Effects Analysis Of Transcription Factor GmC2H2 In Transpgenic Arabidopsis

Zinc finger protein is a kind of transcription factor that has a "finger" structural domain, and it can regulate the gene expression. There are various types of zinc finger proteins, but most of them belong to the C2H2-type. C2H2 type zinc finger protein was first discovered in Xenopus tropicalis, and a C2H2 zinc finger protein gene named ZPT2-1 which was isolated from the petunia was first found in plants, and the gene of this type zinc finger protein have gradually been isolated from the petunia, Arabidopsis thaliana, cotton, soybeans, rice and other plants. And a highly conserved sequence of QALGGH was found in plantkingdom, while it did not exist in zinc finger proteins of animal. Previous studies have found that C2H2 zinc finger protein mainly involved in plant growth and development and regulation of plant stress resistance.In this study, based on the conserved sequence of soybean EST database in GenBank, the GmC2H2 transcription factor gene is isolated from soybean by PCR and RT-PCR techniques using specific primers, and it contains a cDNA open reading frame encoding classic C2H2 type zinc finger protein, and the accession number in Genbank is DQ055134. The full length of this gene is 765 bp with an open reading frame of 516 bp length which encodes 172 amino acids with 19 kD of molecular weight.On the basis of previous studies in this laboratory, the full-length GmC2H2 gene coding sequences were constructed to 16318hGFP vector for subcellular localization, then 16318hGFP-GmC2H2 was transformed to protoplast, finally analyzed the subcellular localization. Data showed that the GmC2H2 zinc finger protein expressed in the nucleus. The full-length GmC2H2 cDNA sequence was constructed to the plant expression vector of pCAMBIA 1304, and with the optimization of the floral-dip method into the model plant Arabidopsis, 19 transgenic seedlings were generated by using of Hygromycine (45-50mg?L-1) resistance screening and the PCR amplification test. According to the Mendel's law, some lines with the ratio of nearly 3:1 were isolated, in which the foreign gene may be inserted as single copy. There were three homozygous lines named line-1, line-4 and line-7. The results of histochemical staining for GUS showed that transgenic Arabidopsis seedlings of 12-day carrying the target vector pCAMBIA1304, the GUS gene were expressed throughout the whole plant, and were mainly concentrated in the roots of the transgenic Arabidopsis carrying the target GmC2H2 gene. In addition, the mutants of GmC2H2 gene which were named MZF1,MZF2,MZF4,MZF8,MZF10 were respectively constructed to the expression vectors to explore the function of elements of GmC2H2 transcription factor in regulating plant growth and development or plant stress resistance.For further identifying the function of GmC2H2 gene, wild-type and transgenic genotypes of Arabidopsis thaliana plants were treated with low temperature (1°C), abscisic acid (ABA, 200μmol?L-1) and high salt (NaCl, 250mmol?L-1) treatment. Studies showed that under the treatment of low temperature, ABA and salt treatment, transgenic plants grew better than the wild-type plants, and the contents of MDA in both of them were increased, but the contents of MDA in the wild-type Arabidopsis were more significant. The contents of soluble sugar and proline in the transgenic Arabidopsis were significantly higher than that in the wild type, and cell injury rates of transgenic plants was lower compared to the wild type Arabidopsis, indicating that the insertion of GmC2H2 gene may lead to a significant higher resistance of stress in transgenic Arabidopsis plants than wild-type Arabidopsis.The results of real-time qPCR showed that expression of GmC2H2 gene in the transgenic Arabidopsis was significantly higher after the treatment of low temperature and ABA treatment than untreated transgenic Arabidopsis both in roots and leaves. Under the treatment of low temperature and ABA, the accumulation of antifreeze gene cor6.6 in leaves and roots of those Arabidopsis plants was higher than that in the wild-type plants. Under low temperature conditions, the accumulation of EPSPS gene in leaves of the transgenic Arabidopsis was higher than that of the wild-type, while it was not obvious in the roots. For ABA treatment, the accumulation of EPSPS gene was higher in transgenic Arabidopsis than the wild-type both in leaves and roots.