茶树谷胱甘肽还原酶基因CsGRs的克隆与表达分析

doi:10.3864/j.issn.0578-1752.2014.16.013

摘要/Abstract

摘要： 【目的】克隆谷胱甘肽还原酶基因CsGRs，研究CsGRs在茶树抵御不同逆境胁迫中的作用。【方法】在茶树转录组数据库中搜索茶树CsGRs，根据获得的基因片段，设计反转录PCR（RT-PCR）引物和RACE-PCR特异引物，从茶树中克隆CsGRs的cDNA全长序列，并利用在线生物信息学软件对其进行分析。采用实时荧光定量PCR（qRT-PCR）分析CsGRs在茶树不同组织间的表达差异及其在低温、干旱、高盐胁迫和ABA处理下的表达模式，利用分光光度计测定低温胁迫和干旱胁迫下叶片中还原型谷胱甘肽（GSH）的含量变化。【结果】RT-PCR克隆获得CsGR1的cDNA全长，其长度为1 827 bp，包含1 482 bp开放阅读框（ORF），编码493个氨基酸；RACE扩增获得712 bp和1 624 bp的5′和3′末端序列，拼接并进行RT-PCR验证后得到CsGR2序列，全长2 282 bp，包含1 698 bp ORF，编码565个氨基酸；CsGR1和CsGR2的GenBank登录号分别为KF906411和KF418080。CsGR1和CsGR2编码的蛋白质分子量分别为53.9 kD和61.0 kD，无信号肽位点，均为非分泌性蛋白。亚细胞定位预测CsGR1主要定位在细胞质等亚细胞中，无叶绿体锚定信号肽位点；CsGR2中N-端的71个氨基酸残基具有叶绿体转运信号功能，主要定位在叶绿体上。序列相似性比较显示，CsGR1与其他植物中胞质GR的相似性均在80％以上，而与叶绿体GR的相似性低于60％；CsGR2与其他叶绿体GR的相似性70％以上，与胞质GR的相似性在50％左右。二者在核酸序列和氨基酸序列水平上分别有63.4％和49.9％的相似性，且蛋白质二级结构也具有较高的相似性。系统发育树显示，CsGR1与胞质GR聚为一类，而CsGR2与叶绿体GR聚在一起，且都与葡萄的亲缘关系最近。二者均含有氧化还原二硫键活性位点、谷胱甘肽结合位点以及NADPH结合的Arg保守位点等结构域。CsGR1为胞质GR，CsGR2为双向定位在叶绿体和线粒体上的叶绿体GR。CsGR1在花和根中表达量较高，在叶片和茎中表达量低；CsGR2在叶片和茎中的表达量比在根和花中的表达量高。在短时胁迫处理24 h过程中，成熟叶片在100 μmol•L-1 ABA处理后，CsGR1和CsGR2的表达均被抑制，且CsGR2的抑制作用较显著；在4℃低温胁迫下，CsGR1的表达被抑制，而CsGR2的表达随处理时间延长逐渐被诱导；250 mmol•L-1 NaCl盐胁迫抑制CsGRs的表达，但胁迫24 h时CsGR2的表达被诱导。10%（w/v）PEG胁迫处理茶树8 h，叶片中的CsGRs均被诱导表达，且在复水48 h后CsGR1的表达被显著上调；根中CsGRs的表达在处理和复水48 h过程中均被抑制。随低温胁迫时间延长，茶树叶片中的GSH含量逐渐升高；干旱胁迫也能促进GSH在叶片中的积累，在复水48 h后又恢复到处理前的水平。【结论】克隆了2个茶树CsGRs，2个基因对4℃低温、NaCl盐、10％PEG和ABA处理均具有响应。推测CsGRs在茶树抵御逆境胁迫中起作用。

