Cloning And Function Analysis Of A Salt-Stress-Induced HD-Zip Transcription Factor MsHB2 From Alfalfa

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. The RACE primers were disigned 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 assemblied by DNAMAN program. A full length of 1126 bp sequence was obtained by assembly of 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 ORF of MsHB2, and the isoelectric point, molecular weight, subcellular localization, phylogenetic tree of the encoding protein were analyzed by some bioimformatics 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. The subcellular localization result suggested MsHB2 located in the nucleus of onion epidermal cell. After being treated with 300 mmol·L-1 NaCl or 0.1 mmol-L"1 ABA for 0,2, 4,10 and 24 h, the 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. MsHB2 mRNA was induced by NaCl and ABA stresses in alfalfa root and shoot. The overexpression vector of MsHB2 was also contructed and transformed into GV3101 Agrobacterium. The phenotype of transgenic Arabidopsis plants was analyzed in salt and ABA stresses. Its overexpression driven by a constitutive cauliflower mosaic virus-35S promoter in Arabidopsis plants confered salinity and ABA sensitivity, as compared with WT plants. 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.