茶树磷转运蛋白基因CsPT4的克隆、亚细胞定位及表达分析

摘要/Abstract

摘要： 茶园中磷肥的利用效率取决于茶树体内与磷元素吸收、转运及生理利用等相关蛋白的协同调控,而磷转运蛋白（Phosphate transporter proteins,Phts）在此过程中起着关键的调控作用。本研究以茶树品种龙井长叶（Camellia sinensis cv. Longjing-changye）为试验材料,采用同源克隆的方法首次克隆获得茶树磷转运蛋白编码基因CsPht1:4（CsPT4）的全长cDNA。该基因全长1β642βbp,开放阅读框（ORF）1β620βbp（GenBank登录号：KY132100）,编码539个氨基酸。生物信息学分析显示,CsPT4基因编码蛋白分子量为59.12 kD,理论等电点（pI）为8.51;具有典型的Pht1家族特性：“6-亲水-6”跨膜结构。亚细胞定位结果显示,该蛋白分布于质膜上,与Softberry软件预测结果一致。荧光定量PCR表明：CsPT4在正常生长的茶树根、茎、嫩叶、老叶中均有表达,在老叶中的表达量较高,在根部表达量最低。低磷处理,根和叶中CsPT4上调表达水平均先上升后下降;根部CsPT4表达量48βh内各个时间点均高于叶部。缺磷处理,根和叶中CsPT4上调表达水平均升高;根部和叶部分别在72βh和48βh达最大值。本研究为茶树响应低磷的分子机制提供了参考。

关键词: 茶树, 磷转运蛋白, 基因克隆, 亚细胞定位, 表达分析

Abstract: Phosphate transporter proteins (Phts) play important roles in plant phosphorus (P) absorption and transportation. Furthermore, Phts affect usage efficiency of the tea garden fertilizer. A full-length phosphate transporter complementary DNA (cDNA) CsPht1:4 (also named CsPT4) was cloned from tea plant (Camellia sinensis cv. Longjingchangye) by rapid amplification of cDNA ends (RACE) techniques. CsPT4 had an open reading frame of 1β620βbp (GenBank accession No. KY132100) and encoded a 539 amino acid polypeptide. Bioinformatic analyses showed that CsPT4 had a molecular weight of 59.12βkD and a theoretical isoelectric point of 8.51. The protein secondary structure was a “6+Hydrophilic+6” configuration,which was consistent with the typical structure of Phts. Subcellular localization assay showed that the CsPT4 protein localized in plasma membrane, which was consistent with the predicted results of Softberry. The expression pattern of CsPT4 gene was tissue-specific. Its transcript abundance in old leaves was much higher than that in tender leaves, stems and roots. The lowest expression of CsPT4 gene was identified in roots. Quantitative real-time PCR showed that the gene expression trends in root and leaves were different under low-P and P-deficiency treatments. Under low-P treatment, its induced level was first increased and then decreased, with higher expression in roots than leaves. While under P-deficiency treatment, the induced expression of CsPT4 gene kept stable, with its peak in roots and leaves at 72 h and 48 h, respectively. The results of this study provided a reference for the study of the molecular mechanism of tea adaptation to low P.

Key words: tea plant (Camellia sinensis), phosphate transporter protein, gene cloning, subcellular localization, expression analysis

中图分类号:

S571.1
Q51

辛华洪, 王伟东, 王明乐, 马青平, 甘玉迪, 黎星辉. 茶树磷转运蛋白基因CsPT4的克隆、亚细胞定位及表达分析[J]. 茶叶科学, 2017, 37(5): 493-502.

XIN Huahong, WANG Weidong, WANG Mingle, MA Qingping, GAN Yudi, LI Xinghui. Molecular Cloning, Subcellular Localization and Expression Analysis of CsPT4 Gene in Tea Plant (Camellia sinensis)[J]. Journal of Tea Science, 2017, 37(5): 493-502.

