茶树磷酸烯醇式丙酮酸转运子CsPPT2基因的克隆和分析

茶叶科学 ›› 2017, Vol. 37 ›› Issue (2): 149-159.

茶树磷酸烯醇式丙酮酸转运子CsPPT2基因的克隆和分析

纪志芳¹, 甘玉迪¹, 陈常颂², 杨鼎俊¹, 孙康¹, 黎星辉¹, 陈暄^1,*

1. 南京农业大学茶叶科学研究所, 江苏南京 210095;
2. 福建省农业科学院茶叶研究所,福建福安 355015

收稿日期:2017-02-14 修回日期:2017-02-22 出版日期:2017-04-15 发布日期:2019-08-22
通讯作者: *chenxuan@njau.edu.cn
作者简介:纪志芳,女,硕士研究生,主要从事茶树遗传育种和茶叶生化研究。
基金资助:
现代农业产业技术体系建设专项资金（CARS-23）、江苏高校优势学科建设工程资助项目、国家自然科学基金（31570691）、江苏省科技支撑计划（BE2009313-1）

Cloning and Expression Analysis of Phosphoenolpyruvate Transporter Gene CsPPT2 in Tea Plant(Camellia sinensis)

JI Zhifang¹, GAN Yudi¹, CHEN Changsong², YANG Dingjun¹, SUN Kang¹, LI Xinghui¹, CHEN Xuan^1,*

1. Tea Research Institute, Nanjing Agricultural University, Nanjing 210095, China;
2. Tea Research Institute, Fujian Academy of Agricultural Sciences, Fu′an 355015, China;

Received:2017-02-14 Revised:2017-02-22 Online:2017-04-15 Published:2019-08-22

摘要/Abstract

摘要： 以茶树（Camellia sinensis）白叶1号为材料,应用RT-PCR和RACE技术克隆获得茶树磷酸烯醇式丙酮酸转运子家族一个基因CsPPT2的cDNA序列,并与茶树体内另一个磷酸烯醇式丙酮酸转运子家族的基因CsPPT1在不同时期和不同部位的表达进行比较。CsPPT2的cDNA全长1β469βbp,其中开放阅读框（ORF）为1β218βbp,编码406个氨基酸。通过生物学信息分析表明,CSPPT2蛋白分子量为44.6βkD,理论等电点为9.90,CsPPT2有6个跨膜区,属于疏水性叶绿体跨膜蛋白。聚类分析显示,茶树磷酸烯醇式丙酮酸转运子家族分为2个亚组,而CsPPT1与CsPPT2属于不同亚组。荧光定量结果分析表明,两个基因可能在茶树体内功能不同,CsPPT2在所考察的组织中均有表达,且在根和成熟叶片中表达量远大于CsPPT1;CsPPT1在茎和复绿前的幼嫩芽叶中表达量高于CsPPT2。在白化期起始时CsPPT2有短暂的升高,但伴随白化受到较大的抑制,表明CsPPT2的表达抑制可能是白叶1号低儿茶素高氨基酸品质形成的关键因素。

关键词: 茶树, 白叶1号, 磷酸烯醇式丙酮酸转运子, CsPPT2, 表达分析

Abstract: A full length cDNA sequence of phosphoenolpyruvate transporter gene (CsPPT2) was obtained from tea plant(Camellia sinensis) cultivar ‘Baiye 1’ by polymerase chain reaction (PCR) and rapid amplification of cDNA ends PCR(RACE-PCR). The nucleotide sequence length of this gene was 1469 bp, containing a complete open reading frame (1218 bp) encoding 406 amino acids. Bioinformatic analysis showed that the predicted molecular weight of the protein was 44.6 kD, and the theoretic isoelectric point was 9.90. CsPPT2 has 6 trans-membrane regions, which may belong to the hydrophobic chloroplast trans-membrane protein. Phylogenetic tree analysis showed the phosphoenolpyruvate transporter family in tea plant could be divided into two subgroups, with CsPPT1 and CsPPT2 belonged to different subgroups. The expression of CsPPT1 and CsPPT2 were compared in different periods and plant organs. It showed that CsPPT2 was expressed in all tested tissues, with higher expression level in roots and mature leaves than those of CsPPT1. But the expression level of CsPPT1 in young shoots was higher than CsPPT2. CsPPT2 was transiently elevated at the beginning of the albino, but inhibited during the development of albino, indicates that the inhibition of the expression of CsPPT2 may act as a key factor for low-catechins and high-amino acids in ‘Baiye 1’.

Key words: Camellia sinensis, Baiye 1, phosphoenolpyruvate transporter, CsPPT2, expression analysis

中图分类号:

S571.1
Q52

纪志芳, 甘玉迪, 陈常颂, 杨鼎俊, 孙康, 黎星辉, 陈暄. 茶树磷酸烯醇式丙酮酸转运子CsPPT2基因的克隆和分析[J]. 茶叶科学, 2017, 37(2): 149-159.

