Studies On The Stress Resistance Of Transcription Factors PdPapERF34 And PdPapWRKY22 Of Populus Davidiana × P.alba Var.pyramidalis

Populus davidiana × P.alba var.pyramadalis(Shanxin poplar),as an excellent tree species in the northern region,has high ornamental value and no flying flocs.ERF and WRKY are two major transcription factor families in plants that regulate plant growth and response to stress.In this study,based on the previously constructed transcriptome,the PdPapERF34 gene and the PdPapWRKY22 gene of Shanxin poplar were cloned and analyzed for bioinformatics.The qRT-PCR method was used to analyze the expression patterns of PdPapERF34 and PdPapWRKY22 in different tissue sites and under various stresses.The overexpression vectors p ROK2-ERF34 and p ROK2-WRKY22 were constructed and transformed by Agrobacteriummediated method to obtain the overexpression strains of Shanxin poplar,so as to study the resistance function of PdPapERF34 and PdPapWRKY22 genes.This study provides a reference for clarifying the functions of transcription factors PdPapERF34 and PdPapWRKY22 and using transgenic technology to cultivate new poplar varieties with high resistance.The following results were obtained:1.The target genes PdPapERF34 and PdPapWRKY22 were cloned.Bioinformatics analysis showed that PdPapERF34 had 792 bases encoding 263 amino acids,and there was an AP2 superfamily conserved domain.Subcellular localization predicted its location in the nucleus;It belongs to unstable,acidic and hydrophilic protein;No transmembrane structure,no signal peptide.It is predicted that random curling is the main component of the protein in its secondary structure.The tertiary structure prediction was most consistent with the ethylene response transcription factor WRI1.Online prediction by MEME showed that PdPapERF34 had highly conserved motifs with the amino acid homologous sequence with high consistency;Genetic evolution analysis showed that PdPapERF34 had the closest genetic relationship with P.alba(XP＿034905682.1)and the farthest genetic relationship with P.trichocarpa(ABQ62974.1).PdPapWRKY22 gene contains 1002 bases encoding 333 amino acids and has a WRKY superfamily conservative domain.Subcellular localization predicts its location in the nucleus.It belongs to unstable,acidic and hydrophilic protein;No transmembrane structure,no signal peptide.Random curling is the main component of the secondary structure of the protein.The tertiary structure prediction was most consistent with WRKY transcription factor 1.Online prediction by MEME showed that PdPapWRKY22 had highly conserved motifs with the amino acid homologous sequence with high consistency;Genetic evolution analysis showed that PdPapWRKY22 had the closest genetic relationship with P.tomentosa(KAG6782825.1)and the farthest genetic relationship with P.trichocarpa(KAI5595172.1).2.The qRT-PCR method was used to analyze the expression patterns of PdPapERF34 and PdPapWRKY22 genes in different tissues and under different stresses.The results showed that the expression levels of PdPapERF34 and PdPapWRKY22 were the highest in roots.Under salt,alkali and drought stresses,the expression of PdPapERF34 was significantly up-regulated in roots.Under stresses from Cytospora chrysosperma,Sclerotinia sclerotiorum and Fusarium oxysporum,the expression of PdPapERF34 was significantly up-regulated in the roots,while under stresses from Alternaria alternata and Rhizoctonia solani,the expression of PdPapERF34 was significantly up-regulated in the shoot tips and leaves;The expression of PdPapERF34 was up-regulated by JA and ABA in the shoot tips and leaves.The expression level of PdPapWRKY22 was significantly up-regulated in shoot tips and leaves under salt stress,up-regulated in shoot tips,leaves and roots under alkali stress and up-regulated in roots under drought stress.The expression of PdPapWRKY22 was significantly up-regulated in roots under the stresses of other pathogen except Sclerotinia sclerotiorum.The expression of PdPapWRKY22 was significantly up-regulated by JA and ABA.3.Overexpression vector pROK2-ERF34 and p ROK2-WRKY22 were constructed and transfected into wild type of Shanxin poplar by Agrobacterium-mediated transformation.Through screening,the overexpression strains OE-ERF34(OE-ERF34 L,OE-ERF34 M and OE-ERF34H)and the overexpression strains OE-WRKY22(OE-WRKY22 L and OE-WRKY22H)of Shanxin poplar were obtained.4.The growth indicators and photosynthetic characteristics of 60-day-old overexpression PdPapERF34 and overexpression PdPapWRKY22 Shanxin poplar strains were studied.The results showed that compared with the control group,the growth,leaf development and internode elongation of transgenic Shanxin poplar were inhibited,but the photosynthetic capacity was improved.5.The stress resistance of 60-day-old overexpression PdPapERF34 and overexpression PdPapWRKY22 Shanxin poplar strains to saline-alkali stress,high temperature and drought stress and pathogen stress was studied.The results showed that transgenic Shanxin poplar had better ability to resist biological and abiotic stress.In conclusion,the transcription factors PdPapERF34 and PdPapWRKY22 of Shanxin poplar play an important role in improving plant photosynthetic capacity and resisting biological and abiotic stresses.