Scientia Agricultura Sinica ›› 2012, Vol. 45 ›› Issue (14): 2904-2912.doi: 10.3864/j.issn.0578-1752.2012.14.013

• HORTICULTURE • Previous Articles     Next Articles

Cloning and Expression Analysis of Metallothionein Gene (MhMT2) in Malus hupehensis

 WANG  Shun-Cai, LIANG  Dong, MA  Feng-Wang   

  1. 1.天水师范学院生命科学与化学学院，甘肃天水748100
    2.西北农林科技大学园艺学院，陕西杨凌712100
  • Received:2012-03-21 Online:2012-07-15 Published:2012-05-08

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Abstract: 【Objective】The objective of this study is to clone the full-length cDNA of metallothionein (MT) gene from rootstock (Malus hupehensis), to understand the mechanism of MT in response to environmental stress and provide fundamental evidence for molecular breeding of apple trees under stress conditions.【Method】The full-length cDNA sequence of MhMT2 gene was amplified from the leaves of M. hupehensis using in silico cloning and RT-PCR. Bioinformatics methods were used to analyze cDNA sequence obtained and putative amino acid sequence. The expression profiles of MhMT2 transcript in control seedlings and those exposed to various abiotic stresses and hormone treatments were detected by quantitative real-time RT-PCR.【Result】Sequence analysis indicated that MhMT2 cDNA was 534 bp in length and contained an intact open reading frame of 243 bp, encoding a polypeptide of 80 amino acids with a theoretical molecular mass of 7.87 kD. Homology comparison analysis showed that the deduced MhMT2 protein was highly homologous to other MT2 proteins from plant species, sharing a 96.4% homology with M. xiaojinensis. Bioinformatic analysis revealed that the putative MhMT2 protein contained 14 cysteine residues occurring as C-C, C-X-C, and C-X-X-C motifs in the N-terminal, and of C-X-C in the C-terminal region (X are other amino acids other than cysteine). Phylogenetic analysis suggested that this gene belonged to type 2 plant MT genes family. Quantitative real-time RT-PCR analysis indicated that MhMT2 showed the highest transcript abundance in leaves, moderate level in roots and the lowest level in shoots. MhMT2 transcripts in leaves were markedly increased by abscisic acid (ABA), hydrogen peroxide (H2O2), mechanical wounding, and NaCl, especially H2O2 treatment.【Conclusion】The induced expression profiling of MhMT2 gene under ABA, H2O2, mechanical wounding, and salt stress indicates that the MhMT2 gene may play an important role in response to these abiotic stresses.

Key words: M. hupehensis, metallothionein gene, cloning, expression

[1]Margoshes M, Vallee B L. A cadmium protein from equine kidney cortex. Journal of the American Chemical Society, 1957, 79(17): 4813-4814.

[2]Casterline J L, Barnett N M. Isolation and characterization of cadmium binding components in soybean plants. Plant Physiology, 1977, 59: (Suppl.): 124.

[3]Hassinen V H, Tervahauta A I, Schat H, Karenlampi S O. Plant metallothioneins--metal chelators with ROS scavenging activity? Plant Biology (Stuttg), 2011, 13(2):225-232.

[4]Vasak M, Meloni G. Chemistry and biology of mammalian metallothioneins. Journal of Biological Inorganic Chemistry, 2011, 16(7):1067-1078.

[5]Freisinger E. Plant MTs-long neglected members of the metallothionein superfamily. Dalton Transactions, 2008, 47: 6663-6675.

[6]Yuan J, Chen D, Ren Y, Zhang X, Zhao J. Characteristic and expression analysis of a metallothionein gene, OsMT2b, down- regulated by cytokinin suggests functions in root development and seed embryo germination of rice. Plant Physiology, 2008, 146(4): 1637-1650.

[7]Xue T T, Li X Z, Zhu W, Wu C G, Yang G D, Zheng C C. Cotton metallothionein GhMT3a, a reactive oxygen species scavenger, increased tolerance against abiotic stress in transgenic tobacco and yeast. Journal of Experimental Botany, 2009, 60(1):339-349.

[8]Mir G, Domenech J, Huguet G, Guo W J, Goldsbrough P, Atrian S, Molinas M. A plant type 2 metallothionein (MT) from cork tissue responds to oxidative stress. Journal of Experimental Botany, 2004, 55(408):2483-2493.

[9]Choi D, Kim H M, Yun H K, Park J A, Kim W T, Bok S H. Molecular cloning of a metallothionein-like gene from Nicotiana glutinosa L. and its induction by wounding and tobacco mosaic virus infection. Plant Physiology, 1996, 112(1):353-359.

[10]Zhou G, Xu Y, Li J, Yang L, Liu J Y. Molecular analyses of the metallothionein gene family in rice (Oryza sativa L.). Journal of Biochemistry and Molecular Biology, 2006, 39(5):595-606.

