欢迎访问作物学报，今天是
作物学报

作物学报 ›› 2015, Vol. 41 ›› Issue (02): 240-250.doi: 10.3724/SP.J.1006.2015.00240

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

茶树叶绿素合成相关基因克隆及在白叶1号不同白化阶段的表达

马春雷1,2,姚明哲1,王新超1,金基强1,2,马建强1,陈亮1,*   

  1. 1 中国农业科学院茶叶研究所 / 国家茶树改良中心 / 农业部茶树生物学与资源利用重点实验室, 浙江杭州 310008; 2 中国农业科学院研究生院, 北京 100081
  • 收稿日期:2014-06-18 修回日期:2014-09-30 出版日期:2015-02-12 网络出版日期:2014-11-18
  • 通讯作者: 陈亮，E-mail: liangchen@mail.tricaas.com
  • 基金资助:

    本研究由国家现代农业产业技术体系建设专项(CARS-23)，国家自然科学基金项目(31100504, 31170624, 30901159), 浙江省自然科学基金项目(Y3100291)和国家科技支撑计划项目(2011BAD01B01)资助。

Cloning and Expression of Three Genes Involved in the Biosynthesis of Chlorophyll in Different Albescent Stages of “Baiye 1”

MA Chun-Lei1,2,YAO Ming-Zhe1,WANG Xin-Chao1,JIN Ji-Qiang1,2,MA Jian-Qiang1,CHEN Liang1,*   

  1. 1 Tea Research Institute of Chinese Academy of Agricultural Sciences / National Center for Tea Improvement / Key Laboratory of Tea Biology and Resources Utilization, Ministry of Agriculture, Hangzhou 310008, China; 2 Graduate School of Chinese Academy of Agriculture Sciences, Beijing 100081, China
  • Received:2014-06-18 Revised:2014-09-30 Published:2015-02-12 Published online:2014-11-18
  • Contact: 陈亮，E-mail: liangchen@mail.tricaas.com

RichHTML

PDF

1303

被引次数

2

摘要：

高等植物叶绿素的生物合成对其正常光合作用起关键作用。本文根据前期芯片杂交结果, 采用RT-PCR和RACE技术克隆了3个茶树叶绿素合成相关基因, 分别为谷氨酸-tRNA还原酶(CsGluTR)、叶绿素合酶(CsChlS)、叶绿素酸醋氧化酶(CsCAO), 对应的GenBank的登录号为HQ660371、HQ660370、HQ660369。序列分析表明, CsGluTR基因全长2165 bp, 开放阅读框长1665 bp, 编码554个氨基酸, 推测的蛋白分子量约为60.6 kD, 理论等电点为8.78；CsChlS基因全长1463 bp, 其中开放阅读框长1125 bp, 编码374个氨基酸, 推测的蛋白分子量约为40.5 kD, 理论等电点为8.58；CsCAO基因全长2146 bp, 其中开放阅读框长1611 bp, 编码536个氨基酸, 推测的蛋白分子量约为60.8 kD, 理论等电点为8.03。比对分析表明, 3个基因编码的氨基酸序列与其他植物中同源基因的相似性均在70%以上。利用荧光定量PCR技术检测3个基因在不同白化阶段的表达,表明, CsChlS和CsCAO基因具有明显的表达协同性, 它们在叶片中的表达量与叶片的颜色变化高度同步；而CsGluTR在白化叶片和正常叶片中的表达差异相对较小, 同时在新生芽叶转绿过程中最先恢复正常表达水平。说明在白化叶片中, 叶绿素的合成机制受到较大影响, 叶绿素合成受阻导致的叶片内色素类物质含量降低或消失是叶片白化的直接原因。

关键词: 茶树, 谷氨酸-tRNA还原酶, 叶绿素合酶, 叶绿素酸醋氧化酶, 基因克隆, 表达分析

Abstract:

