Cloning And Identification Of The Gene MsRBP From Alfalfa (Medicago Sativa L.)

Post-transcriptional modulation of gene expression plays a vital role in the normal growth and development of plants. As one of the key acting factors that involve in the post-transcriptional regulation, RNA binding proteins (RBPs) play a vital role in RNA processes. Up to now, various RBPs have been cloned and analyzed in many organisms, but there is still no report about RBPs in alfalfa, although it is one of the most valuable forage legumes and has a distribution all over the world.In this study, we cloned a new gene from Medicago sativa L.cv "Zhongmu No.1" on the basis of an EST (GenBank accession No. FE896907.1). And we also did preliminary appraisal research on its structure and function through bioinformatics analysis and molecular biology methods. The main results are as follows:1) Using RT-PCR and RACE methods, a novel gene was cloned. It was tentatively named MsRBP (GenBank accession No. JN986878.1). The full-length of MsRBP cDNA sequence was1551bp, which contained a1230bp open reading frame (ORF) and a69bp5’-UTR and a252bp3’-UTR. It encoded a protein of409amino acids. The theoretical pI of the putative protein was6.31and its MV was45.6kDa. The functional site prediction result showed that there was three RRM and a Glu-Pro-rich domain at the C-terminal ends of the protein.2) To examine the subcellular localization of MsRBP, the fusion vector pA7-GFP-MsRBP was constructed. Then the fusion vector and pA7-GFP vector were transformed into onion epidermal cells using a gene gun. The results indicated that MsRBP is a nuclear-localized protein.3) The expression pattern of MsRBP gene under different kinds of stresses, real-time Q-PCR was carried out to examine the transcript levels of MsRBP in alfalfa seedlings under various treatments. The results showed that transcripts of MsRBP were found in all alfalfa organs tested, including roots and shoots. When treated by300mM NaCl,20%PEG6000or0.1mM ABA, the expression levels of MsRBP were up-regulated compared to control. Treatment of NaCl increased the ralative level of MsRBP mRNA in root significantly after10h. When treated by ABA or PEG, the relative level increased sharply within a short time (2h-4h) and then began to descend, but still remained at a high level. It indicated that MsRBP was sensitive to ABA or PEG4) Plant expression vector over-expression pBI121-MsRBP was constructed and it was heterogeneously expressed in NC89or "Zhongmu No.1"via Agrobacterium mediation. The MsRBP transformants were screened on media with kanamycin (50mg/L) and identified by PCR, RT-PCR and GUS detection. A total of three transgenic tabacco lines and two transgenic alfalfa lines were obtained.5) We also expressed pBI121-MsRBP in Arabidopsis theliana via Agrobacterium mediation using traditional flower dip transformation method. The MsRBP transformants were screened with kanamycin (50mg/L) and identified by PCR, RT-PCR and GUS detection. Six T2transgenic lines were obtained and some of them were selected and used for physiological analysis. Results showed that the transgenic plants of Arabidopsis had higher sensitivity to NaCl stress. MsRBP had a negative impact on seed germination and seedling growth under salt stress conditions. Over-expression of MsRBP reduced the salt tolerance of the transgenic plants.