关键词: 茶树 , 谷胱甘肽还原酶（GR） , 逆境胁迫 , 表达分析

Abstract: 【Objective】 The objectives of the present study were to clone the genes of glutathione reductase family (CsGRs) from tea plant (Camellia sinensis) and investigate their functions under different abiotic stresses. 【Method】 According to the sequences of CsGR genes obtained from the transcriptome database of tea plant, specific primers were designed for reverse transcription-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends PCR (RACE-PCR) to clone the full-length sequences of CsGRs. The bioinformatic characteristics of the cloned CsGRs were analyzed using online service. The expression profiles of CsGRs in various tissues and in response to cold, drought, salt and abscisic acid (ABA) treatment were investigated using quantitative real-time PCR (qRT-PCR). Spectrophotometer technique was employed to determine the content of glutathione (GSH) upon cold and drought stress. 【Result】 CsGR1 was isolated from tea plant using RT-PCR and had 1 827 bp in length containing a 1 482 bp open reading frame (ORF) encoded 493 amino acid residues. For CsGR2 isolating, 712 bp and 1 624 bp in length of 5′ and 3′ terminal sequences were amplified by RACE-PCR, respectively, after sequences assembling and verified by RT-PCR, CsGR2 with 2 282 bp in length containing a 1 698 bp ORF encoded polypeptide of 565 amino acids was obtained. CsGR1 and CsGR2 were submitted to GenBank with accession number KF906411 and KF418080, respectively. The molecular weights of CsGR1 and CsGR2 encoded protein were 53.9 and 61.0 kD, respectively, and both of them did not contain the signal peptide sites, indicating that they were not the secretory proteins. Subcellular localization prediction showed that CsGR1 might localize in cytoplasm without chloroplast transit peptides (cTP), whereas CsGR2, containing a putative cTP of 71 amino acid residues at the N-terminal, was most likely to target to chloroplast. Comparison of sequences similarity with reported GRs showed that CsGR1 had more than 80% similarity with cytosolic-GRs and less than 60% similarity with chloroplastic-GRs, whereas CsGR2 shared over 70% identity with other chloroplastic-GRs and about 50% similarity with cytosolic-GRs. CsGR1 and CsGR2 shared 63.4% and 49.9% sequence identity in nucleotide and amino acid, respectively, and had high similarity with each other in secondary structure of protein. Phylogenetic tree analysis showed that CsGR1 and CsGR2 could be clustered into cytosolic-GRs and chloroplastic-GRs, respectively, and they had the closest genetic relationship with Vitis vinifera. The redox-active disulfide bridge, the glutathione-binding residues and the conserved arginine residues for NADPH binding were found in both of them. Hence, CsGR1 encodes a putative cytosolic isoform, and CsGR2 belongs to chloroplastic GR which dual-targeted to both chloroplasts and mitochondria. Transcript abundance of CsGR1 was higher in flowers and roots than that in leaves and stems. And CsGR2 displayed the opposite expression pattern in tissues compared to CsGR1. Analysis of the expression patterns in response to abiotic stress revealed that CsGR1 and CsGR2 were down-regulated by ABA treatement in leaves and the suppression level of CsGR2 was higher than CsGR1. Furthermore, the expression of CsGR1 was repressed by cold (4℃) stress, whereas CsGR2 could be gradually induced with the extension of treatment time. Similarly, under salt stress, the expression of CsGR1 was suppressed, and CsGR2 was up-regulated after 24 h treatment. Under PEG treatment, both of CsGRs were up-regulated in leaves and down-regulated in roots. Analysis of the concentration of GSH showed that GSH content was gradually induced by cold and PEG treatment in leaves. Moreover, the GSH concentration could be returned to and kept at constant level after 48 h recovery in PEG treatment. 【Conclusion】 In this study, two CsGRs were cloned from tea plant, and their expressions were regulated by ABA, cold (4℃), salt and PEG stress. The results demonstrated that GR might play a role in abiotic stresses tolerance of tea plant.

Key words: tea plant (Camellia sinensis) , glutathione reductase (GR) , abiotic stress , expression analysis

岳川，曹红利，周艳华，王璐，郝心愿，王新超，杨亚军. 茶树谷胱甘肽还原酶基因CsGRs的克隆与表达分析[J]. 中国农业科学, 2014, 47(16): 3277-3289.

YUE Chuan, CAO Hong-li, ZHOU Yan-hua, WANG Lu, HAO Xin-yuan, WANG Xin-chao, YANG Ya-jun. Cloning and Expression Analysis of Glutathione Reductase Genes(CsGRs) in Tea Plant (Camellia sinensis)[J]. Scientia Agricultura Sinica, 2014, 47(16): 3277-3289.

0
/ / 推荐

导出引用管理器 EndNote|Reference Manager|ProCite|BibTeX|RefWorks

链接本文: https://www.chinaagrisci.com/CN/10.3864/j.issn.0578-1752.2014.16.013

https://www.chinaagrisci.com/CN/Y2014/V47/I16/3277

参考文献

[1]Pastori G, Foyer C H, Mullineaux P. Low temperature-induced changes in the distribution of H2O2 and antioxidants between the bundle sheath and mesophyll cells of maize leaves. Journal of Experimental Botany, 2000, 51(342): 107-113.