Chlorophyll is one of the main pigments participating in photosynthesis in plant chloroplasts, and its biosynthesis is crucial for higher plant. In this article, we cloned and characterized three important genes involved in the biosynthesis of chlorophyll which were CsGluTR, CsChlS, and CsCAO (GenBank accession number HQ660371, HQ660370, and HQ660369) lead on the results of cDNA microarray hybridization. The full-length cDNA of CsGluTR was 2165 bp, containing a 1665 bp ORF encoding a 554 amino acids protein, and its 3′untranslated region had an obvious polyadenylation signal. The deduced protein molecular weight was 60.6 kD and its theoretical isoelectric point was 8.78. The obtained cDNA of CsChlS was 1463 bp in length, containing a 1125 bp ORF which encoded 374 amino acid residues. The deduced protein molecular weight was 40.5 kD and its theoretical isoelectric point was 8.58. The full-length of CsCAO was 2146 bp, containing a 1611 bp ORF encoding a 536 amino acids protein. The deduced amino acid sequence of CsGluTR, CsChlS, and CsCAO from tea plant shared high identity with those of other species, for instance the similarity of 79%, 90% and 77 % with Vitis vinifera, respectively. The result of Real-time RT-PCR analysis showed a coordinated expression of CsChlS and CsCAO, which was corresponded with the change of the albino phenotype. However, there were small changes in the expression level of CsGluTR between the normal and albino leaves. These results implied that the biosynthesis of chlorophyll is completely hindered in albino leaves, causing the decline of pigment content and the albino phenotype.

Key words: Tea plant (Camellia sinensis), Glutamyl-tRNA reductase, Chlorophyll synthase, Chlorophyllide a oxygenase, Gene cloning, Expression analysis

[1]Nagata N, Tanaka R, Satoh S, Tanaka A. Identifieation of a vinyl reduetase gene for chlorophyll synthesis in Arabidopsis thaliana and implications for the evolution of Prochlorococcus Species. Plant Cell, 2005, 17: 233–240

[2]Beale S I. Green genes gleaned. Trends Plant Sci, 2005, 10: 309–312

[3]吴自明, 张欣, 万建民. 叶绿素生物合成的分子调控. 植物生理学通讯, 2008, 44: 1064–1070

Wu Z M, Zhang X, Wan J M. Molecular regulation of chlorophyll biosynthesis. Plant Physiol Commun, 2008, 44: 1064–1070 (in Chinese with English abstract)

[4]欧立军. 水稻叶色突变体的高光合特性. 作物学报, 2011, 37: 1860–1867

Ou L J. High photosynthetic efficiency of leaf colour mutant of rice (Oryza sativa L.). Acta Agron Sin, 2011, 37: 1860–1867 (in Chinese with English abstract)

[5]Zhao H B, Guo H J, Zhao L S, Gu J Y, Zhao S R, Li J H, Liu L X. Agronomic traits and photosynthetic characteristics of chlorophyll-deficient wheat mutant induced by spaceflight environment. Acta Agron Sin, 2011, 37: 119–126

[6]Zhang H, Zhang D, Han S, Zhang X, Yu D. Identification and gene mapping of a soybean chlorophyll-deficient mutant. Plant Breed, 2011, 130: 133–138

[7]张立科, 李志彬, 刘海燕, 李如海, 陈满元, 陈爱国, 钱益亮, 华泽田, 高用明, 朱苓华, 黎志康. 两个新的水稻叶色突变体形态结构与遗传定位研究. 中国农业科学, 2010, 43: 223–229

Zhang L K, Li Z B, Liu H Y, Li R H, Chen M Y, Chen A G, Qian Y L, Hua Z T, Gao Y M, Zhu L H, Li Z K. Study on morphological structure and genetic mapping of two novel leaf color mutants in rice. Sci Agric Sin, 2010, 43: 223–229 (in Chinese with English abstract)

[8]Karaca M, Saha S, Callahan F E, Jenkins J N, Read J J, Percy R G. Molecular and cytological characterization of a cytoplasmic-specific mutant in pima cotton (Gossypium barbadense L.). Euphytica, 2004, 139: 187–197

[9]Kurata N, Miyoshi K, Nonomura K, Yamazaki Y, Ito Y. Rice mutants and genes related to organ development, mor-phogenesis and physiological traits. Plant Cell Physiol, 2005, 46: 48–62

[10]刘元义, 游继芳. “安吉白茶”可持续发展的调查与研究. 茶叶, 2013, 39: 75–78

Liu Y Y, You J F. A study on sustainable development of ‘Anji Baicha’ industry. J Tea, 2013, 39: 75–78 (in Chinese with English abstract)

