45(7): 1347-1354.

SONG Yang, ZHANG Yan-Min, LIU Mei-Yan, WANG Chuan-Zeng, LIU Jin, FENG Shou-Qian, WANG Yan-Ling, CHEN Xue-Sen. Cloning and Prokaryotic Expression of MdRGL Gene from Spur-Type Apple (Malus domestica Borkh.)[J]. Scientia Agricultura Sinica, 2012, 45(7): 1347-1354.

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