Cloning And Identification Of The Salt-Induced Helicase Gene MsRH From Medicago Sativa L.

RNA helicases have been shown to participate in every aspect of RNA metabolism. Members of the DExD/H-box family of RNA helicases are involved in many processes and complexes within the cell. In response to abiotic stress, RNA helicase have been proved to regulate downstream genes expression on transcription, post-transcription and translation level. Recently, an Arabidopsis DEAD box RNA helicase LOS4localized in the cytoplasm and enriched at the nuclear rim was shown to be essential for mRNA export and important for development and stress responses in Arabidopsis.In this study, a full length of1678bp cDNA was isolated by RACE, based on an EST (GenBank accession No. FE896924.1)in a salt stress induced SSH library of Medicago sativa L. cv "Zhongmu No.1", This gene was predicted to be an RNA helicase gene and named as MsRH (GenBank accession No. JX508648). To investigate the function of MsRH, based on MsRH gene structure through bioinformatics analysis and molecular biology methods, the main results are as follows:1) MsRH gene sequence was code a protein of426amino acids, homology to Arabidopsis thaliana DEAD-box ATP-dependent RNA helicase (RH56, RH15) and Zea mays BAT1, which has been identified as an essential splicing factor. Indeed, All of them possesses eight conserved motifs.2) Transient expression of PA7-GFP-MsRH fusion in onion epidermis cell indicated that MsRH localized in the nucleus.3) Real time PCR was showed that the expression levels of MsRH gene were up-regulated compared to control under300mM NaCl,20%PEG6000or0.1mM ABA stress. Treatment of20%PEG6000increased the ralative level of MsRH mRNA significantly after2h.4) To investigate the fuction of MsRH gene, the plant over-expression vector pBI-MsRH was constructed and transferred into tobacco and Arabidopsis. The MsRH gene transformants were screened with kanamycin and identified by PCR, RT-PCR and GUS detection. Finally, Five positive tobaccoa lines and eight positive Arabidopsis lines were obtained respectively.5) After being treated with250mM NaCl for seven days, the proline contents of transgenic plants were lower than those of the WT plants, and the malondialdehyde contents and relative electric conductivity were higher than those of the WT tobacco. Taken together, these results demonstrated that MsRH gene increased the salt sensitivity of tobacco plants under salt stress condition. Furthermore, over expression of MsRH gene of Arabidopsis plants reduced root length under salt and drought stress.