AfNAC52 Gene Involved In Drought Regulation In Amorpha Fruticosa

The current drought stress seriously affects the growth and development of plants,and genetic improvement of plants with genetic engineering methods has become an effective means to improve plant drought resistance.Stress can induce the expression of many stress-related plant transcription factors,and these transcription factors can activate the expression of more stress-resistant functional genes,thereby improving plant stress resistance.Many attempts have been made to improve the resistance of plants to various transcription factors.An up-regulated sequence tag was obtained from PEG6000 transcriptome response sequence simulation of drought stress,which may be related to drought stress response.In this study,the full-length cDNA of the gene was isolated from Amorpha fruticosa seedlings by RT-PCR technology.After BLAST alignment analysis,it was highly homologous to Cicer arietinum NAC52 gene,so it was named AfNAC52 gene(GeneBANK accession number MN180266).In addition,the spatiotemporal expression pattern of AfNAC52 gene in Amorpha fruticosa was studied;meanwhile,the bioinformatics prediction and subcellular localization of AfNAC52 gene were performed;the promoter sequence of AfNAC52 gene was analyzed;the growth tolerance of yeast overexpressing AfNAC52 gene under different stress conditions was detected Acceptable changes;the prokaryotic expression vector GST-AfNAC52 protein was constructed and purified protein was obtained;plant expression vector was constructed and heterologous overexpression studies were conducted using tobacco.The results are as follows:1.Using 2 month old Amorpha fruticosa seedlings as test material,AfNAC52 gene fragment was cloned by RT-PCR.After sequencing.bio informatics analysis was performed to predict that the full length of the open reading frame sequence(ORF)of AfNAC52 gene was 576bp,encoding 191 amino acids,the relative molecular weight of the protein is predicted to be 22.04kDa,the theoretical isoelectric point(p1 9.70).AfNAC52 protein is a hydrophilic stable protein.It has the closest relationship to Cicer arietinum with a similarity of 99%.There are NAM functional domains that are more conserved across species.And.AfNAC52 gene is located in the nucleus.In addition to the basic cis-acting elements such as CAAT-box in the promoter sequence of the.AfNAC52 gene,it also has regulatory elements for the MEJA response,regulatory elements necessary for anaerobic induction;auxin response elements;Type acting element;cis acting element participating in low temperature response.2.The expression characteristics of AfNAC52 gene in different tissues and organs of Amorpha fruticosa were analyzed,and it was found that AfNAC52 gene was expressed in roots.stems,leaves and flowers,and AfNAC52 gene was the most expressed in flowers and the least expressed in roots.The expression characteristics of AfNAC52 gene in leaves and roots of Amorpha fruticosa under different stress conditions were also analyzed.It was found that under drought stress,the expression level of AfNAC52 gene increased,especially in roots.Under NaCl stress,the expression of AfNAC52 gene did not change significantly;under NaHCO3 stress,the expression of AfNAC52 gene increased,especially in roots.3.The yeast expression vector PYES2-AfNAC52 was successfully constructed,and the growth tolerance of AfNAC52 gene yeast under different stress conditions was analyzed.It was found that AfNAC52 gene yeast under mannitol and Sorbitol drought stress,especially in 2M and 3M mannitol and 2M Sorbitol YPG medium,the growth condition of yeast transferred into pYES2-AfNAC52 was significantly stronger than that of the control yeast transferred to pYES2 empty vector,showing strong drought tolerance;under NaHCO3 stress,the growth of the two yeasts was slightly different,Showed slight alkali resistance;under NaCl stress,the growth status of yeast transferred into pYES2-AfNAC52 was not significantly different from that of yeast transferred into pYES2 empty vector.4.The recombinant prokaryotic expression vector PGEX-6P-3-AfNAC52 was successfully constructed,and the AfNAC52 protein was purified.5.The plant over-expression vector pBI121-AfNAC52-GFP was successfully constructed,and T3 transgenic lines of transgenic tobacco were obtained.Overexpression of AfNAC52 gene made tobacco have higher survival rate in drought stress test,and improved resistance to tobacco stress.