[11]李素芳. 安吉白茶返白机理的研究. 中国计量学院学报, 2002, 13: 214–217

Li S F. Studies on the mechanism of the leaf color change in Anji Baicha (Camellia sinensis). J China Inst Metrol, 2002, 13: 214–217 (in Chinese with English abstract)

[12]成浩, 李素芳, 陈明. 安吉白茶特异性状的生理生化本质. 茶叶科学, 1999, 19: 87–92

Cheng H, Li S F, Chen M. Physiological and biochemical essence of the extraordinary characters of Anji Baicha. J Tea Sci, 1999, 19: 87–92 (in Chinese with English abstract)

[13]陆建良, 梁月荣, 倪雪华, 汪湖北. 安吉白茶阶段性返白过程中的生理生化变化. 浙江农业大学学报, 1999, 25: 245–247

Lu J L, Liang Y R, Ni X H, Wang H B. Changes of physiological and biochemical characters during stage albescent process of Anji Baicha. J Zhejiang Agric Univ, 1999, 25: 245–247 (in Chinese with English abstract)

[14]Ma C L, Chen L, Wang X C, Jin J Q, Ma J Q, Yao M Z, Wang Z L. Differential expression analysis of different albescent stages of ‘Anji Baicha’(Camellia sinensis (L.) O. Kuntze) using cDNA microarray. Sci Hort, 2012, 148: 246–254

[15]Wang X C, Zhao Q Y, Ma C L, Zhang Z H, Cao H L, Kong Y M, Yue C, Hao X Y, Chen L, Ma J Q, Jin J Q, Li X and Yang Y J. Global transcriptome profiles of Camellia sinensis during cold acclimation. BMC Genom, 2013, 14: 415

[16]马春雷, 姚明哲, 王新超, 金基强, 陈亮. 茶树2个MYB转录因子基因的克隆及表达分析. 林业科学, 2012, 48: 31–37

Ma C L, Yao M Z, Wang X C, Jin J Q, Chen L. Cloning and expression of two MYB transcription factors in tea plant (Camellia sinensis). Sci Silvae Sin, 2012, 48: 31–37 (in Chinese with English abstract)

[17] Livak K J, Schmittgen T D. Analysis of relative gene expression data using Real-time quantitative PCR and the 2?ΔΔCt method. Methods, 2001, 25: 402–408

[18]Marchler-Bauer A, Zheng C, Chitsaz F, Derbyshire M K, Geer L Y, Geer R C, Gonzales N R, Gwadz M, Hurwitz D I, Lanczycki C J, Lu F, Lu S, Marchler G H, Song J S, Thanki N, Yamashita R A, Zhang D, Bryant S H. CDD: a conserved domain database for the functional annotation of proteins. Nucl Acids Res, 2013, 41: D348–352

[19]Br?uer L, Brandt W, Schulze D, Zakharova S, Wessjohann L. A structural model of the membrane-bound aromatic prenyltransferase UbiA from E. coli. Chembiochem, 2008, 9: 982–992

[20]Tanaka R, Yoshida K, Nakayashiki T, Masuda T, Tsuji H, Inokuchi H, Tanaka A. Differential expression of two hemA mRNAs encoding glutamyl-tRNA reductase proteins in greening cucumber seedlings. Plant Physiol, 1996, 110: 1223–1230

[21]徐培洲, 李云, 袁澍, 张红宇, 彭海, 林宏辉, 汪旭东, 吴先军. 叶绿素缺乏水稻突变体中光系统蛋白和叶绿素合成特性的研究. 中国农业科学, 2006, 39: 1299–1305

Xu P Z, Li Y, Yuan S, Zhang H Y, Peng H, Lin H H, Wu X J. Studies of photosystem complexes and chlorophyll synthesis in chlorophyll-deficient rice mutant W1. Sci Agric Sin, 2006, 39: 1299–1305 (in Chinese with English abstract)

[22]马建强, 姚明哲, 陈亮. 茶树遗传图谱研究进展. 茶叶科学, 2010, 30: 329–335

Ma J Q, Yao M Z, Chen L. Research progress in genetic map of tea plant (Camellia sinensis). J Tea Sci, 2010, 30: 329–335 (in Chinese with English abstract)

