月季丁香酚合成酶基因RhEGS1的克隆及表达分析

doi:10.3864/j.issn.0578-1752.2012.03.023

摘要/Abstract

摘要： 【目的】克隆月季丁香酚合成酶基因RhEGS1的全长cDNA序列，分析其序列及表达特征，并对该基因编码的蛋白进行原核表达分析，为深入探讨丁香酚合成酶的生化特性奠定基础。【方法】根据已发表的其它植物EGS基因序列的保守结构域设计简并引物，结合RACE技术，获得RhEGS1的全长cDNA序列，并进行生物信息学分析；利用半定量RT-PCR对不同组织及不同花发育时期RhEGS1进行表达分析。采用Gateway克隆技术，构建原核表达载体，并进行原核蛋白表达。【结果】月季RhEGS1 的cDNA全长为1 207 bp，包含一个927 bp的ORF，编码309个氨基酸。同源序列比对发现RhEGS1与仙女扇的CbEGS2有83.87%的同源性，与矮牵牛PhEGS1具有81.55%同源性。表达谱分析表明，RhEGS1主要在雄蕊中表达, 且在花盛开期表达最强，而在花蕾期及凋谢期表达较弱。原核表达分析发现，在37℃、0.5 mmol•L-1 IPTG 诱导4 h 后，携带RhEGS1 ORF 的原核表达载体在大肠杆菌中生成了大量的分子量约为35 kD 的蛋白质，其分子量大小与预测的理论值相一致。【结论】从月季雄蕊中克隆到丁香酚合成酶基因RhEGS1，具有EGS基因的结构特征和完整的编码框，且在盛开期雄蕊中表达量最高。

关键词: 月季, 丁香酚合成酶基因, RT-PCR, Gateway克隆, 原核表达

Abstract: 【Objective】 The objective of this study was to clone and identify eugenol synthase gene (RhEGS1) related to flower scent metabolism in rose. The results will provide a foundation for further studying the gene’s function and biochemical characters. 【Method】 The full-length cDNA sequence of RhEGS1 was amplified by degenerate primers based on EGS from other plants and RACE cloning technology. The expression patterns of RhEGS1 in different tissues and different developmental stages were analyzed by semi-quantitative RT-PCR. Furthermore, prokaryotic expression vector was successfully constructed by Gateway cloning technology. 【Result】 The full-length of RhEGS1 was 1 207 bp, containing a 927 bp ORF which encoded a 83.87% and 81.55% homology putative EGS protein with 309 amino acids. Phylogeny analysis showed that RhEGS1 shared with CbEGS2 in Clarkia breweri and PhEGS1 in Petunia hybrida, respectively. RhEGS1 had a highest transcript level in stamens at blooming stage, and lower levels in those of bud at senescence stage. The expression of predicted 35 kD recombinant protein was induced by isopropyl β-D-thiogalacto-pyranoside (IPTG) at a final concentration of 0.5 mmol•L-1 for 4 h at 37℃. 【Conclusion】 The RhEGS1 was isolated from rose stamen and shared the conserved domains with other EGS. It showed the highest transcript level in stamens at blooming stage, and demonstrated active expression in prokaryotic cells.

Key words: rose (Rosa hybrida), RhEGS1, RT-PCR, Gateway clone, prokaryotic expression

晏慧君, 张颢, 蹇洪英, 王其刚, 邱显钦, 张婷, 唐开学. 月季丁香酚合成酶基因RhEGS1的克隆及表达分析[J]. 中国农业科学, 2012, 45(3): 590-597.

YAN Hui-Jun, ZHANG Hao, JIAN Hong-Ying, WANG Qi-Gang, QIU Xian-Qin, ZHANG Ting, TANG Kai-Xue. Cloning and Expression Analysis of Eugenol Synthase Gene (RhEGS1) in Cut Rose (Rosa hybrida)[J]. Scientia Agricultura Sinica, 2012, 45(3): 590-597.

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