茶树GGPS基因家族的克隆、生物信息学和表达分析

茶叶科学 ›› 2017, Vol. 37 ›› Issue (2): 130-138.

茶树GGPS基因家族的克隆、生物信息学和表达分析

方洁, 李春芳, 马春雷, 陈亮^*

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

收稿日期:2016-09-14 修回日期:2016-10-28 出版日期:2017-04-15 发布日期:2019-08-22
通讯作者: *liangchen@tricass.com
作者简介:方洁,女,硕士研究生,主要从事茶树资源育种研究。
基金资助:
国家茶叶产业技术项目（CARS-023）、中国农业科学院科技创新工程（CAAS-ASTIP-2014-TRICAAS）

Molecular Cloning, Bioinformatics and Expression Analysis of GGPS Gene Family in Tea Plant

FANG Jie, LI Chunfang, MA Chunlei, CHEN Liang^*

Tea Research Institute of the Chinese Academy of Agricultural Sciences, National Center for Tea Improvement/Key Laboratory of Tea Biology and Resources Utilization, Ministry of Agriculture, Hangzhou 310008, China

Received:2016-09-14 Revised:2016-10-28 Online:2017-04-15 Published:2019-08-22

摘要/Abstract

摘要： 牻牛儿基牻牛儿基焦磷酸合酶（Geranylgeranyl diphosphate synthase, GGPS）是萜类合成途径的结构酶,对植物生长发育具有重要意义。本研究通过RACE和RT-PCR方法克隆得到5条潜在的茶树GGPS序列,分别命名为CsGGPS1-4和CsGGPS9,其中CsGGPS9存在3条等位基因,分别是CsGGPS9-1、CsGGPS9-2和CsGGPS9-3,在系统进化树上与其他基因分成两支。蛋白质序列分析表明,茶树GGPS家族成员都具有polyprenyl_synt结构域,不存在信号肽序列。亚细胞定位预测结果显示,CsGGPS1、CsGGPS2和CsGGPS4定位在叶绿体上,CsGGPS3和CsGGPS9定位在线粒体上。通过Swiss Model进行三维建模,结合“three-floor”模型对茶树GGPS家族成员的功能进行预测,预测结果显示,CsGGPS1、CsGGPS2和CsGGPS4是GGPS;CsGGPS3是异源二聚体形式的牻牛儿基焦磷酸合酶的小亚基;CsGGPS9的催化主产物是碳链数大于30的异戊烯基焦磷酸。qRT-PCR分析表明,CsGGPS1整体表达丰度较低,仅在一芽二叶中表达量稍高;CsGGPS2在茶树各个组织中均有表达,在花中表达量最高,且花发育过程中表达量先上升后下降;CsGGPS3在叶和幼根中的表达量高于花,花发育过程中表达平稳;CsGGPS4在茶树各个组织中表达量数值相近,在花发育过程中表达量变化趋势与CsGGPS2相同;CsGGPS9的表达量在成熟叶中显著低于幼嫩叶片。

关键词: 茶树, 牻牛儿基牻牛儿基焦磷酸合酶, 三维结构模型, 表达分析

Abstract: Geranylgeranyl diphosphate synthase (GGPS) is a constitutive enzyme in the terpenoid biosynthesis pathway and plays an important role in plant growth. Five putative gene sequences of GGPS were cloned by RACE and RT-RCR, and named as CsGGPS1-4 and CsGGPS9, respectively. Three allelic variants (CsGGPS9-1, CsGGPS9-2 and CsGGPS9-3) were detected for CsGGPS9, and phylogenetic analysis indicated CsGGPS9 was different from the others. Protein sequence analysis revealed that all CsGGPSs had a conserved polyprenyl_synt domain but no N-terminal signal peptide. Subcellular location predication showed that CsGGPS1, CsGGPS2 and CsGGPS4 might be located in chloroplast while CsGGPS2 and CsGGPS4 might be located in mitochondria. The 3D model of CsGGPSs were predicted by Swiss Model and combined with the ‘three floor’ model indicated that CsGGPS1, CsGGPS2 and CsGGPS4 were bona fide GGPS. CsGGPS3 was the small subunit of heterodimer GPS. Although CsGGPS9 shared a similar structure with the others, the main product of it could be isopentenyl pyrophosphate with chain length longer than 30. The qRT-PCR analysis showed that CsGGPS1 had low expression levels in all tissues except the ‘two and a bud’. By contrast, CsGGPS2 was highly expressed in all tissues with the highest levels in flowers, where in the expression levels increased first and then decreased during flower blooming. CsGGPS3 exhibited higher expression levels in the leaves and tender root than the flower. The expression levels of CsGGPS4 were similar in different tissues, with the same pattern as CsGGPS2 during flower blooming. CsGGPS9 was significantly higher expressed in the young leaves than the mature leaves.

