《植物生理学报》 2017, 53(7): 1234-1244

紫花苜蓿叶酸合成关键酶GCHI和ADCS编码基因的克隆及分析

杨宝萍^*, 郭欢^*, 施颖, 冯文锦, 李婵, 包爱科^**

兰州大学草地农业科技学院, 草地农业生态系统国家重点实验室, 兰州730020

通信作者：杨宝萍；E-mail: baoaik@lzu.edu.cn

摘　要：

GTP环化水解酶I (GCHI)与氨基脱氧分支酸合酶(ADCS)是植物叶酸合成途径的限速酶, 对叶酸的合成起着至关重要的作用。本研究利用cDNA末端快速扩增技术(RACE)结合逆转录PCR方法, 克隆获得了紫花苜蓿(Medicago sativa)的GCHI和ADCS编码基因, 分别命名为MsGCHI与MsADCS。序列分析结果表明, MsGCHI基因全长1 752 bp, 包含1 371 bp的开放阅读框(ORF), 编码456个氨基酸; MsADCS基因全长3 324 bp, ORF为2 613 bp, 编码870个氨基酸。经同源比对与进化树分析, MsGCHI和MsADCS基因编码的氨基酸与蒺藜苜蓿(M. truncatula)的MtGCHI和MtADCS氨基酸序列具有最高的同源性, 分别达到98%和96%, 与其他植物的氨基酸序列相似度也均在58%以上。实时定量荧光PCR分析结果表明, MsGCHI与MsADCS基因在紫花苜蓿的根、茎、叶中均有表达, 但在叶中的表达量都为最高。

关键词：紫花苜蓿; 叶酸; GCHI; ADCS; 基因克隆

收稿：2017-03-16   修定：2017-05-05

资助：国家自然科学基金(31670405和31372360)和兰州大学中央高校基本科研业务费专项资金(lzujbky-2016-4)。

Cloning and analysis of genes encoding key enzymes GCHI and ADCS in folate biosynthesis from Medicago sativa

YANG Bao-Ping^*, GUO Huan^*, SHI Ying, FENG Wen-Jin, LI Chan, BAO Ai-Ke^**

State Key Laboratory of Grassland Agro-ecosystems, College of Pastoral Agriculture Science and Technology, Lanzhou University, Lanzhou 730020, China

Corresponding author: YANG Bao-Ping; E-mail: baoaik@lzu.edu.cn

Abstract:

GTP cyclohydrolase I (GCHI) and aminodeoxychorismate synthase (ADCS) are the rate-limiting enzymes and play vital roles in the biosynthesis process of folate. In this study, the genes encoding GCHI and ADCS were isolated from alfalfa (Medicago sativa) by reverse transcription PCR and rapid amplification of cDNA ends (RACE) methods, and were named as MsGCHI and MsADCS, respectively. MsGCHI and MsADCS contain the open reading frame (ORF) of 1 371 and 2 613 bp, which encode 456 and 870 amino acid residues, respectively. The putative amino acid sequences of MsGCHI and MsADCS shared more than 58% amino acid sequence similarity with homologous proteins in other plant species, and especially, the highest amino acid sequence similarity of 98% and 96% with MtGCHI and MtADCS from M. truncatula, respectively. The expression pattern analysis indicated that MsGCHI and MsADCS exhibited significant higher levels of expression in leaf, though they also express in root and stem.

Key words: Medicago sativa; folate; GCHI; ADCS; gene clone