[2]Gill S S, Anjum N A, Hasanuzzaman M, Gill R, Trivedi D K, Ahmad I, Pereira E, Tuteja N. Glutathione and glutathione reductase: A boon in disguise for plant abiotic stress defense operations. Plant Physiology and Biochemistry, 2013, 70: 204-212.

[3]Le Martret B, Poage M, Shiel K, Nugent G D, Dix P J. Tobacco chloroplast transformants expressing genes encoding dehydroascorbate reductase, glutathione reductase, and glutathione- S-transferase, exhibit altered anti-oxidant metabolism and improved abiotic stress tolerance. Plant Biotechnology Journal, 2011, 9(6): 661-673.

[4]Noctor G, Mhamdi A, Chaouch S, Han Y I, Neukermans J, Marquez-Garcia B, Queval G, Foyer C H. Glutathione in plants: an integrated overview. Plant, Cell and Environment, 2012, 35(2): 454-484.

[5]Foyer C H, Souriau N, Perret S, Lelandais M, Kunert K J, Pruvost C, Jouanin L. Overexpression of glutathione reductase but not glutathione synthetase leads to increases in antioxidant capacity and resistance to photoinhibition in poplar trees. Plant Physiology, 1995, 109(3): 1047-1057.

[6]Noctor G, Foyer C H. Ascorbate and glutathione: Keeping active oxygen under control. Annual Review of Plant Physiology and Plant Molecular Biology, 1998, 49: 249-279.

[7]Chen Y P, Xing L P, Wu G J, Wang H Z, Wang X E, Cao A Z, Chen P D. Plastidial Glutathione Reductase from Haynaldia villosa is an Enhancer of Powdery Mildew Resistance in Wheat (Triticum aestivum). Plant and Cell Physiology, 2007, 48(12): 1702-1712.

[8]Stevens R G, Creissen G P, Mullineaux P M. Cloning and characterisation of a cytosolic glutathione reductase cDNA from pea (Pisum sativum L.) and its expression in response to stress. Plant Molecular Biology, 1997, 35(5): 641-654.

[9]Creissen G, Edwards E A, Enard C, Wellburn A, Mullineaux P. Molecular characterization of glutathione reductase cDNAs from pea (Pisum sativum L.). The Plant Journal,1992, 2(1): 129-131.

[10]陆建良, 梁月荣. 铝对茶树等植物超氧化物歧化酶的影响. 茶叶科学, 1997(02): 38-41.

Lu J L, Liang Y R. Effect of aluminium stress on superoxide dismutase in tea and other plants. Journal of Tea Science, 1997(02): 38-41. (in Chinese)

[11]朱政, 蒋家月, 江昌俊, 李雯. 低温胁迫对茶树叶片SOD、可溶性蛋白和可溶性糖含量的影响. 安徽农业大学学报, 2011(01): 24-26.

Zhu Z, Jiang J Y, Jiang C J, Li W. Effects of low temperature stress on SOD activity, soluble protein content and soluble sugar content in Camellia sinensis leaves. Journal of Anhui Agricultural University, 2011(01): 24-26. (in Chinese)

[12]屠幼英, 杨秀芳, 杨贤强. 茶树抗逆境生理与超氧歧化酶(SOD)的相关性. 茶叶, 1996(02): 40-43.

Tu Y Y, Yang X F, Yang X Q. The correlation of anti-stress physiology and superoxide dismutase (SOD) in tea plant. Journal of Tea, 1996(02): 40-43. (in Chinese)

[13] Vyas D, Kumar S. Tea (Camellia sinensis (L.) O. Kuntze) clone with lower period of winter dormancy exhibits lesser cellular damage in response to low temperature. Plant Physiology and Biochemistry, 2005, 43(4): 383-388.

[14]Chang S, Puryear J, Cairney J. A simple and efficient method for isolating RNA from pine trees. Plant Molecular Biology Reporter, 1993, 11(2): 113-116.

[15]曹红利, 岳川, 郝心愿, 王新超, 杨亚军. 茶树胆碱单加氧酶CsCMO的克隆及甜菜碱合成关键基因的表达分析. 中国农业科学, 2013, 46(15): 3087-3096.

