茶树GDP-D-甘露糖焦磷酸化酶基因cDNA全长的克隆与表达分析

doi:10.13305/j.cnki.jts.2015.01.010

茶叶科学 ›› 2015, Vol. 35 ›› Issue (1): 55-63.doi: 10.13305/j.cnki.jts.2015.01.010

茶树GDP-D-甘露糖焦磷酸化酶基因cDNA全长的克隆与表达分析

肖瑶¹, 周天山¹, 李佼^1,2, 张佳欣¹, 余有本^1,*

1. 西北农林科技大学茶叶研究所,陕西杨凌 712100;
2. 汉中市农业科学研究所,陕西汉中 723000

收稿日期:2014-08-05 修回日期:2014-10-13 出版日期:2015-02-15 发布日期:2019-08-23
通讯作者: *yyben@163.com
作者简介:肖瑶,女,硕士研究生,主要从事茶叶生物技术研究。
基金资助:
中央高校基本科研业务费专项（QN 2013017）、唐仲英育种基金项目（10YZ034）、西北农林科技大学农业科技推广专项（TGZX2012-08）

Cloning and Expression Analysis of GDP-D-mannose Pyrophosphorylase cDNA in Camellia sinensis

XIAO Yao¹, ZHOU Tianshan¹, LI Jiao^1,2, ZHANG Jiaxin¹, YU Youben^1,*

1. College of Horticulture, Northwest Agriculture & Forestry University, Yangling 712100, China;
2. Hanzhong Agricultural Science Institute, Hanzhong 723000, China

Received:2014-08-05 Revised:2014-10-13 Online:2015-02-15 Published:2019-08-23

摘要/Abstract

摘要： 抗坏血酸（Vc）是重要的抗氧化剂,在茶树体内的各种生理代谢及抵御非生物胁迫中起重要作用,Vc含量关系到绿茶品质优劣。GDP-D-甘露糖焦磷酸化酶（GMP）是茶树Vc生物合成途径中的关键酶之一。本研究通过RACE-PCR方法从茶树中克隆了GMP cDNA全长序列,共计1510βbp,其中包含1β086βbp的开放阅读框（ORF）,编码361个氨基酸,推测蛋白分子量为39.599βkDa。BLAST分析表明,茶树GMP基因氨基酸序列与猕猴桃的亲缘性最高,达到96%。实时定量PCR分析表明,茶树新梢芽下第三叶中GMP基因表达量最高,嫩茎中最低,不同品种茶树新梢叶片中表达量存在明显差异。高温胁迫初期,GMP基因表达量及抗坏血酸含量迅速升高,然后逐渐下降且均低于同期对照。

关键词: 茶树, GDP-D-甘露糖焦磷酸化酶, 基因克隆, 表达分析

Abstract: Ascorbic acid (Vitamin C, Vc) is an important antioxidant in tea plant, playing important roles in metabolism and responses to abiotic stress, and tea quality is positive correlation with the content of Vc. GDP-D-mannose pyrophosphorylase (GMP) is an important enzyme in the synthesis of ascorbic acid. The full-length cDNA sequence of GMP gene was isolated from the shoots of Camellia sinensis by RT-PCR and RACE. The entire GMP cDNA was 1β510βbp, containing a 1β086βbp complete open reading frame which encoding a protein with 361 amino acids and a calculated molecular weight of 39.599βkDa. Blast analysis showed that GMP gene in Camellia sinensis was most closely to Actinidia with 96% amino acids similarity. Quantitative real-time PCR analysis showed that the expression levels of GMP gene in the third leaves were highest while the stem is the lowest, and the different varieties also existed an obvious differences. High temperature stress stimulate the expression of GMP gene and the accumulation of Vc in incipient stage, then reduced rapidly.

Key words: Camellia sinensis, GDP-D-mannose pyrophosphorylase (GMP), gene clonging, expression analysis

中图分类号:

肖瑶, 周天山, 李佼, 张佳欣, 余有本. 茶树GDP-D-甘露糖焦磷酸化酶基因cDNA全长的克隆与表达分析[J]. 茶叶科学, 2015, 35(1): 55-63. doi: 10.13305/j.cnki.jts.2015.01.010.

XIAO Yao, ZHOU Tianshan, LI Jiao, ZHANG Jiaxin, YU Youben. Cloning and Expression Analysis of GDP-D-mannose Pyrophosphorylase cDNA in Camellia sinensis[J]. Journal of Tea Science, 2015, 35(1): 55-63. doi: 10.13305/j.cnki.jts.2015.01.010.

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茶树GDP-D-甘露糖焦磷酸化酶基因cDNA全长的克隆与表达分析

Cloning and Expression Analysis of GDP-D-mannose Pyrophosphorylase cDNA in Camellia sinensis

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