一个橡胶树谷胱甘肽-S-转移酶基因的克隆和表达特性分析

doi:10.3864/j.issn.0578-1752.2011.20.003

摘要/Abstract

摘要： 【目的】从橡胶树中克隆Glutathione-S-transferase（GST）基因的cDNA和基因组DNA，进行序列、结构和系统进化分析，研究胁迫条件和激素处理下的表达谱。【方法】利用产排胶机理课题组构建的胶乳EST数据库，通过PCR技术克隆橡胶树GST基因的cDNA和基因组DNA；在线预测蛋白质保守功能域和亚细胞定位，构建系统进化树，利用实时荧光定量PCR分析割胶、伤害、低温、激素和死皮病等因素对该基因表达的影响。【结果】从橡胶树胶乳中克隆到1个GST基因的全长cDNA（793 bp），编码25.4 kD（219aa）蛋白，命名为HbGSTU1；克隆了HbGSTU1的基因组DNA（1 313 bp），包含1个内含子（520 bp）和2个外显子。HbGSTU1属于GST Tau家族，推测是一种无信号肽的细胞质蛋白，其N端结构域包含结合GSH的G位点，C端结构域包含特异结合底物的H位点；HbGSTU1与一个蓖麻GST蛋白的亲缘关系最近，氨基酸序列的一致性为85%；HbGSTU1的表达受割胶、伤害、低温、2,4-D、乙烯利和死皮病等因素调控，但对JA、SA和ABA处理的应答不明显。【结论】HbGSTU1是一个典型的GST Tau家族蛋白，可能在橡胶树抗逆过程中起作用，可作为橡胶树抗逆分子育种的靶标基因。

关键词: 橡胶树, 谷胱甘肽-S-转移酶, 基因表达, 胁迫, 激素

Abstract: 【Objcetive】The objective of this study is to clone the cDNA and genomic clone of a glutathione-S-transferase (GST) from rubber tree, and characterize its structural and phylogenetic characteristics, investigate its expression patterns under various abiotic stresses and hormone treatments. 【Method】Combined with the latex EST database, RT-PCR and PCR were used to clone the full length cDNA and genomic DNA of a Hevea GST, respectively. On-line bioinformatics tools were used to predict the conserved functional domains and subcellular localization. Clustal X was used to construct the phylogenetic tree. Real-time RT-PCR (qRT-PCR) was used to characterize the expression patterns of the Hevea GST gene under various treatments (tapping, wounding, chilling, hormone and tapping panel dryness). 【Result】The full-length cDNA (793 bp) encoding a 25.4 kD (219 aa) Tau GST protein was firstly cloned from the latex of H. brasiliensis, named HbGSTU1 and deposited in GenBank (JF729318). The genomic DNA (1 313 bp) of HbGSTU1 was also cloned, which contains one intron (520 bp) and two exons. HbGSTU1 protein contained the Tau GST-specific N-terminal domain (G site) and the C-terminal domain (H site). HbGSTU1 was predicted to be a cytoplasmic protein, which shared the highest amino acid identity (85%) with a GST from Ricinus communis. The qRT-PCR results show that HbGSTU1 expression was regulated by tapping, wounding, 4℃ chilling, 2,4-D and Ethrel, but not by the three hormones JA, SA and ABA. 【Conclusion】HbGSTU1 is a typical Tau GST protein, and is implicated in stress-responses in Hevea tree, which may serve as a target gene for Hevea molecular breeding.

Key words:

Heveabrasiliensis, glutathione-S-transferases, geneexpression, stress, hormones

范玉洁, 林飞鹏, 安泽伟, 唐朝荣. 一个橡胶树谷胱甘肽-S-转移酶基因的克隆和表达特性分析[J]. 中国农业科学, 2011, 44(20): 4150-4158.

FAN Yu-Jie, LIN Fei-Peng, AN Ze-Wei, TANG Chao-Rong. Cloning and Expression Analysis of A Glutathione-S-transferase Gene in The Latex of Hevea brasiliensis (para rubber tree)[J]. Scientia Agricultura Sinica, 2011, 44(20): 4150-4158.

