Study On The Response Function Of PdbLOL2 Gene To Salt Stress In Populus Davidiana×P.bolleana

LSD1-Like,a family of genes containing zinc finger domains,played an important role in plant abiotic stress.LOL2 was one of the members of this gene family.Previous transcriptome of Populus davidiana×P.bolleana under salt stress results showed that the expression of PdbLOL2 gene was significantly induced by salt stress,indicating that it could respond to salt stress in P.davidiana×P.bolleana.In this study,the function of PdbLOL2 gene in the regulation of salt stress in P.davidiana×P.bolleana were further analyzed.The specific results were as follows:（1）The PdbLOL2 gene with a complete open reading frame（ORF）was cloned and identified from P.davidiana×P.bolleana.The ORF length of the PdbLOL2 gene was 426 bp,and the coding amino acid residue number was 141 aa.The PdbLOL2 gene had typical structural characteristics of the LSD1 family.The results of subcellular localization and transcriptional activation activity analysis showed that the PdbLOL2 protein was located in the nucleus and had transcriptional activation activity.The transcriptional activation domain was located in 1-42 aa without a conservative domain.（2）The expression of PdbLOL2 was obvious in different tissues,with the highest expression level in young stems.After Na Cl,PEG₆₀₀₀,and ABA treatment,the expression level of PdbLOL2 gene in roots and leaves of P.davidiana×P.bolleana changed significantly at most time points.Especially during the entire process of Na Cl treatment,the expression of PdbLOL2 gene in the root was significantly induced,indicating that it may play an important role in the salt stress responsed of P.davidiana×P.bolleana.（3）In order to further explore the function of PdbLOL2 gene in response to salt stress in P.davidiana×P.bolleana,an overexpression vector pROKII-PdbLOL2 was constructed and stably transformed into P.davidiana×P.bolleana by Agrobacterium mediated leaf-disk method,resulting in 8 overexpression poplar lines.After 15 days of salt stress treatment,the leaves of the overexpressed lines withered more severely than those of the wild type.The results of physiological indicators showed that compared with the wild type,the overexpression PdbLOL2 strain has lower the total antioxidant capacity,higer ROS,H₂O₂,electrolyte permeability,and MDA content.（4）In order to further study the salt sensitive mechanism of PdbLOL2 gene,RNA-seq was performed on wild-type and overexpressed transgenic P.davidiana×P.bolleana.A total of 211DEGs were obtained,of which 139 were up-regulated and 72 were down-regulated.The GO classification results of DEGs showed that they are mainly involved in the process of lipid biosynthesis,the response to extracellular stimuli,and the response of cells to auxin stimulation in biological processes.（5）The results showed that PdbLOL2 can bind the element W1"TTGCCAAAAC",and the series of deletion results determined that"GCCAAAAC"is the core element LBE（LOL2binding element）of PdbLOL2 transcription factor recognition.LBE elements were found in the promoter regions of six stress related DEGs（PdbND2,PdbABCG11,PdbNLP2,PdbWAT1,PdbSP1L5,and Pdbb HLH）.The results of Ch IP-PCR showed that the PdbLOL2 only specifically binds to the promoter region of PdbWAT1,indicating that PdbLOL2 might participates in the salt stress response of P.davidiana×P.bolleana by directly regulating the expression of PdbWAT1.In summary,PdbLOL2 might regulate the expression of PdbWAT1 gene by binding to the LBE element under salt stress,thereby reducing the total antioxidant capacity of transgenic P.davidiana×P.bolleana,increasing the ROS content in the body of P.davidiana×P.bolleana,causing cell damage in the plant,thereby reducing its salt tolerance.This study lays a foundation for further exploring the salt stress regulatory mechanism of the PdbLOL2transcription factor in P.davidiana×P.bolleana.