A Preliminary Study On A Putative Plant Transcription Factors Family

Transcription factors (TF)were some kinds of proteins which could bind with the specific DNA sequence with the promoter of a gene in eukaryotes. They played central roles in plant development, differentiation and response to environmental stresses. We identified a on a new transcription factors family by bioinformatics analysis and carried out some preliminary studies. The mainly results were as follows:(1)After bioinformatics analysis of the transcription factors family by the database searches, multiple sequence alignments and related software of bioinformatics, it was found that two highly conserved regions were existed in the protein sequence of the transcription factors family. Next, the expressed producation of these genes were divided into four subfamilies by phylogenetic analysis. Then the expression information of these genes was obtained after scanning the Genbank EST database, it was found that most genes of this family in A. thaliana were expressed in flower and seed, most genes of this family in Oryza sativa were expressed in flower, bud and callus tissue. Finally, low complexity, ZnF_C2H2 , RING finger and DUF1644 Domain were found in protein of these transcription factors family thought secondary structure forecast. These results indicated that the genes of this family may have the function of transcription factors.(2)At1g15430 , At5g38370 gene were cloned from the genome of A. thaliana, and Os09g27860, Os01g42700, Os02g35840 were cloned from the genome of Oryza sativa. The prokaryotic expression vector pGEX-4T-2 was constructed and was transformed into E.coli BL21(DE3) or Rosetta^TM to induce the prokaryotic expression. It was found that the expressed proteins mainly existed as inclusion bodies. The optimal factors for expression of soluble fusion protein was 28.5℃, 1.0mmol/L IPTG, OD₆₀₀ equals to 0. 6 and 4h induction.(3)The fusion gene fragments of At1g15430-GFP,At5g38370-GFP,Os01g42700-GFP and Os09g27860-GFP were cloned by overlap extension PCR (OE PCR) and was ligated with pMD18-T to be sequenced. Then it was cut from pMD18-T with the restriction enzyme and inserted into the expression vector pBPF. Then plasmids were transformed into onion inner epidermal cells by particle bombardment to get the subcellular localization information of the expressed proteins. Green fluorescence was observed in the cytoplasm and nucleolus of onion inner epidermal cells transformed gene Os01g42700-GFP. In the nucleolus of onion inner epidermal cells transformed gene At5g38370-GFP can observe green fluorescence. Cell transformed gene At1g15430-GFP can observe green fluorescence in cytoplasm. In cells transformed gene Os09g27860-GFP could not show any green fluorescence. Theses results showed that the expressed Os01g42700-GFP was transferred into nucleolus partly, the expressed At5g38370-GFP was transferred into nucleolus completely, the expressed At1g15430-GFP was not transferred into nucleolus and the gene Os09g27860-GFP was not expressed.