参考文献 34

[1]	Liu J, Yang L, Luan M, et al.A vacuolar phosphate transporter essential for phosphate homeostasis in Arabidopsis[J]. Proceedings of the National Academy of Sciences, 2015, 112(47): E6571-E6578.
[2]	Rausch C, Bucher M.Molecular mechanisms of phosphate transport in plants[J]. Planta, 2002, 216(1): 23-37.
[3]	Rae A L, Cybinski D H, Jarmey J M, et al.Characterization of two phosphate transporters from barley, evidence for diverse function and kinetic properties among members of the Pht1 family[J]. Plant Mol Biol, 2003, 53(1/2): 27-36.
[4]	苏顺宗, 吴锋锴, 刘丹, 等. 一个玉米Pht1家族磷转运蛋白基因克隆和功能分析[J]. 核农学报, 2013, 27(7): 885-894.
[5]	Liu J, Versaw W K, Pumplin N, et al.Closely related members of the medicago truncatula PHT1 phosphate transporter gene family encode phosphate transporters with distinct biochemical activities[J]. Journal of Biological Chemistry, 2008, 283(36): 24673-24681.
[6]	Catarecha P, Segura M D, Franco-zorrilla J M, et al. A mutant of the arabidopsis phosphate transporter PHT1:1 displays enhanced arsenic accumulation[J]. The Plant Cell Online, 2007, 19(3): 1123-1133.
[7]	Miao J, Sun J, Liu D, et al.Characterization of the promoter of phosphate transporter TaPHT1.2, differentially expressed in wheat varieties[J]. Journal of Genetics and Genomics, 2009, 36(8): 455-466.
[8]	Preuss C P, Huang C Y, Gilliham M, et al.Channel-like characteristics of the low-affinity barley phosphate transporter PHT1:6 when expressed in xenopus oocytes[J]. Plant Physiology, 2010, 152(3): 1431-1441.
[9]	Park M R, Baek S, Los Reyes B G, et al. Overexpression of a high-affinity phosphate transporter gene from tobacco (NtPT1) enhances phosphate uptake and accumulation in transgenic rice plants[J]. Plant and Soil, 2007, 292(1/2): 259-269.
[10]	Tan Z, Hu Y, Lin Z.Expression of NtPT5 is correlated with the degree of colonization in tobacco roots inoculated with glomus etunicatum[J]. Plant Molecular Biology Reporter, 2012, 30(4): 885-893.
[11]	Shin H, Shin H, Dewbre G R, et al.Phosphate transport in Arabidopsis: Pht1:1 and Pht1:4 play a major role in phosphate acquisition from both low- and high-phosphate environments[J]. The Plant Journal, 2004, 39(4): 629-642.
[12]	Misson J, Thibaud M C, Bechtold N, et al.Transcriptional regulation and functional properties of Arabidopsis, Pht1;4, a high affinity transporter contributing greatly to phosphate uptake in phosphate deprived plants[J]. Plant Molecular Biology, 2004, 55(5): 727.
[13]	高佳, 刘雄伦, 刘玲, 等. 水稻磷酸盐转运蛋白Pht1家族研究进展[J]. 中国农学通报, 2009, 25(15): 31-34.
[14]	Ye Y, Yuan J, Chang X, et al.The phosphate transporter gene OsPht1:4 is involved in phosphate homeostasis in rice[J]. Plos One, 2015, 10(5): e126186.
[15]	Ren F, Zhao C, Liu C, et al.A brassica napus PHT1 phosphate transporter, BnPht1:4, promotes phosphate uptake and affects roots architecture of transgenic arabidopsis[J]. Plant Molecular Biology, 2014, 86(6): 595-607.
[16]	Song H, Yin Z, Chao M, et al.Functional properties and expression quantitative trait loci for phosphate transporter GmPT1 in soybean[J]. Plant, Cell & Environment, 2014, 37(2): 462-472.
[17]	Peng L, Chen S, Song A, et al.A putative high affinity phosphate transporter, CmPT1, enhances tolerance to Pi deficiency of chrysanthemum[J]. Bmc Plant Biology, 2014, 14(1): 1-9.
[18]	周俊琴, 谭晓风, 袁军, 等. 油茶Pht1;2的克隆及生物信息学分析[J]. 生物技术, 2013(1): 37-42.
[19]	周俊琴, 谭晓风, 袁军, 等. 油茶Phtl;1基因克隆及其表达分析[J]. 植物遗传资源学报, 2013, 14(3): 512-517.
[20]	Akn Z, Loganathan P, Hedley M J.Phosphorus utilisation efficiency and depletion of phosphate fractions in the rhizosphere of three tea (Camellia sinensis L.) clones[J]. Nutrient Cycling in Agroecosystems, 1999, 53(2): 189-201.
[21]	Salehi S Y, Hajiboland R.A high internal phosphorus use efficiency in tea (Camellia sinensis L.) plants[J]. Asian Journal of Plant Sciences, 2008, 7(1): 30-36.
[22]	Lin Z, Qi Y, Chen R, et al.Effects of phosphorus supply on the quality of green tea[J]. Food Chemistry, 2012, 130(4): 908-914.
[23]	Wan Q, Xu RK, Li XH.Proton release by tea plant (Camellia sinensis L.) roots as affected by nutrient solution concentration and pH[J]. Plant Soil Environ, 2012, 58(9): 429-434
[24]	Raghothama K G.Phosphate acquisition[J]. Annual Review of Plant Biology, 1999, 50(1): 665-693.
[25]	Schachtman D P, Reid R J, Ayling S M.Phosphorus uptake by plants: from soil to cell[J]. Plant Physiology, 1998, 116(2): 447-453.
[26]	何飞顶, 李华昌, 冯先进. 电感耦合等离子体原子发射光谱法(ICP-AES)测定红土镍矿中的Cd、Co、Cu、Mg、Mn、Ni、Pb、Zn、Ca 9种元素[J]. 中国无机分析化学, 2011, 1(2): 39-41.
[27]	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.
[28]	杨亦扬, 胡雲飞, 万青, 等. 茶树硝态氮转运蛋白NRT1.1基因的克隆及表达分析[J]. 茶叶科学, 2016, 36(5): 505-512.
[29]	王明乐, 王伟东, 赵真, 等. 茶树NADPH氧化酶基因的克隆、亚细胞定位与表达分析[J]. 园艺学报, 2015, 42(1): 95-103.
[30]	Wang W, Wang Y, Du Y, et al.Overexpression of Camellia sinensis H1 histone gene confers abiotic stress tolerance in transgenic tobacco[J]. Plant Cell Reports, 2014, 33(11): 1829-1841.
[31]	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.
[32]	Livak K J, Schmittgen T D.Analysis of relative gene expression data using real-time quantitative PCR and the 2-ΔΔCT method[J]. Methods, 2001, 25(4): 402-408.
[33]	李慧, 丛郁, 常有宏, 等. 豆梨磷转运蛋白质基因(PcPht1)的克隆、表达及启动子分析[J]. 江苏农业学报, 2013, 29(4): 842-850.
[34]	杨存义, 刘灵, 沈宏, 等. 植物Pht1家族磷转运子的分子生物学研究进展[J]. 分子植物育种, 2006, 4(2): 153-159.