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作物学报 ›› 2016, Vol. 42 ›› Issue (09): 1332-1341.doi: 10.3724/SP.J.1006.2016.01332

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

甘蔗捕光叶绿素a/b 结合蛋白基因ScLhca3 的克隆及表达

翟玉山,邓宇晴,董萌,徐倩,程光远,彭磊,林彦铨*,徐景升*   

  1. 福建农林大学 / 农业部福建甘蔗生物学与遗传改良重点实验室, 福建福州 350002
  • 收稿日期:2016-01-11 修回日期:2016-05-09 出版日期:2016-09-12 网络出版日期:2016-05-30
  • 通讯作者: 林彦铨, E-mail: zwlinyq@163.com; 徐景升, E-mail: xujingsheng@126.com, Tel: 13959174787
  • 基金资助:

    本研究由国家高技术研究发展计划(863 计划)项目(2013AA102604), 国家自然科学基金项目(31171605, 31371688)和国家现代农业产业技术体系建设专项(CARS-20-1-1)资助。

Cloning and Characterization of Light Harvesting Chlorophyll a/b-Binding Protein Coding Gene (ScLhca3) in Sugarcane

ZHAI Yu-Shan, DENG Yu-Qing, DONG Meng, XU Qian, CHENG Guang-Yuan, PENG Lei, LIN Yan-Quan*,XU Jing-Sheng*   

  1. Fujian Agriculture and Forestry University / Key Laboratory of Sugarcane Biology and Genetic Breeding, Ministry of Agriculture, Fuzhou 350002,China
  • Received:2016-01-11 Revised:2016-05-09 Published:2016-09-12 Published online:2016-05-30
  • Contact: 林彦铨, E-mail: zwlinyq@163.com; 徐景升, E-mail: xujingsheng@126.com, Tel: 13959174787
  • Supported by:

    This study was supported by the National High-tech Research and Development Program of China (2013AA102604), the National Natural Science Foundation of China (31171605, 31371688), and the China Agriculture Research System (CARS-20-1-1).

摘要:

捕光叶绿素a/b结合蛋白是植物光系统I (photosystem I, PSI)中与色素分子结合的膜蛋白, 由Lhca基因家族编码, 主要参与光合作用中光能的捕获与传递。本研究对甘蔗叶片cDNA文库测序, 获得甘蔗PSI中Lhca3基因的cDNA序列, 命名为ScLhca3 (GenBank登录号为KU215669)。生物信息学分析表明, ScLhca3的开放读码框(opening reading frame, ORF)长度为804 bp, 编码267个氨基酸, 分子量为28.91 kD, 等电点为8.96; ScLhca3被定位于叶绿体, 无信号肽, 存在3个明显的跨膜区域, 含有典型的捕光叶绿素a/b结合蛋白功能域(chlorophyll a/b binding domain), 为亲水性非分泌蛋白。多序列比对和进化分析表明, ScLhca3在不同物种间具有较强的保守性, 具有种属特性。构建原核表达载体pGEX-6P-1-ScLhca3, 通过IPTG诱导表明, ScLhca3蛋白与预测大小一致。亚细胞定位试验显示, ScLhca3与报告基因GFP的融合蛋白定位于叶绿体中。实时定量PCR分析表明, ScLhca3在成熟叶片中的相对表达量最高, 根中几乎不表达, 具有明显的组织特异性; 在CdCl2、ABA和H2O2外源胁迫下, ScLhca3均上调表达; 在黑暗、NaCl和PEG胁迫下则下调表达。

关键词: 甘蔗, 光系统I, 捕光叶绿素a/b 结合蛋白, 实时定量PCR

Abstract:

The Lhca gene family in green plants encodes several light-harvesting chlorophyll a/b-binding proteins that harvest and transfer light energy to the reaction center of photosystem I (PSI) in photosynthesis. The cDNA sequence of Lhca3 gene was firstly obtained from sugarcane leaf full-length cDNA library through sequencing and validated by homology comparison. It was designated ScLhca3 and submitted to the GenBank (accession number: KU215669). ScLhca3 contains an 804 bp open reading frame (ORF) and encodes a deduced protein of 267 amino acids, with a molecular weight and pI of 28.91 kD and 8.96, respectively. Bioinformatics analysis showed that ScLhca3 is a hydrophilic non-secretory protein with three transmembrane domains and a chlorophyll a/b binding domain. Sequence multi-alignment and phylogenetic analysis demonstrated that the ScLhca3 protein sequence shared a high identity with Lhca3 from other plants, with the specificity of species. The protokaryotic expression vector pGEX-6P-1-ScLhca3 was constructed and expressed in E. coli cells under the induction of IPTG. The subcellular localization experiment showed that the ScLhca3 fused with GFP, a reporter protein, was located on chloroplast. Real-time quantitative PCR analysis showed the expression of ScLhca3 had a clear tissue specificity, and was upregulated by CdCl2, ABA, and H2O2, but downregulated by darkness, NaCl, and PEG.

Key words: Sugarcane, Photosystem I, Light harvesting chlorophyll a/b-binding protein, Real-time quantitative PCR

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