Cao H L, Yue C, Hao X Y, Wang X C, Yang Y J. Cloning of choline monooxygenase (CMO) gene and expression analysis of the key glycine betaine biosynthesis-related genes in tea plant (Camellia sinensis). Scientia Agricultura Sinica, 2013, 46(15): 3087-3096. (in Chinese)

[16]Wang X C, Zhao Q Y, Ma C L, Zhang Z H, Cao H L, Kong Y M, Yue C, Hao X Y, Chen L, Ma J Q, Jin J Q, Li X, Yang Y J. Global transcriptome profiles of Camellia sinensis during cold acclimation. BMC Genomics, 2013, 14(1): 415.

[17]岳川, 曾建明, 曹红利, 郝心愿, 章志芳, 王新超, 杨亚军. 茶树赤霉素受体基因CsGID1a的克隆与表达分析. 作物学报, 2013(04): 599-608.

Yue C, Zeng J M, Cao H L, Hao X Y, Zhang Z F, Wang X C, Yang Y J. Cloning and expression analysis of gibberellin receptor gene CsGID1a in tea plant (Camellia sinensis). Acta Agronomica Sinica, 2013(04): 599-608. (in Chinese)

[18]Bhattacharya A, Saini U, Joshi R, Kaur D, Pal A K, Kumar N, Gulati A, Mohanpuria P, Yadav S K, Kumar S, Ahuja P S. Osmotin-expressing transgenic tea plants have improved stress tolerance and are of higher quality. Transgenic Research, 2013: 1-13.

[19]Chew O, Rudhe C, Glaser E, Whelan J. Characterization of the targeting signal of dual-targeted pea glutathione reductase. Plant Molecular Biology, 2003, 53(3): 341-356.

[20]Creissen G, Reynolds H, Xue Y, Mullineaux P. Simultaneous targeting of pea glutathione reductase and of a bacterial fusion protein to chloroplasts and mitochondria in transgenic tobacco. The Plant Journal, 1995, 8(2): 167-175.

[21]Romero-Puertas M C, Corpas F J, Sandalio L M, Leterrier M, Rodríguez-Serrano M, Del Río L A, Palma J M. Glutathione reductase from pea leaves: response to abiotic stress and characterization of the peroxisomal isozyme. New Phytologist, 2006, 170(1): 43-52.

[22]Kaminaka H, Morita S, Nakajima M, Masumura T, Tanaka K. Gene cloning and expression of cytosolic glutathione reductase in rice (Oryza sativa L.). Plant and Cell Physiology, 1998, 39(12): 1269-1280.

[23]Edwards E A, Rawsthorne S, Mullineaux P. Subcellular distribution of multiple forms of glutathione reductase in leaves of pea (Pisum sativum L.). Planta, 1990, 180(2): 278-284.

[24]Trivedi D K, Gill S S, Yadav S, Tuteja N. Genome-wide analysis of glutathione reductase (GR) genes from rice and Arabidopsis. Plant Signaling & Behavior, 2013, 8(2): e23021.

[25]Ding S H, Lei M, Lu Q T, Zhang A H, Yin Y, Wen X G, Zhang L X, Lu C M. Enhanced sensitivity and characterization of photosystem II in transgenic tobacco plants with decreased chloroplast glutathione reductase under chilling stress. Biochimica et Biophysica Acta, 2012, 1817(11): 1979-1991.

[26]Li G Z, Peng X Q, Wei L T, Kang G Z. Salicylic acid increases the contents of glutathione and ascorbate and temporally regulates the related gene expression in salt-stressed wheat seedlings. Gene, 2013, 529(2): 321-325.

[27]Liu Y J, Yuan Y, Liu Y Y, Liu Y, Fu J J, Zheng J, Wang G Y. Gene families of maize glutathione-ascorbate redox cycle respond differently to abiotic stresses. Journal of Plant Physiology, 2012, 169(2): 183-192.

[28]Contour-Ansel D, Torres-Franklin M L, Cruz DECMH, D'Arcy- Lameta A, Zuily-Fodil Y. Glutathione reductase in leaves of cowpea: cloning of two cDNAs, expression and enzymatic activity under progressive drought stress, desiccation and abscisic acid treatment. Annals of Botany, 2006, 98(6): 1279-1287.

[29]Jiang M Y, Zhang J H. Effect of abscisic acid on active oxygen species, antioxidative defence system and oxidative damage in leaves of maize seedlings. Plant and Cell Physiology, 2001, 42(11): 1265-1273.

[30]Lau O S, Deng X W. Plant hormone signaling lightens up: integrators of light and hormones. Current Opinion in Plant Biology, 2010, 13(5): 571-577.