[23]邢才, 王贵学, 黄俊丽, 吴金钟. 植物叶绿素突变体及其分子机理的研究进展. 生物技术通报, 2008, (5): 10–13

Xing C, Wang G X, Huang J L, Wu J Z. Research on chlorophyll mutation of plants and molecular mechanism. Biotech Bull, 2008, (5): 10–13 (in Chinese with English abstract)

[24]王让剑. 茶树叶绿素生物合成及其检测与提取研究进展. 福建农业学报, 2012, 27: 1401–1408

Wang R J. Biosynthesis, detection and extraction of tea chlorophyll. Fujian J Agric Sci, 2012, 27: 1401–1408 (in Chinese with English abstract)

[25]Moser J, Schubert W D, Beier V, Bringemeier I, Jahn D, Heinz D W. V-shaped structure of glutamyl-tRNA reductase, the first enzyme of tRNA-dependent tetrapyrrole biosynthesis. EMBO J, 2001, 20: 6583–6590

[26]Kumar A M, Soll D. Antisense HEMA1 RNA expression inhibits heme and chlorophyll biosynthesis in Arabidopsis. Plant Physiol, 2000, 122: 49–56

[27]Tanaka A, Ito H, Tanaka R, Tanaka N K, Yoshida K, Okada K. Chlorophyll a oxygenase (CAO) is involved in chlorophyll b formation from chlorophyII a. Proc Natl Acad Sci USA, 1998, 95: 12719–12723

[28]Bougri O, Grimm B. Members of a low-copy number gene family encoding glutamyl-tRNA reductase are differentially expressed in barley. Plant J, 1996, 9: 867–878

[29]Rudiger W. Chlorophyll metabolism: From outer space down to the molecular level. Phytochem, 1997, 46: 1151–1167

[30]Paulsen H, Rümler U, Rüdiger W. Reconstitution of pigment-containing complexes from light-harvesting chlorophyll a/b-binding protein overexpressed in Escherichia coli. Planta, 1990, 181: 204–211

[31]Gaubier P, Wu H J, Laudie M, Delseny M, Grellet F. A chlorophyll synthetase gene from Arabidopsis thaliana. Mol General Genet, 1995, 249: 58–64

[32]Lopez J C, Ryan S, Blankenship R E. Sequence of the bchG gene from Chloroflexus aurantiacus: relationship between chlorophyll synthase and other polyprenyltransferases. J Bacteriol, 1996, 178: 3369–3373

[33]Schmid H C, Oster U, Koegel J, Lenz S, Ruediger W. Cloning and characterisation of chlorophyll synthase from Avena sativa. Biol Chem, 2001, 382: 903–911

[34]Wu Z, Zhang X, He B, Diao L, Sheng S, Wang J, Guo X, Su N, Wang L, Jiang L, Wang C, Zhai H, Wan J. A chlorophyll-deficient rice mutant with impaired chlorophyllide esterification in chlorophyll biosynthesis. Plant Physiol, 2007, 145: 29–40

[35]Oster U, Tanaka R, Tanaka A, Rüdiger W. Cloning and functional expression of the gene encoding the key enzyme for chlorophyll b biosynthesis (CAO) from Arabidopsis thaliana. Plant J, 2000, 21: 305–310

[36]Lee S, Kim J H, Yoo E S, Lee C H, Hirochika H, An G. Differential regulation of chlorophyll a oxygenase genes in rice. Plant Mol Biol, 2005, 57: 805–818

[37] 王新超, 赵丽萍, 姚明哲, 陈亮, 杨亚军. 安吉白茶正常与白化叶片基因表达差异的初步研究. 茶叶科学, 2008, 28: 50–55

Wang X C, Zhao L P, Yao M Z, Chen L, Yang Y J. Preliminary study on gene expression differences between normal leaves and albino leaves of Anji Baicha (Camellia sinensis cv. Baiye1). J Tea Sci, 2008, 28: 50–55 (in Chinese with English abstract)

[38]李娟, 刘硕谦, 刘仲华, 李勤, 吴扬, 邓婷婷, 黄建安. ‘安吉白茶’抑制消减杂交cDNA文库的构建及初步分析. 中国农学通报, 2011, 27: 96–101

