Scientia Agricultura Sinica ›› 2014, Vol. 47 ›› Issue (4): 622-632.doi: 10.3864/j.issn.0578-1752.2014.04.002

• CROP GENETICS & BREEDING·GERMPLASM RESOURCES·MOLECULAR GENETICS • Previous Articles     Next Articles

Cloning and Function Analysis of a Salt-Stress-Induced HD-Zip Trascription Factor MsHB2 from Alfalfa

 LI  Ming-Na-1, 2 , LONG  Rui-Cai-1, YANG  Qing-Chuan-1, 2 , SHEN  Yi-Xin-2, KANG  Jun-Mei-1, ZHANG  Tie-Jun-1   

  1. 1、Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, Beijing 100193;
    2、College of Animal Science and Technology, Nanjing Agriculture University, Nanjing 210095
  • Received:2013-09-05 Online:2014-02-15 Published:2013-11-01

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Abstract: 【Objective】Based on an EST of unknown gene, cloning and function analysis of a salt-induced gene (MsHB2) from alfalfa (Medicago sativa L. cv. Zhongmu-1) were conducted to further research the salt tolerance mechanism in alfalfa. 【Method】The RACE primers were designed according to the known EST sequence. The 3′- and 5′-end of the MsHB2 were amplificated by RACE method. The full length of the gene was assembled by DNAMAN program. The ORF of MsHB2, and the isoelectric point, molecular weight, molecular weight, subcellular localization, phylogenetic tree of the encoding protein were analyzed by some bioinformatics programs. The subcellular localization transient expression vector was constructed and transformed into onion epidermal cell by particle gun. MsHB2 and GFP were expressed in a fusion, which could be used to analyze the subcellular localization by the fluorescence signal. After being treated with 300 mmol•L-1 NaCl or 0.1 mmol•L-1 ABA for 0, 2, 4, 10 and 24 h, total RNA was extracted from root and shoot of 30-day-old Medicago sativa L. cv. Zhongmu-1 to analyze the expression pattern of MsHB2. The overexpression vector of MsHB2 was also contructed and transformed into GV3101 Agrobacterium. The phenotype of transgenic Arabidopsis plants was analyzed under salt and ABA stresses. 【Result】 A full length of 1 126 bp sequence was obtained by assembling 3′- and 5′-end RACE sequence. The sequence analysis result indicated that MsHB2 encoded 247 amino acid and contained a homeobox domain and a leucine zipper domain. MsHB2 had a high similarity with ATHB12. The phylogenetic tree analysis indicated that MsHB2 belonged to the class Ⅰ of plant homeobox domain protein. The subcellular localization result suggested that MsHB2 located in the nucleus of onion epidermal cell. MsHB2 mRNA was induced by NaCl and ABA stresses in alfalfa root and shoot. Its overexpression driven by a constitutive cauliflower mosaic virus-35S promoter in Arabidopsis plants conferred salinity and ABA sensitivity, as compared with WT plants. 【Conclusion】 A homeobox domain and leucine zipper domain protein gene (MsHB2) was cloned from alfalfa, which is induced by NaCl and ABA stresses. MsHB2 also retards the growth of transgenic Arabidopsis after NaCl and ABA stresses. These imply that MsHB2 may affect growth through an ABA dependent regulation pathway. MsHB2 may play a negetive role in salt and some other abiotic stress regulation in alfalfa.

Key words: alfalfa , homeobox domain , leucine zipper , subcellular localization , function analysis , transcription factor

[1]Liu Q, Kasuga M, Sakuma Y, Abe H, Miura S, Yamaguchi-Shinozaki K, Shinozaki K. Two transcription factors, DREB1 and DREB2, with an EREBP/AP2 DNA binding domain separate two cellular signal transduction pathways in drought- and low-temperature-responsive gene expression, respectively, in Arabidopsis. The Plant Cell, 1998, 10(8): 1391-1406.

[2]Choi H I, Hong J H, Ha J O, Kang J Y, Kim S Y. ABFs, a family of ABA-responsive element binding factors. Journal of Biological Chemistry, 2000, 275(3): 1723-1730.

[3]Kang J Y, Choi H I, Im M Y, Kim S Y. Arabidopsis basic leucine zipper proteins that mediate stress-responsive abscisic acid signaling. The Plant Cell, 2002, 14(2): 343-357.

[4]Uno Y, Furihata T, Abe H, Yoshida R, Shinozaki K, Yamaguchi- Shinozaki K. Arabidopsis basic leucine zipper transcription factors involved in an abscisic acid-dependent signal transduction pathway under drought and high-salinity conditions. Proceedings of the National Academy of Sciences of the United States of America, 2000, 97(21): 11632-11637.

[5]Abe M, Katsumata H, Komeda Y, Takahashi T. Regulation of shoot epidermal cell differentiation by a pair of homeodomain proteins in Arabidopsis. Development, 2003, 130(4): 635-643.