Key words: tea plant (Camellia sinensis), geranylgeranyl diphosphate synthase, 3D structure model, expression analysis

中图分类号:

S571.1
Q52

方洁, 李春芳, 马春雷, 陈亮. 茶树GGPS基因家族的克隆、生物信息学和表达分析[J]. 茶叶科学, 2017, 37(2): 130-138.

FANG Jie, LI Chunfang, MA Chunlei, CHEN Liang. Molecular Cloning, Bioinformatics and Expression Analysis of GGPS Gene Family in Tea Plant[J]. Journal of Tea Science, 2017, 37(2): 130-138.

参考文献 15

[1]	Wang C, Chen Q, Fan D, et al.Structural analyses of short-chain prenyltransferases identify an evolutionarily conserved GFPPS clade in Brassicaceae plants[J]. Molecular Plant, 2016, 9(2): 195-204.
[2]	Shi J, Ma C, Qi D, et al.Transcriptional responses and flavor volatiles biosynthesis in methyl jasmonate-treated tea leaves[J]. BMC Plant Biology, 2015, 15: 233.
[3]	张祯岘. UV-B对茶叶挥发性化合物形成的影响[D]. 杭州: 浙江大学, 2008: 1-51.
[4]	Li CF, Xu YX, Ma JQ, et al.Biochemical and transcriptomic analyses reveal different metabolite biosynthesis profiles among three color and developmental stages in ‘Anji Baicha’ (Camellia sinensis)[J]. BMC Plant Biology, 2016, 16(1): 195.
[5]	张秀云. 乌龙茶香气形成机理研究进展[J]. 福建茶叶, 1999(3): 15-17.
[6]	Tarshis LC, Yan M, Poulter CD, et al.Crystal structure of recombinant farnesyl diphosphate synthase at 2.6. Ang. Resolution[J]. Biochemistry, 1994, 33: 10871-10877.
[7]	Hosfield DJ, Zhang Y, Dougan DR, et al.Structural basis for bisphosphonatemediated inhibition of isoprenoid biosynthesis[J]. Biological Chemistry, 2004, 279: 8526-8529.
[8]	Kloer DP, Welsch R, Beyer P, et al.Structure and reaction geometry of geranylgeranyl diphosphate synthase from Sinapis alba [J]. Biochemistry, 2006, 45: 15197-15204.
[9]	Wallrapp FH, Pan JJ, Ramamoorthy G, et al.Prediction of function for the polyprenyl transferase subgroup in the isoprenoid synthase superfamily[J]. Proceedings of the National Academy of Sciences, 2013, 110(13): E1196-E1202.
[10]	Ohara K, Sasaki K, Yazaki K.Two solanesyl diphosphate synthases with different subcellular localizations and their respective physiological roles in Oryza sativa[J]. Journal of Experimental Botany, 2010, 61(10): 2683-2692.
[11]	Galpaz N, Ronen G, Khalfa Z, et al.A chromoplast-specific carotenoid biosynthesis pathway is revealed by cloning of the tomato whiteflower locus[J]. Plant Cell, 2006, 18: 1947-1960.
[12]	Ament K, Van Schie CC, Bouwmeester HJ, et al.Induction of a leaf specific geranylgeranyl pyrophosphate synthase and emission of (E, E)-4,8,12-trimethyltrideca-1,3,7,11-tetraene in tomato are dependent on both jasmonic acid and salicylic acid signaling pathways[J]. Planta, 2006, 224: 1197-1208.
[13]	Okada K, Saito T, Nakagawa T, et al.Five geranylgeranyl diphosphate synthases expressed in different organs are localized into three subcellular compartments in Arabidopsis[J]. Plant Physiology, 2000, 122(4): 1045-1056.
[14]	Ruiz-Sola MÁ, Coman D, Beck G, et al.Arabidopsis geranylgeranyl diphosphate synthase 11 is a hub isozyme required for the production of most photosynthesis-related isoprenoids[J]. New Phytologist, 2016, 209(1): 252-264.
[15]	Rai A, Smita SS, Singh AK, et al.Heteromeric and homomeric geranyl diphosphate synthases from Catharanthus roseus and their role in monoterpene indole alkaloid biosynthesis[J]. Molecular Plant, 2013, 6(5): 1531-1549.