JI Zhifang, GAN Yudi, CHEN Changsong, YANG Dingjun, SUN Kang, LI Xinghui, CHEN Xuan. Cloning and Expression Analysis of Phosphoenolpyruvate Transporter Gene CsPPT2 in Tea Plant(Camellia sinensis)[J]. Journal of Tea Science, 2017, 37(2): 149-159.

参考文献 21

[1]	Fischer K, Kammerer B, Gutensohn M, et al.A new class of plastidic phosphate translocators: a putative link between primary and secondary metabolism by the phosphoenolpyruvate/phosphate antiporter[J]. Plant Cell, 1997, 9: 453-462.
[2]	Voll L, Husler R E, Hecker R, et al.The phenotype of the Arabidopsis cue1 mutant is not simply caused by a general restriction of the shikimate pathway[J]. Plant Journal, 2003, 36: 301-317.
[3]	Kerbarh O, Bulloch E M M, Payne R J, et al. Mechanistic and inhibition studies of chorismate-utilizing enzymes[J]. Biochemical Society Transactions, 2005, 33: 763-766.
[4]	Li H, Culligan K, Dixon RA, et al.Expression of the functional mature chloroplast triose phosphate translocator in yeast internal membranes and purification of the histidine-tagged proteion by a single metalaffinity chromatography step[J]. Proc Natl Acad Sci, 1993, 90: 2155-2159.
[5]	Streatfield SJ, Weber A, Kinsman EA, et al.The phosphoenolpyruvate/phosphater translocator is required for phenolic metabolism, plastid cell development and plastid-dependent nuclear gene expression[J]. Plant Cell, 1999, 11: 1609-1621.
[6]	Knappe S, Flugge UI, Fischer K.Analysis of the plastidic phosphate translocator gene family in Arabidopsis and identification of new phosphate translocator-homologous transporters, classified by their putative substrate-binding site[J]. 2003, 131(3): 1178-1190.
[7]	Knappe S, Löttgert T, Schneider A, et al.Characterization of two functional phosphoenolpyruvate/phosphate translocator (PPT) genes in Arabidopsis-AtPPT1 may be involved in the provision of signals for correct mesophyll development[J]. The Plant Journal, 2003, 36(3): 411-420.
[8]	Staehr P, Löttgert T, Christmann A, et al.Reticulate leaves and stunted roots are independent phenotypes pointing at opposite roles of the phosphoenolpyruvate/phosphate translocator defective in cue1 in the plastids of both organs[J]. Front Plant Sci, 2014, 5: 126.
[9]	杨坤, 吴学龙, 郎春秀, 等. 甘蓝型油菜PEP转运子BnPPT1基因的克隆、序列分析和表达模式[J]. 浙江农业学报, 2011, 23(1): 1-7.
[10]	吴学龙. PPT1基因调控植物生长发育的研究及叶脉特异表达增强子的分离应用[D]. 杭州: 浙江大学, 2013: 12-131.
[11]	成浩, 李素芳, 陈明, 等. 安吉白茶特异性状的生理生化本质[J]. 茶叶科学, 1999, 19(2): 87-92.
[12]	李素芳, 成浩, 虞富莲, 等. 安吉白茶阶段性返白过程中氨基酸的变化[J]. 茶叶科学, 1996, 16(2): 153-154.
[13]	李素芳, 陈树尧, 成浩. 茶树阶段性返白现象的初步研究[J]. 中国茶叶, 1994, 16(2): 26-27.
[14]	赵真, 陈暄, 王明乐, 等. 茶树磷酸烯醇式丙酮酸转运子基因CsPPT的克隆与表达分析[J]. 茶叶科学, 2015, 35(5): 491-500.
[15]	孙美莲, 王云生, 杨冬青, 等. 茶树实时荧光定量PCR分析中内参基因的选择[J]. 植物学报, 2010, 45(5): 579-587.
[16]	Bagge P, Larsson C.Biosynthesis of aromatic amino acids by highly purified spinach chloroplasts-Compartmentation and regulation of the reactions[J]. Physiol Plantarum, 1986, 68(4): 641-647.
[17]	Van Der Straeten D, Rodrigues-Pousada RA, Goodman HM, et al. Plant enolase: Gene structure, expression and evolution[J]. Plant Cell, 1991, 3: 719-735.
[18]	Journet EP, Douce R.Enzymic capacities of purified cauliflower bud plastids for lipidsynthesis and carbohydrate metabolism[J]. Plant Physiol, 1985, 79: 458-467.
[19]	Schulze-Siebert D, Heineke D, Scharf H, et al.Pyruvate-derived amino acids in spinach chloroplasts: Synthesis and regulation during photosynthetic carbon metabolism[J]. Plant Physiol, 1984, 76: 465-471.
[20]	Prabhaka V, Löttgert T, Gigolashvili T, et al.Molecular and functional characterization of the plastid-localized Phosphoenolpyruvate enolase (ENO1) from Arabidopsis thaliana[J]. FEBSLet, 2009, 583: 983-991.
[21]	Li HM, Culligan K, Dixon RA, et al.CUE1: a mesophyll cell-specific positive regulator of light-controlled gene expression in Arabidopsis[J]. Plant Cell, 1995, 7: 1599-1610.