Functional Analysis Of High Temperature Stress Transcriptome And Small Molecular Heat Shock Protein Gene In Paeonia Suffruticosa

Tree peony(Paeonia suffruticosa)is a traditional famous flower in China,which has both beautiful flowers and leaves.It has been deeply loved by the people for many years and is widely used in landscaping.Tree peony is not tolerant to high temperature,especially the high temperature and sunburn in summer limit the growth and ornamental quality of peony.In this study,P.suffruticosa‘Yuhong’ was selected as the research object.The transcriptome of high temperature stress was sequenced and analyzed to screen the HSP genes related to high temperature stress response.Three small heat shock protein genes PsHSP17.8,PsHSP21 and PsHSP27.4,were cloned for bioinformatics and gene expression analysis,and genetic transformation of Arabidopsis(Arabidopsis thaliana)to explore the heat tolerance.The main results are as follows:1.Through the transcriptome sequencing of tree peony ‘Yuhong’ under high temperature stress,a total of 54 935 Unigenes were obtained,including 7 673 differential genes,of which 4 220 were upregulated and 3 453 down-regulated.The GO functional annotation and KEGG pathway analysis of the differential genes showed that the metabolic process,cells and binding activity in GO functional group had more sequences,while carbon metabolism and endoplasmic reticulum protein processing in the KEGG pathway were the two pathways with the most gene enrichment.In addition,18 heat shock protein HSP genes were obtained by differential analysis,all of which were significantly up-regulated under high temperature treatment.2.Based on the s HSP gene sequence date obtained from transcriptome sequencing,three small heat shock protein genes(PsHSP17.8,PsHSP21 and PsHSP27.4)with ORF length of 459,714 and 693 bp were cloned and obtained,encoding 152,237 and 230 amino acids respectively,all of which contained HSP20 domain.Phylogenetic tree analysis showed that PsHSP17.8 belonged to cytoplasmic Class I type,PsHSP21 and PsHSP27.4 belonged to chloroplast small heat shock protein.The fluorescence quantitative results showed that PsHSP17.8 and PsHSP21 were significantly up-regulated at 2 hours after high temperature treatment at 40 ℃,and the expression of PsHSP27.4 was significantly upregulated at 6 hours after high temperature stress,indicating that PsHSP17.8,PsHSP21 and PsHSP27.4may be involved in the response of tree peony to heat stress.3.PsHSP17.8,PsHSP21 and PsHSP27.4 were digested by double endonuclease to construct expression vector,and the restriction sites were Xho I and Cla I.Agrobacterium tumefaciens were heterologous transformed into Arabidopsis thaliana.Tissue quantitative analysis showed that the expression levels of PsHSP17.8,PsHSP21 and PsHSP27.4 genes were the highest in the leaves of transgenic lines.The transgenic Arabidopsis thaliana lines of PsHSP17.8,PsHSP21 and PsHSP27.4were treated with high temperature,and the survival rates after the recovery period were 43%,36% and31%,respectively.After high temperature stress,the leaf length and leaf width of transgenic lines were larger than those of wild type.After high temperature treatment,the related physiological indexes showed that the activity of superoxide dismutase,the accumulation of free proline in transgenic lines and chlorophyll level were higher than those in wild-type lines,while MDA content and the electrical conductivity in transgenic plants were lower than those in wild-type lines,and the membrane lipid peroxidation in wild-type plants was more intense.It is suggested that the small heat shock protein genes PsHSP17.8,PsHSP21 and PsHSP27.4 of tree peony can improve the high temperature tolerance of Arabidopsis thaliana.In summary,small heat shock proteins PsHSP17.8,PsHSP21 and PsHSP27.4 play a role in the regulation of high temperature stress in tree peony.This study provides a reference basis for analyzing the high temperature response of tree peony and the innovation of high temperature tolerant peony germplasm.