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

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