[6]Chinnusamy V, Ohta M, Kanrar S, Lee B H, Hong X H, Agarwal M, Zhu J K. ICE1: A regulator of cold-induced transcriptome and freezing tolerance in Arabidopsis. Genes & Development, 2003, 17(8): 1043-1054.

[7]Gilmour S J, Zarka D G, Stockinger E J, Salazar M P, Houghton J M, Thomashow M F. Low temperature regulation of the Arabidopsis CBF family of AP2 transcriptional activators as an early step in cold- induced COR gene expression. The Plant Journal, 1998, 16(4): 433-442.

[8]Kmita M, Duboule D. Organizing axes in time and space; 25 years of colinear tinkering. Science, 2003, 301(5631): 331-333.

[9]Morgan R. Hox genes: A continuation of embryonic patterning? Trends in Genetics, 2006, 22(2): 67-69.

[10]Wang K C, Helms J A, Chang H Y. Regeneration, repair and remembering identity: The three Rs of Hox gene expression. Trends in Cell Biology, 2009, 19(6): 268-275.

[11]Ariel F D, Manavella P A, Dezar C A, Chan R L. The true story of the HD-Zip family. Trends in Plant Science, 2007, 12(9): 419-426.

[12]Ciarbelli A R, Ciolfi A, Salvucci S, Ruzza V, Possenti M, Carabelli M, Fruscalzo A, Sessa G, Morelli G, Ruberti I. The Arabidopsis homeodomain-leucine zipper II gene family: Diversity and redundancy. Plant Molecular Biology, 2008, 68(4/5): 465-478.

[13]Henriksson E, Olsson A S B, Johannesson H, Johansson H, Hanson J, Engstrom P, Soderman E. Homeodomain leucine zipper class I genes in Arabidopsis. Expression patterns and phylogenetic relationships. Plant Physiology, 2005, 139(1): 509-518.

[14]Frank W, Phillips J, Salamini F, Bartels D. Two dehydration-inducible transcripts from the resurrection plant Craterostigma plantagineum encode interacting homeodomain-leucine zipper proteins. The Plant Journal, 1998, 15(3): 413-421.

[15]Johannesson H, Wang Y, Engstrom P. DNA-binding and dimerization preferences of Arabidopsis homeodomain-leucine zipper transcription factors in vitro. Plant Molecular Biology, 2001, 45(1): 63-73.

[16]Meijer A H, Scarpella E, vanDijk E L, Qin L, Taal A J C, Rueb S, Harrington S E, McCouch S R, Schilperoort R A, Hoge J H C. Transcriptional repression by Oshox1, a novel homeodomain leucine zipper protein from rice. The Plant Journal, 1997, 11(2): 263-276.

[17]Gago G M, Almoguera C, Jordano J, Gonzalez D H, Chan R L. Hahb-4, a homeobox-leucine zipper gene potentially involved in abscisic acid-dependent responses to water stress in sunflower. Plant Cell and Environment, 2002, 25(5): 633-640.

[18]Wang Y, Henriksson E, Soderman E, Henriksson K N, Sundberg E, Engstrom P. The Arabidopsis homeobox gene, ATHB16, regulates leaf development and the sensitivity to photoperiod in Arabidopsis. Developmental Biology, 2003, 264(1): 228-239.

[19]Olsson A S B, Engstrom P, Soderman E. The homeobox genes ATHB12 and ATHB7 encode potential regulators of growth in response to water deficit in Arabidopsis. Plant Molecular Biology, 2004, 55(5): 663-677.

[20]Himmelbach A, Hoffmann T, Leube M, Hohener B, Grill E. Homeodomain protein ATHB6 is a target of the protein phosphatase ABI1 and regulates hormone responses in Arabidopsis. The EMBO Journal, 2002, 21(12): 3029-3038.

[21]Sessa G, Carabelli M, Sassi M, Ciolfi A, Possenti M, Mittempergher F, Becker J, Morelli G, Ruberti I. A dynamic balance between gene activation and repression regulates the shade avoidance response in Arabidopsis. Genes & Development, 2005, 19(23): 2811-2815.

[22]Morelli G, Ruberti I. Light and shade in the photocontrol of Arabidopsis growth. Trends in Plant Science, 2002, 7(9): 399-404.

[23]Prigge M J, Otsuga D, Alonso J M, Ecker J R, Drews G N, Clark S E. Class III homeodomain-leucine zipper gene family members have overlapping, antagonistic, and distinct roles in Arabidopsis development. The Plant Cell, 2005, 17(1): 61-76.

[24]Kubo H, Peeters A J M, Aarts M G M, Pereira A, Koornneef M. ANTHOCYANINLESS2, a homeobox gene affecting anthocyanin distribution and root development in Arabidopsis. The Plant Cell, 1999, 11(7): 1217-1226.

[25]Otsuga D, DeGuzman B, Prigge M J, Drews G N, Clark S E. REVOLUTA regulates meristem initiation at lateral positions. The Plant Journal, 2001, 25(2): 223-236.

