Cloning And Stress Resistance Analysis Of Transcription Factor GmTGA In Glycine Max

The transcription factor TGA is a member of the D family of the bZIP family subfamily,and the TGA transcription factor has a bZIP domain,including an N-terminal basic domain,which can bind N-X7-R/K-X9 directly to DNA so that it can determine DNA specificity and nuclear localization.In the current study,the TGA family has a STDXDT phosphorylation site at the N-terminus and a glutamine binding site at the C-terminus.These binding sites are involved in the response of TGA to different abiotic stresses.TGA also has a specific domain,TGACG,which binds to the promoter as-1 to induce expression of the promoter.In order to explore the TGA function of soybean,the GmTGA gene was cloned from Jiyu 72 soybean,and its bioinformatics analysis,transcription self-activation,subcellular localization and other experiments were carried out.For the soybean seed root,NaCl,SA,PEG was used for abiotic stress,the stress resistance index and TGA gene expression were determined.And the stress conditions of GmTGA soybean roots were established.The function of GmTGA gene soybean roots under abiotic stress conditions such as NaCl,SA and PEG was studied.This study provides candidate genes and theoretical basis for the application of soybean GmTGA in stress-resistant breeding.The main results of this study are as follows:1?According to the amino acid sequences of Arabidopsis thaliana AtTGA1(AT5G65210)and AtTGA4(AT5G10030)proteins,the TGA gene with higher soybean similarity was obtained by Blast in Phytonzome,which was named as GmTGA;GmTGA was cloned by reverse transcription PCR(RT-PCR).The fragment length of GmTGA was 1089 bp encoding a complete open reading frame(ORF)of 362 amino acid residues.Predicted by online bioinformatics software,the predicted results indicate that GmTGA is neither a secreted protein nor a hydrophilic protein;alignment analysis of soybean GmTGA and Arabidopsis TGA protein sequences revealed that they all have the domain of the bZIP family.2?The yeast self-activation vector pGBKT7-GmTGA was constructed and then it was transformed into the yeast strain Y2 HGold.The positive transcript was obtained in the deficient medium SD/-Trp-His.The results showed that the yeast strain with the GmTGA could not grow normally in the defect medium,which proved that GmTGA hasn't transcriptional activation activity.3?The pPSN-GmTGA transient epitope vector was constructed and then it was transformed into Arabidopsis protoplasts.Using a fluorescence inverted microscope,we find that the GmTGA transcription factor is located on the nucleus.4?The seedling soybean roots were used as experimental materials,and abiotic stress experiment was carried out with different concentrations of NaCl,SA and PEG.The expression of GmTGA gene,the activity of antioxidant enzyme system enzyme and the content of membrane lipid peroxide MDA were determined.The expression of GmTGA gene increased firstly and then decreased with the increase of stress time and stress concentration.When the concentration of NaCl was 150 mM,the highest expression of GmTGA gene reached 4.83 after 12 hours of treatment;When the concentration of SA was 1.5 mM,the highest expression of GmTGA gene reached 2.96 after 6 hours of treatment;When the concentration of PEG was 15%,the highest expression of GmTGA gene reached 7.58 after 12 hours of treatment.5?In the experiment of transgenic GmTGA soybean rooting,with the treatment of soybean hair roots under 150 mM NaCl,1.5 mM SA,15% PEG,the changes of antioxidant enzyme system(SOD,POD,CAT)enzyme activity and MDA content are consistent with the changes of various indexes of root of seedling soybean.The expression of GmTGA gene increased first and then decreased with the increase of NaCl and PEG stress time,and reached the maximum at 12 h,which were 9.43 and 10.75,respectively.SA is shown as a maximum of 8.28 at 6h.This indicates that the transcription factor GmTGA has a certain response to abiotic stress.