Studies On The Function Of Hydroxyproline-Rich Glycoprotein Gene (PdPapHRGP1) In Stress Resistansce Of Poplar

Populus davidiana × P.alba var.Pyramidalis(Shanxin poplar)is not only a tree species for ecological protection,but also an excellent tree species for greening today.Hydroxyproline-rich glycoprotein(HRGPs)are unique structural proteins in the cell walls of higher plants,which are widely present in plants and play an important role in resisting pathogens.In this study,the PdPapHRGP1 gene(Potri.003G129400.v3.1)was screened and cloned based on the transcriptome data constructed in the previous study.Through bioinformatics analysis and q RT-PCR technology,the tissue expression patterns of PdPapHRGP1 gene in different tissues and under various stress conditions were clarified,and the transgenic technology was used to obtain PdPapHRGP1 gene overexpression lines(O1-1,O1-3 and O1-6),verified the anti-stress function of PdPapHRGP1.The results of this study laid the foundation for revealing the function of PdPapHRGP1 gene and obtaining high-resistance germplasm resources of Shanxin poplar through genetic engineering technology.The main findings are as follows:1.The target gene PdPapHRGP1 was obtained.The results of bioinformatics analysis showed that the open reading frame of PdPapHRGP1 gene was 1363 bp,encoding 453 amino acids.PdPapHRGP1 protein has a molecular weight of 49.079 k Da and a theoretical isoelectric point of 5.13.It is a stable hydrophilic non-transmembrane protein.The protein has a conserved domain "PHA02682".Subcellular localization is predicted to be in the nucleus,accounting for about 73.9%.The predicted secondary structure of PdPapHRGP1 protein was α-helix accounting for 19.43%,extended chain accounting for16.78%,β-turn fold accounting for 1.77% and random coil accounting for 62.03%.Phylogenetic tree analysis showed that this protein is the closest relative to KAB5564519.1(Salix brachista).2.The expression patterns of this gene in different tissues and under different stresses were analyzed by q RT-PCR technology.The gene is expressed in shoot tips,leaves,stems and roots,and its expression level is highest in mature leaves,which is 19.8 times higher than that in young stems(S1).There were changes in expression after induction.Among them,the expression of this gene was significantly up-regulated in various tissue parts under salt and alkali stress;the expression of this gene was significantly up-regulated in leaves and roots under drought treatment,which was 2.71 and 1.37 times that of the control;the gene was significantly up-regulated in roots and It was most affected by Fusarium oxysporum,which was 1.61 times that of the control;Jasmonic acid and abscisic acid could significantly up-regulate the expression of PdPapHRGP1 gene in roots,which were 5.67 and 9.76 times that of the control,respectively,and were only significantly up-regulated in leaves induced by salicylic acid The expression was 8.97 times that of the control;Trichoderma aculeatus Ta536 could induce the expression of this gene to be significantly up-regulated at the shoot tip,which was 10 times that of the control.3.The p ROK2-HRGP1 overexpression recombinant vector was constructed,and wild Shanxin poplar was transformed by Agrobacterium-mediated leaf disc method,and three positive overexpression lines with different expression levels were obtained.The growth characteristics,photosynthetic characteristics,co-expression of specific genes,resistance to external abiotic stress and stress tolerance of pathogenic bacteria were analyzed in60-day-old overexpressed Shanxin poplar transplanted seedlings.The results showed that,compared with the wild type,PdPapHRGP1 gene overexpression showed significant growth advantages,stronger photosynthetic capacity,and higher resistance to abiotic and biotic stresses.And found that PdPapHRGP1 co-expression relationship with WAK,ASPG,ERF34,WRKY22 and BBE and other genes.