茶树胡萝卜素合成关键基因CsLCYb 和CsLCYe 的克隆与功能鉴定

doi:10.13305/j.cnki.jts.2019.03.003

茶叶科学 ›› 2019, Vol. 39 ›› Issue (3): 257-266.doi: 10.13305/j.cnki.jts.2019.03.003

茶树胡萝卜素合成关键基因CsLCYb 和CsLCYe 的克隆与功能鉴定

刘关华^1,2,3，杨梅^1,2,3，付建玉^1,2*

1. 中国农业科学院茶叶研究所，浙江杭州 310008；2. 农业农村部茶叶质量安全控制重点实验室，浙江杭州 310008；3. 中国农业科学院研究生院，北京 100081

收稿日期:2019-01-23 修回日期:2019-02-13 出版日期:2019-06-15 发布日期:2019-06-15
通讯作者: 刘关华，E-mail: m15079683172@163.com
作者简介:刘关华，男，主要从事茶树栽培与育种的研究，E-mail: m15079683172@163.com。*通信作者：mybatigoal@mail.tricaas.com
基金资助:
国家自然科学基金（31470693，31100503）、浙江省自然科学基金（LY18C160006）、中央级公益性科研院所基本科研业务费专项（1610212018004，1610212016017）、中国农业科学院科技创新工程（CAAS-ASTIP-2014-TRICAAS）

Cloning and Functional Analysis of CsLCYb and CsLCYe for Carotene Biosynthesis in Tea Plant (Camellia sinensis)

LIU Guanhua1,2,3, YANG Mei1,2,3, FU Jianyu1,2*

1. Tea Research Institute, Chinese Academy of Agricultural Sciences, Hangzhou 310008, China; 2. Key Laboratory of Tea Quality and Safety Control, Ministry of Agriculture and Rural Affairs, Hangzhou 310008, China; 3. Graduate School of Chinese Academy of Agricultural Sciences, Beijing 100081, China.

Received:2019-01-23 Revised:2019-02-13 Online:2019-06-15 Published:2019-06-15
Contact: LIU Guanhua，E-mail: m15079683172@163.com

摘要/Abstract

摘要： 胡萝卜素是茶树叶片色素重要的组分之一，具有参与光合作用、保护光合系统的功能。从茶树叶片转录组中获得两个胡萝卜素合成关键基因：番茄红素-β-环化酶基因（CsLCYb）和番茄红素-ε-环化酶基因（CsLCYe），它们分别含含1515bp和1524bp的开放阅读框，编码504和507个氨基酸残基，与其他植物的同源基因高度相似。利用多基因串联表达证明CsLCYb能够将番茄红素环化成胡萝卜素，而CsLCYe则无此活性。通过ELISA分析胡萝卜素生成效率，发现含有CsLCYb表达载体的菌落能够产生大量胡萝卜素，与阴性对照差异极显著，而含有CsLCYe表达载体的菌落则与阴性对照一致，无胡萝卜素产生。这与茶树中胡萝卜素的种类和含量水平一致，说明茶树主要是通过LCYb途径产生胡萝卜素。qRT-PCR分析表明，CsLCYb在黄化品种中黄2号的不同叶位中表达水平与其黄化程度呈正相关，与胡萝卜素含量水平相一致，并且该基因在正常叶色品种龙井43和中黄2号的相对表达结果也符合这一规律。这充分证明CsLCYb在黄化茶树叶片的胡萝卜素合成和叶色变化过程中扮演了重要的角色。本研究阐明了茶树胡萝卜素合成的关键基因和主要途径，为揭示茶树黄化的分子机理提供了重要的遗传基础。

关键词: 茶树, 番茄红素环化酶, 胡萝卜素, 基因表达, 黄化

Abstract: Carotene, participating in photosynthesis and protecting photosynthetic system, is one of the important pigments of tea leaves. Two key genes [lycopene β-cyclase gene (CsLCYb) and lycopene ε-cyclase gene (CsLCYe)] of carotene biosynthesis in tea plant were cloned from transcriptome. They are 1 515 bp and 1 524 bp in length, and encode 504 and 507 amino acid residues, respectively. Based on sequences alignment, CsLCYb and CsLCYe are highly conserved as compared with the homologous genes from other plants. Multiple-gene tandem expression indicated that CsLCYb can cyclize lycopene to β-carotene while CsLCYe was inactive. The enzymatic activities of the two genes were further confirmed by ELISA method in E. coli. It showed that CsLCYb was able to generate a large amount of carotene, which was significantly different from the negative control. However, no carotene was detected in the colonies with the expression plasmid of pAC-LYC-CsLCYe. The type and content of carotene in tea foliage were consistent with previous reports, which proved that carotene was mainly produced by the LCYb pathway in tea plant. The qRT-PCR analysis revealed that the expression level of CsLCYb in buds and different leaves of Zhonghuang 2 was positively correlated with the degree of chlorisis and the content of carotene. Furthermore, its relative expression in normal cultivar Longjing 43 and chlorisis cultivar Zhonghuang 2 also showed similar pattern. These findings confirmed that CsLCYb plays a key role in the carotene biosynthesis and leaf color changing in tea plant, which also provided an important genetic basis for uncovering the molecular mechanism of chlorisis in tea cultivars.

Key words: tea plant, lycopene cyclase, carotene, gene expression, chlorisis cultivars

中图分类号:

S571.1
Q52

刘关华, 杨梅, 付建玉. 茶树胡萝卜素合成关键基因CsLCYb 和CsLCYe 的克隆与功能鉴定[J]. 茶叶科学, 2019, 39(3): 257-266. doi: 10.13305/j.cnki.jts.2019.03.003.

LIU Guanhua, YANG Mei, FU Jianyu, . Cloning and Functional Analysis of CsLCYb and CsLCYe for Carotene Biosynthesis in Tea Plant (Camellia sinensis) [J]. Journal of Tea Science, 2019, 39(3): 257-266. doi: 10.13305/j.cnki.jts.2019.03.003.

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茶树胡萝卜素合成关键基因CsLCYb 和CsLCYe 的克隆与功能鉴定

Cloning and Functional Analysis of CsLCYb and CsLCYe for Carotene Biosynthesis in Tea Plant (Camellia sinensis)

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