Li J, Liu S Q, Liu Z H, Li Q, Wu Y, Deng T T, Huang J A. Construction and preliminary analyses of SSH cDNA libraries of Anji Baicha (Camellia sinensis). Chin Agric Sci Bull, 2011, 27: 96–101 (in Chinese with English abstract)
[1] 左同鸿, 张贺翠, 曾静, 朱利泉. 甘蓝自交不亲和相关基因BoPUB3L的克隆与表达分析[J]. 作物学报, 2026, 52(6): 1698-1710.
[2] 胡赵, 钱润, 谢丰璞, 应素平. 水稻SPX基因家族鉴定及响应磷处理的表达分析[J]. 作物学报, 2026, 52(6): 1902-1912.
[3] 徐苗苗, 邸太妹, 王洁, 吴叶蝶, 刘恩贝, 王玉春, 王新超, 王璐. 外源槲皮素增强茶树抗寒性的分子机制[J]. 作物学报, 2026, 52(5): 1418-1429.
[4] 田立涛, 丁宁, 王树林, 齐恩芳, 张荣, 王蕊蕊, 马利雯, 李建武, 杨江伟. 马铃薯Argonaute基因家族鉴定及晚疫病诱导表达分析[J]. 作物学报, 2026, 52(4): 1116-1126.
[5] 杨影, 郝豫皖, 张学宁, 方佳璐, 马月华, 杨伟龙, 孙文清, 王新超, 王玉春, 黄建燕. 乙烯响应因子CsERF9调控茶树炭疽病抗性的分子机制研究[J]. 作物学报, 2026, 52(4): 1103-1115.
[6] 杨宗桃, 杨婷, 王禹童, 艾静, 李燕烨, 刘家勇, 邓军, 赵勇, 张跃彬. 甘蔗CLC基因家族鉴定与表达分析[J]. 作物学报, 2026, 52(3): 722-734.
[7] 孟成, 王哲. 玉米ZmPFK基因家族全基因组鉴定及响应胁迫表达分析[J]. 作物学报, 2026, 52(3): 764-779.
[8] 张力岚, 杨军, 王让剑. 基于WGCNA发掘茶树糖苷类香气前体含量性状相关的候选基因[J]. 作物学报, 2026, 52(2): 494-513.
[9] 余开航, 周洪斌, 罗亮扎, 王玫郦, 姜瑞梅, 董陈文华, 李仕金, 毛孝强, 陈升位. 大麦亮氨酸富集重复型类受体激酶基因HvLRR-RLK-510的克隆和表达分析[J]. 作物学报, 2026, 52(2): 421-432.
[10] 梅飘, 刘丁丁, 叶圆圆, 张晨禹, 丁诗琦, 李亚奇, 王培鑫, 梅菊芬, 马春雷. 基于茶树液相功能芯片的白化茶树资源遗传多样性分析[J]. 作物学报, 2025, 51(9): 2358-2370.
[11] 薛晓菲, 戴云静, 李熙林, 丁艳艳, 王翔, 雷长英, 韩焕勇, 贺道华. 陆地棉杜松烯合酶基因GhCDN10的特征及其在棉酚合成中功能分析[J]. 作物学报, 2025, 51(8): 2060-2076.
[12] 王彬, 蒙姜宇, 邱浩良, 贺亚军, 钱伟. 甘蓝型油菜BnaDUF579基因家族的鉴定与表达模式分析[J]. 作物学报, 2025, 51(8): 2100-2110.
[13] 郭腾达, 崔梦杰, 陈琳杰, 韩锁义, 郭敬坤, 吴晨迪, 付留洋, 黄冰艳, 董文召, 张新友. 花生磷脂酰肌醇转运蛋白基因AhSFH的克隆及其响应黄曲霉菌侵染的表达特征分析[J]. 作物学报, 2025, 51(6): 1489-1500.
[14] 潘炬忠, 韦萍, 朱德平, 邵胜雪, 陈珊珊, 韦雅倩, 高维维. 水稻转录因子OsERF104的克隆和功能研究[J]. 作物学报, 2025, 51(4): 900-913.
[15] 王林, 陈晓雨, 张文梦龙, 汪思琦, 程冰云, 程靖秋, 潘锐, 张文英. 大麦HvMYB2分子特性及响应干旱胁迫的功能分析[J]. 作物学报, 2025, 51(4): 873-887.
Viewed
Full text


Abstract

Cited
  Shared   
  Discussed   
No Suggested Reading articles found!
京ICP备15008789号-2