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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

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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

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