Functional Analysis Of The Salt Response Gene PtrAGL74 In Populus Trichocarpa

Currently,salt stress has become one of the most pressing problems worldwide due to the increasing salinity of the land.Soil salinity,drought and other abiotic stresses are important factors limiting the normal growth and development of trees,therefore,the selection of resistant tree species is of great importance in reforestation practices on difficult sites.Poplar(Populus L.)is a fast-growing and productive tree with high adaptability and is widely used in plant genetic transformation and as material for plant stress resistance studies.In this experiment,a member of the salt stress-responsive MADS-box gene family,Ptr AGL74,was screened and its overexpression vector,p BI121-Ptr AGL74-GFP,was constructed.The transgenic plants were genetically transformed using Agrobacterium-mediated method using Populus trichocarpa as the recipient material.The main findings were as follows:(1)In this study,the Ptr AGL74(Potri.012G110100)gene was cloned from Populus trichocarpa,which has a CDS fragment length of 1032 bp and encodes 343 amino acids.The protein contains a typical conserved structural domain at the N-terminal end and belongs to the MADS-box family.(2)RT-q PCR was used to analyse the spatio-temporal expression patterns of the Ptr AGL74 gene under salt and drought treatments in three different tissues and at six different time points.The results showed that the Ptr AGL74 gene was expressed in roots,stems and leaves under salt stress compared to the control,with the highest relative expression levels in roots,followed by stems and leaves,while the relative expression levels were higher in roots and leaves under drought stress(simulated by PEG-6000 treatment),indicating that Ptr AGL74 responded to both salt and drought stresses.(3)Cis-acting element analysis of the first 2500 bp of the Ptr AGL74 start codon using Plant Care showed that the Ptr AGL74 promoter region contains a large number of abiotic stress response elements: salt stress response element(GT-1 motif),MYB drought-inducible binding site(MBS),stress response element(STRE),light response element(G-Box);and elements for phytohormone response: ethylene response element(ERE),abscisic acid response element(ABRE),methyl jasmonate induction element(TGACG-motif),involved in the growth hormone response element(TGA-element),salicylic acid induction element(TCA-element).(4)The results of subcellular localization and transcriptional activation activity analysis indicated that Ptr AGL74 is localized in the nucleus and had transcriptional activation activity,with its transcriptional activation structural domain at the C-terminus of the protein(274-343aa).(5)Under normal conditions,the growth status of wild-type and transgenic plants was the same.In contrast,under salt stress,the growth status of the transgenic was better than that of the wild type(WT);POD,SOD,CAT activity and proline content were significantly higher than that of WT,and MDA content and electrolyte permeability were lower than that of WT;the transgenic leaves were lighter in colour than WT by NBT,DAB and Evans Blue histochemical staining.(6)Under normal conditions,the growth status of wild-type and transgenic plants was the same.In contrast,under salt stress,the growth status of the transgenic was better than that of the wild type(WT);POD,SOD,CAT activity and proline content were significantly higher than that of WT,and MDA content and electrolyte permeability were lower than that of WT;the transgenic leaves were lighter in colour than WT by NBT,DAB and Evans Blue histochemical staining.The above results indicated that the Ptr AGL74 gene can be induced to be expressed by salt stress and enhance the salt tolerance of plants by improving the scavenging ability of reactive oxygen species and reducing the content of membrane lipid peroxides,thereby reducing the damage to the cell membrane structure.This experiment analyzed the basic characteristics and biological functions of the Ptr AGL74 gene for salt stress tolerance,and provided an experimental basis for studying the resistance mechanism of this gene under salt stress.