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参考文献

[1]Gong H B, Hu W W, Jiao Y X, Pua E C. Molecular characterization of a Phi-class mustard (Brassica juncea) glutathione-S-transferase gene in Arabidopsis thaliana by 5’-deletion analysis of its promoter. Plant Cell Reports, 2005, 24(7): 439-447.

[2]Wilce M C, Parker M W. Structure and function of glutathione- S-transferases. Biochimica et Biophysica Acta-Biomembranes, 1994, 1205(1): 1-18.

[3]Edwards R, Dixon D P. The role of glutathione transferases in herbicide metabolism. Herbicides and Their Mechanisms of Action. Sheffield: Sheffield Academic Press, 2000: 38-71.

[4]Moons A. Regulatory and functional interactions of plant growth regulators and plant glutathione-S-transferases (GSTs). Vitamins and Hormones, 2005, 72: 155-202.

[5]Marrs K A. The functions and regulation of glutathione S-transferases in plants. Annual Review of Plant Physiology and Plant Molecular Biology, 1996, 47(1): 127-158.

[6]Droog F. Plant glutathione S-transferases, a tale of theta and Tau. Journal of Plant Growth Regulation, 1997, 16(2): 95-107.

[7]Dixon D P, Lapthorn A, Edwards R. Plant glutathione transferases. Genome Biology, 2002, 3(3): 3004. 1-3004.10.

[8]Basantani M, Srivastava A. Plant glutathione transferases: A decade falls short. Canadian Journal of Botany, 2007, 85(5): 443-456.

[9]Chronopoulou E G, Labrou N E. Glutathione transferases: Emerging multidisciplinary tools in red and green biotechnology. Recent Patents on Biotechnology, 2009, 3(3): 211-223.

[10]Conn S, Curtin C, Bézier A, Franco C, Zhang W. Purification, molecular cloning, and characterization of glutathione S-transferases (GSTs) from pigmented Vitis vinifera L. cell suspension cultures as putative anthocyanin transport proteins. Journal of Experimental Botany, 2008, 59(13): 3621-3634.

[11]Sappl P G, Carroll A J, Clifton R, Lister R, Whelan J, Harvey Millar A, Singh K B. The Arabidopsis glutathione transferase gene family displays complex stress regulation and co-silencing multiple genes results in altered metabolic sensitivity to oxidative stress. The Plant Journal, 2009, 58(1): 53-68.

[12]Soranzo N, Sari Gorla M, Mizzi L, De Toma G, Frova C. Organisation and structural evolution of the rice glutathione S-transferase gene family. Molecular Genetics and Genomics, 2004, 271(5): 511-521.

[13]Lan T, Yang Z L, Yang X, Liu Y J, Wang X R, Zeng Q Y. Extensive functional diversification of the Populus glutathione S-transferase supergene family. The Plant Cell, 2009, 21(12): 3749-3766.

[14]Dixon D P, Cummins I, Cole D J, Edwards R. Glutathione-mediated detoxification systems in plants. Current Opinion in Plant Biology, 1998, 1(3): 258-266.

[15]Nutricati E, Miceli A, Blando F, De Bellis L. Characterization of two Arabidopsis thaliana glutathione-S-transferases. Plant Cell Reports, 2006, 25(9): 997-1005.

[16]Wagner U, Edwards R, Dixon D P, Mauch F. Probing the diversity of the Arabidopsis glutathione-S-transferase gene family. Plant Molecular Biology, 2002, 49(5): 515-532.

[17]Dixon D P, Davis B G, Edwards R. Functional divergence in the glutathione transferase superfamily in plants. Identification of two classes with putative functions in redox homeostasis in Arabidopsis thaliana. The Journal of Biological Chemistry, 2002, 277(34): 30859-30869.

