Cloning And Characterization Of MtGT1 Gene From Medicago Truncatula

Trihelix transcription factor is a relatively late family found in plant transcription factor family.It was first recognized as a light regulatory factor,and it was found to play an important role in plant growth and development.In recent years,excavating the family's function in plant abiotic stress has become a hot research direction for this family member.The target of this study was the MTGT1 gene from Medicago truncatula.The primers were designed based on the sequence of MTGT1 gene.The target gene MTGT1 was obtained from Medicago truncatula using the methods of gene cloning,bioinformatics analysis,and transgenesis for preliminary identification.It was found that the gene is related to the abiotic stress of plants,which laid the foundation for the study of the function of the gene and provided the theoretical basis.The specific findings are as follows:1.Total RNA was extracted from Medicago truncatula.The value of OD260/OD280 was between 1.8 and 2.0.The quality of the RNA was better.Reverse transcription into cDNA,using PCR amplification technology to obtain the complete fragment of the target gene MTGT1,named MtGT1.The target gene was ligated Into the vector pMD19-T to construct a cloning vector PMD-MtGT1.2.The target gene was translated into an amino acid sequence using DNAMAN 7.0 for bioinformatics analysis.The full-length cDNA was 1109 bp and the open reading frame was 972 bp,encoding a total of 324 amino acids.Contains a MYB conserved domain belonging to the GT1 subfamily of the Trihelix family.Homologous sequences were found to have good homology with leguminous plants,among which the closest relationship to the GT protein of Trifolium subterraneus.The protein belongs to the stable lipophilic hydrophobin,it has no transmembrane region and no signal peptide.The secondary structure shows that the protein is composed of three structures:a-helix,extended chain and random coil.The tertiary structure predicts its partial fragment.The three-dimensional structure map shows a region very similar to the DNA conserved domain of Arabidopsis thaliana GT1,demonstrating that the gene belongs to the Trihelix family.3.Quantitative qRT-PCR analysis of gene expression of MtGT1 gene.The results showed that there was a significant difference in the expression of this gene in different tissues of Medicago truncatula.Among them,the highest expression level in roots,the lowest expression level in flowers,and the expression level of stems and leaves were in between.Spraying 10 mg/L IAA(Indole-3-acetic acid,Indol-yl-3-acetic Acid)and 10 ?mol/L 6-BA(6-Benzylaminopurine)hormones,the change in the expression level was roughly time-dependent.The increase increases first and then decreases.IAA appeared at the second peak at 12h.After low temperature treatment,the expression level first decreased and then increased with the increase of time;while the drought and salt treatment increased first and then decreased.In conclusion,this gene responds to both IAA and 6-BA phytobiotics and drought,salt,and low temperature abiotic stresses,suggesting that MtGT1 may be involved in some abiotic stresses of plants.4.The target gene MtGT1 was ligated into the 3302Y vector to construct a plant expression vector 3302Y-MtGT1 and then Agrobacterium EHA105 was transformed.Agrobacterium was used to infect the Arabidopsis inflorescences.Next generation plants were screened by glyphosate.Apoptosis and well-growth Arabidopsis thaliana were selected for DNA identification.Four positive plants were identified.The target gene MtGT1 was proved to be successfully transferred into Arabidopsis thaliana and expressed in Arabidopsis thaliana.5.Use the 3302Y-MtGT1 expression vector carrying the 35S promoter and YFP fluorescent protein gene to shake the bacteria and activate Agrobacterium tumefaciens EHA105.After resuspending the bacterial suspension,it was injected into the lower epidermis of Nicotiana benthamiana.After 48 hours of dark culture,it was observed using a laser confocal microscope.The results showed that the protein is a nuclear localization protein.