[26]Guan X Y, Li Q J, Shan C M, Wang S, Mao Y B, Wang L J, Chen X Y. The HD-Zip IV gene GaHOX1 from cotton is a functional homologue of the Arabidopsis GLABRA2. Physiologia Plantarum, 2008, 134(1): 174-182.

[27]Mattsson J, Söderman E, Svenson M, Borkird C, Engström P. A new homeobox-leucine zipper gene from Arabidopsis thaliana. Plant Molecular Biology, 1992, 18(5): 1019-1022.

[28]Schena M, Davis R W. HD-Zip proteins: Members of an Arabidopsis homeodomain protein superfamily. Proceedings of the National Academy of Sciences of the USA, 1992, 89(9): 3894-3898.

[29]Aso K, Kato M, Banks J A, Hasebe M. Characterization of homeodomain-leucine zipper genes in the fern Ceratopteris richardii and the evolution of the homeodomain-leucine zipper gene family in vascular plants. Molecular Biology and Evolution, 1999, 16(4): 544-552.

[30]Sakakibara K, Nishiyama T, Kato M, Hasebe M. Isolation of homeodomain-leucine zipper genes from the moss Physcomitrella patens and the evolution of homeodomain-leucine zipper genes in land plants. Molecular Biology and Evolution, 2001, 18(4): 491-502.

[31]王宏, 李刚波, 张大勇, 蔺经, 盛宝龙, 韩金龙, 常有宏. 植物HD-Zip转录因子的生物学功能. 遗传, 2013, 35(10): 1179-1188.

Wang H, Li G B, Zhang D Y, Lin J, Sheng B L, Han J L, Chang Y H. Biological functions of HD-Zip transcription factors. Hereditas, 2013, 35(10): 1179-1188. (in Chinese)

[32]Cheng W H, Endo A, Zhou L, Penney J, Chen H C, Arroyo A, Leon P, Nambara E, Asami T, Seo M, Koshiba T, Sheen J. A unique short-chain dehydrogenase/reductase in Arabidopsis glucose signaling and abscisic acid biosynthesis and functions. The Plant Cell, 2002, 14(11): 2723-2743.

[33]Himmelbach A, Iten M, Grill E. Signalling of abscisic acid to  regulate plant growth. Philosophical Transactions of the Royal  Society of London Series B-Biological Sciences, 1998, 353(1374): 1439-1444.

[34]Leung J, Giraudat J. Abscisic acid signal transduction. Annual  Review of Plant Physiology and Plant Molecular Biology, 1998, 49: 199-222.

[35]Mccarty D R. Genetic-control and integration of maturation and germination pathways in seed development. Annual Review of Plant Physiology and Plant Molecular Biology, 1995, 46: 71-93.

[36]郝格格, 孙忠富, 张录强, 杜克明. 脱落酸在植物逆境胁迫研究中的进展. 中国农学通报, 2009, 25(18): 212-215.

Hao G G, Sun Z F, Zhang L Q, Du K M. A research overview of the plant resistance to adverse environment by using abscisic acid. Chinese Agricultural Science Bulletin, 2009, 25(18): 212-215. (in Chinese)

[37]Zhang S X, Haider I, Kohlen W, Jiang L, Bouwmeester H, Meijer A H, Schluepmann H, Liu C M, Ouwerkerk P B F. Function of the HD-Zip I gene Oshox22 in ABA-mediated drought and salt tolerances in rice. Plant Molecular Biology, 2012, 80(6): 571-585.

[38]Son O, Hur Y S, Kim Y K, Lee H J, Kim S, Kim M R, Nam K H, Lee M S, Kim B Y, Park J, Park J, Lee S C, Hanada A, Yamaguchi S, Lee I J, Kim S K, Yun D J, Soderman E, Cheon C I. ATHB12, an ABA-inducible homeodomain-leucine zipper (HD-Zip) protein of Arabidopsis, negatively regulates the growth of the inflorescence stem by decreasing the expression of a gibberellin 20-oxidase gene. Plant and Cell Physiology, 2010, 51(9): 1537-1547.

[39]Ariel F, Diet A, Verdenaud M, Gruber V, Frugier F, Chan R, Crespi M. Environmental regulation of lateral root emergence in medicago truncatula requires the HD-Zip I transcription factor HB1. The Plant Cell, 2010, 22(7): 2171-2183.

[40]Song S Y, Chen Y, Zhao M G, Zhang W H. A novel Medicago truncatula HD-Zip gene, MtHB2, is involved in abiotic stress responses. Environmental and Experimental Botany, 2012, 80: 1-9.

[41] Hu X, Zhang C R, Xie H, Huang X, Chen Y F, Huang X L. The expression of a new HD-Zip II gene, MSHB1, involving the inhibitory effect of thidiazuron on somatic embryogenic competence in alfalfa (Medicago sativa L. cv. Jinnan) callus. Acta Physiologiae Plantarum, 2012, 34(3): 1067-1074.
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