[18]Cottingham C K, Hatzios K K, Meredith S. Influence of chemical treatments on glutathione-S-transferases of maize with activity towards metolachlor and cinnamic acid. Zeitschrift fur Naturforschung C: Journal of Biosciences, 1998, 53(11/12): 973-979.

[19]Vollenweider S, Weber H, Stolz S, Chételat A, Farmer E E. Fatty acid ketodienes and fatty acid ketotrienes: Michael addition acceptors that accumulate in wounded and diseased Arabidopsis leaves. The Plant Journal, 2000, 24(4): 467-476.

[20]Seppänen M M, Cardi T, Borg Hyökki M, Pehu E. Characterisation and expression of cold induced glutathione S-transferase in freezing tolerant Solanum commersonii, sensitive S. tuberosum and their interspecific somatic hybrids. Plant Science, 2000, 153(2): 125-133.

[21]Zhou J M, Goldsbrough P B. An Arabidopsis gene with homology to glutathione S-transferases is regulated by ethylene. Plant Molecular Biology, 1993, 22(3): 517-523.

[22]Chen W Q, Singh K B. The auxin, hydrogen peroxide and salicylic acid induced expression of the Arabidopsis GST6 promoter is mediated in part by an ocs element. The Plant Journal, 1999, 19(6): 667-677.

[23]Chen W Q, Chao G, Singh K B. The promoter of a H2O2- inducible, Arabidopsis glutathione S-transferase gene contains closely linked OBF- and OBP1-binding sites. The Plant Journal, 1996, 10(6): 955-966.

[24]安泽伟, 陈根辉, 程汉, 赵彦宏, 谢黎黎, 黄华孙. 橡胶树冷应答转录组cDNA-AFLP分析. 林业科学, 2010, 46(3): 62-67.

An Z W, Chen G H, Cheng H, Zhao Y H, Xie L L, Huang H S. cDNA-AFLP Analysis on transcriptomics of Hevea brasiliensis induced by cold stress. Scientia Silvae Sinicae, 2010, 46(3): 62-67. (in Chinese)

[25]Tang C R, Qi J Y, Li H P, Zhang C L, Wang Y K. A convenient and efficient protocol for isolating high-quality RNA from latex of Hevea brasiliensis (para rubber tree). Journal of Biochemical and Biophysical Methods, 2007, 70(5): 749-754.

[26]Kiefer E, Heller W, Ernst D. A simple and efficient protocol for isolation of functional RNA from plant tissues rich in secondary metabolites. Plant Molecular Biology Reporter, 2000, 18(1): 33-39.

[27]Tang C R, Huang D B, Yang J H, Liu S J, Sakr S, Li H P, Zhou Y H, Qin Y X. The sucrose transporter HbSUT3 plays an active role in sucrose loading to laticifer and rubber productivity in exploited trees of Hevea brasiliensis (para rubber tree). Plant Cell and Environment, 2010, 33(10): 1708-1720.

[28]Moons A. Osgstu3 and osgtu4, encoding tau class glutathione- S-transferases, are heavy metal- and hypoxic-stress induced and differentially salt stress-responsive in rice roots. Federation of European Biochemical Societies, 2003, 553(3): 427-432.

[29]Polidoros A N, Scandaios J G. Role of hydrogen peroxide and different classes of antioxidants in the regulation of catalase and glutathione-S-transferase gene expression in maize (Zea mays L.). Physiologia Plantarum, 1999, 106(1): 112-120.

[30]许闻献, 校现周. 橡胶死皮树过氧化物酶同工酶和超氧化物歧化酶同工酶的研究. 热带作物学报, 1988, 9(1): 31-36.

Xu W X, Xiao X Z. Studies on peroxidase and superoxidase dimutase isozymes in dry rubber trees. Chinese Journal of Tropical Crops, 1988, 9(1): 31-36. (in Chinese)

[31]Darussamin A, Suharyanto S, Chaidamsari T. Changes in the chemical compositions and electrophoretic profile of latex and bark proteins related to tapping panel dryness incidence in Hevea brasiliensis. Menara Perkebunan, 1995, 63(2): 52-59.