Cloning And Charactering Of The Gene Encoding Zinc Finger Protein From Alfalfa

A suppression subtraction hybridization cDNA library induced by salt had been constructed to identify differentially expressed genes. Based on the sequence of an expressed sequence tag homologous to Pisum sativum zinc finger protein mRNA (Accession Number: AF160911), primers were designed for 3'RACE and 5'RACE. A 865bp and a 1186bp cDNA fragments were acquired and the full-length cDNA of 1,676 nucleotides was cloned. The open reading frame of the MsZFN gene was 1254bp in length, encoding 417 amino acid residues. The amino acid sequence compared by Blast revealed there was high homology with zinc finger protein of other plants and the similarity to Pisum sativum zinc finger protein was the highest with 93%. And there were five conserved typical C-x8-C-x5-C-x3-H zinc finger motifs. The gene encoding a Medicago sativa zinc finger protein (MsZFN), and its Genbank accession number is EU624138.The coding region of MsZFN was inserted into the expression vector pBI121 and the recombined vector was named as pBI-ZFN. Then pBI-ZFN was transformed into Agrobacterium tumefaciens LBA4404. Alfalfa and tobacco were transferred, expecting to gain MsZFN overexpression plant. After selection by antibiotics, the regenerated plants were obtained and PCR analysis of them showed they were all PCR positive lines. With vectors pKANNIBAL and pART27, the RNAi expression vector pART-F-R was reconstructed. And pART-F-R was transformated into alfalfa, expecting to obtain MsZFN low-expression plant. Total DNAs of different plants were extrated and PCR analysis was carried on. The results showed that some plants were transformed successfully.Real-time RT-PCR was performed to reveal transcript level of MsZFN in different tissues and under different abiotic stresses. The results indicated MsZFN was abundant in leaf and stem, rather than in root; And the expression of MsZFN was quickly and transiently induced by NaCl treatment; Furthermore, induced by darkness, the transcript level of MsZFN was up-regulated.The open reading framgment of MsZFN were inserted into pET-30α(+) to construct the expression vectors, and the recombinant plasmids were transformed into E.coli BL21(DE3). The fusion proteins were induced to express by IPTG at 37℃. SDS-PAGE analysis revealed that the full-length gene could be expressed and the fusion protein was highly expressed, which made it possible to purify the fusion protein and prepare the corresponding antibody in future study. The coding region of MsZFN was inserted into pA7-GFP to construct the fusion protein expression vector MsZFN-GFP. The fusion was then transformed into onion epidermal cells using a gene gun. Subcellular localization of transiently expressed MsZFN-GFP fusion was detected by a confocal laser scanning microscope. The result revealed that MsZFN localized in nucleus.