[11]Guo W-J, Bundithya W, Goldsbrough P B. Characterization of the Arabidopsis metallothionein gene family: tissue-specific expression and induction during senescence and in response to copper. New Phytologist, 2003, 159:369-381.

[12]Samardzic J T, Nikolic D B, Timotijevic G S, Jovanovic Z S, Milisavljevic M D, Maksimovic V R. Tissue expression analysis of FeMT3, a drought and oxidative stress related metallothionein gene from buckwheat (Fagopyrum esculentum). Journal of Plant Biology, 2010, 167(16):1407-1411.

[13]Freisinger E. The metal-thiolate clusters of plant metallothioneins. Chimia (Aarau), 2010, 64(4):217-224.

[14]Yang J L, Wang Y C, Liu G F, Yang C P, Li C H. Tamarix hispida metallothionein-like ThMT3, a reactive oxygen species scavenger, increases tolerance against Cd2+, Zn2+, Cu2+, and NaCl in transgenic yeast. Molecular Biology Reports, 2011, 38(3):1567-1574.

[15]张玉刚, 韩振海. 小金海棠金属硫蛋白基因MxMT2克隆与生物信息学分析. 华北农学报, 2010, 25(2):60-63.

Zhang Y G, Han Z H. Cloning and bioinformatic analysis of metallothionein gene (MxMT2) in Malus xiaojinensis. Acta Agriculturae Boreali-Sinica, 2010, 25(2):60-63 (in Chinese)

[16] Wang S C, Liang D, Shi S G, Ma F W, Shu H R, Wang R C. Isolation and characterization of a novel drought responsive gene encoding a glycine-rich RNA-binding protein in Malus prunifolia (Willd.) Borkh. Plant Molecular Biology Reporter, 2011, 29:125-134.

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

[18]Fowler B A, Hildebrand C E, Kojima Y, Webb M. Nomenclature of metallothionein. Experientia Supplementum, 1987, 52:19-22.

[19]Binz P A, Kägi J H R. Metallothionein: molecular evolution and classification. In: Klaassen C. ed. Metalloth-ionein Ⅳ. Birkhäuser Verlag Basel, Switzerland, 1999: 7-13.

[20]Cobbett C, Goldsbrough P. Phytochelatins and metallothioneins: roles in heavy metal detoxification and homeostasis. Annual Review of Plant Biology, 2002, 53:159-182.

[21]Wan X, Freisinger E. The plant metallothionein 2 from Cicer arietinum forms a single metal-thiolate cluster. Metallomics, 2009, 1(6):489-500.

[22]Ren Y J, Liu Y, Chen H Y, Li G, Zhang X L, Zhao J. Type 4 metallothionein genes are involved in regulating Zn ion accumulation in late embryo and in controlling early seedling growth in Arabidopsis. Plant, Cell and Environment, 2011, 35(4):770-789.

[23]Yang Z, Wu Y, Li Y, Ling H Q, Chu C. OsMT1a, a type 1 metallothionein, plays the pivotal role in zinc homeostasis and drought tolerance in rice. Plant Molecular Biology, 2009, 70(1-2):219-229.

[24]Chang T J, Chen L, Lu Z X, Chen W X, Liu X, Zhu Z. Cloning and Expression Patterns of a Metallothionein-like Gene htMT2 of Helianthus tuberosus. Acta Botanica Sinica, 2002, 44(10):118-1193.

[25]Zhou Y, Chu P, Chen H, Li Y, Liu J, Ding Y, Tsang E W, Jiang L, Wu K, Huang S. Overexpression of Nelumbo nucifera metallothioneins 2a and 3 enhances seed germination vigor in Arabidopsis. Planta, 2011, 235(3):523-537.

[26]Osobova M, Urban V, Jedelsky P L, Borovicka J, Gryndler M, Ruml T, Kotrba P. Three metallothionein isoforms and sequestration of intracellular silver in the hyperaccumulator Amanita strobiliformis. New Phytologist, 2011, 190(4):916-926.

[27]Zhu W, Zhao D X, Miao Q, Xue T T, Li X Z, Zheng C C. Arabidopsis thaliana metallothionein, AtMT2a, mediates ROS balance during oxidative stress. Journal of Plant Biology, 2009, 52(6):585-592.

[28]周博如, 王  雷, 吴丽丽, 姜廷波. 转金属硫蛋白基因(MT1)烟草耐 NaCl 胁迫能力. 生态学报, 2010, 30(15):4103-4108.

Zhou B R, Wang L, Wu L L, Jiang T B. Characterization of transgenic tobacco overexpressing metallothionein gene (MT1) on NaCl stress. Acta Ecologica Sinica, 2010, 30(15):4103-4108. (in Chinese)

[29]Sutherland D E, Stillman M J. The "magic numbers" of metallothionein. Metallomics, 2011, 3(5):444-463.

[30]Singh R K, Anandhan S, Singh S, Patade V Y, Ahmed Z, Pande V. Metallothionein-like gene from Cicer microphyllum is regulated by multiple abiotic stresses. Protoplasma, 2011, 248(4):839-847.
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