Isolation And Characterization Of C2H2 Type Zinc Finger Transcription Factor In Glycine Max (L.) Merrill

The family of zinc finger proteins is an important member of transcription factors which exists widely in prokaryote and eukaryote. Zinc finger proteins play an important role in growth and development in both animals and plants. The biggest subfamily of zinc finger proteins is C2H2 (Cys2/His2) type zinc finger protein, which functions as a part of DNA-binding and protein-protein interaction domains. C2H2 type zinc finger, also called the classical or IFIIIA-type finger, have been found in a number of eukaryotic transcription factors as DNA-binding motifs. At present, it is reported that their involvement in plant development and their important roles under various abiotic stresses.In this study, based on configuration from two fragments containing conserved motifs from EST databank of soybean, a novel gene, GmC2H2, encoding a typical C2H2 type zinc finger protein, was isolated from soybean by RT-PCR. GmC2H2 gene has an open reading frame with 516 bp length, and encodes a protein of 19 KD with 172 amino acids. GmC2H2 contains two conserved C2H2 motifs with two conserved plant-specific QALGGH sequences, rspectively. GmC2H2 has a region consisting of three acidic residues followed by hydrophobic residues rich in Leu (L-box); and has C-terminal short regions containing DLNL sequences (DLN-box) as well. And the L-box and DLN-box are thought to play roles in protein-protein interactions or in maintaining the folded structure.The RT-PCR analysis showed that both cold and ABA induced the expression of GmC2H2 gne in Glycine max. SDS-PAGE analysis showed that GST::GmC2H2 fusion gene is expressed well in E. coli Using purified fusion protein, electrophoretic mobility shift assay was performed. Analysis showed the fusion proteins can bind strongly to the core sequence EP1S (TGACAGTGTCA), and that the two mutations with two bases substituted in one or both half sites of the inverted repeat of the core sequence greatly affects the specificity of the interaction between transcription factors and the core sequence. We also found that the strength of the interaction between EP1S and the GST::GmC2H2 protein and its mutants with one motif and some of its flanking sequeence delated separately or with various spacing delated between the two motifs is dramaticly different, which suggested that both of the two conserved motifs and its flanking sequeences are required for the specificity of the interaction between transcription factors and the core sequence, and the amino acid sequence between the GST::GmC2H2 protein and EP1S is one of the essential factors which affects the strength of the interaction between transcription factors and the core sequence. It was observed that the root length of transgenic Arabidopsis with a constructed vector GmC2H2::pCAMBIA is much shorter than control with pCAMBIA vector, but transgenic paints with C2H2 gene has better effect on the development of lateral roots. Amino acid assay showed that under the cold stress, the content of Pro which is related with the tolerance of plants in transgenic plant type is much higher than that in control, the content of and some of the other amino acids and NH3 in transgenic plants is higher than that in control.As a novel ranscription factor gene in soybean, GmC2H2 is characterized. Profound research on functionl and regulation mechanism is investigated.