Bioinformatics Analysis Of Heat Stress Related Genes And Functional Characterization Of Hsf And Hsp In Dianthus Caryophyllus L. And Prunus Mume Sieb.et.Zucc.

Carnation（Dianthus caryophyllus L.）is a perennial herb plant which belongs to the genus Dianthus of Caryophyllaceae family.It is one of the four cut flowers in the world with important commercial value because of its long vase life and wide array of colors.Carnation represents of maternal warmth,known as the flower of"Mother’s Day".However,carnation is native to the Mediterranean region,which performs well under cool climate,while high temperature exercises a great influence on its growth and development,and affecting yield and quality of cut-flowers.Mei flower（Prunus mume Sieb.et.Zucc.）is a woody plant which belongs to the genus Prunus of Rosaceae family,and it’s the first of the top ten traditional famous flowers of China.P.mume has a numerous cultivars with rich colors and forms,which is highly ornamental and usually as symbol cultural plant.Nevertheless,the leaves of P.mume will curl and turn yellow under sustained high temperature and drought in summer.Vegetative growth will be severely affected and it will lower the quality of flowering.Therefore,it’s very urgent to understand the thermotolerance mechanism of those two plants.In this study,D.caryophyllus’Fancy’and P.mume’Xue Mei’are used as materials to analyze the function of key genes Hsfs and Hsps in response to heat stress,which can provide information that they invlove in high temperature stress tolerance.It lays on a molecular foundation for mining the heat resistant genes in carnation and Mei flower,and clarifying their function in signal transduction pathways of thermal stress,so that we can improve plants thermotolerance which Hsfs and Hsps involved in by genetic engineering.The main results are as follows:1.A transcriptome analysis was first performed to generate heat stress-responsive gene expression profiles in carnation.A c DNA library constructed from mixed RNA of carnation leaf subjected to 42℃HS（0,0.5,1 and 2 h）and 46℃HS（0.5,1 and 2 h）was sequenced,and 45,604,882 high quality paired-end reads were obtained.After de novo assembly and quantitative assessment,99,255 contigs were generated with an average length of 1053 bp.Functional annotations were obtained by aligning unigenes with public protein databases including NR,Swiss Prot,KEGG and COG.Using the transcriptome as reference,the effects of high temperature（42℃）treatments（0.5,2 or 12h）on carnation aseptic seedlings relative to untreated controls were compared based on the FPKM method.Overall,11,471 genes were identified which showed a significant response to one or more of the three HS treatment times.In addition,based on GO and metabolic pathway enrichment analyses,a series of candidate genes involved in thermo-tolerance responses were selected and characterized.The study of which may advance our understanding of heat response of carnation.2.DcHsf A1d was obtained via homology-based cloning combined with Tail-PCR technology.Its g DNA full-length was 4852bp and open reading frame（ORF）was1368bp encoding 455 amino acids with protein molecular weight（Mw）51.039k D and isoelectric point（p I）4.94.Amino acid sequence alignment and structure analysis showed that DcHsf A1d had five typical functional domains of Hsf A family.Arabidopsis overexpressed DcHsf A1d showed improved thermotolerance,whereas the heat tolerance of Arabidopsis transformed with antisense DcHsf A1d was decreased.Under high temperature stress,lower accumulation of reactive oxygen species（ROS）,higher activities of anti-oxidant enzymes and lower MDA content and relative electrolyte leakage value in Arabidopsis overexpressed DcHsf A1d were observed than WT.The expression level of 9 stress-related genes was significantly changed in transgenic Arabidopsis before and after heat treatment.In addition,the root length and fresh weight of Arabidopsis overexpressed DcHsf A1d were significant higher than WT under salt stress,which showed that the salt tolerance of transgenic Arabidopsis increased at a certain extent.3.DcHsp70 was obtained via homology-based cloning combined with Tail-PCR technology,which contained a full-length of g DNA（ATG-TAA）of 2982bp,an open reading frame（ORF）of 1944bp encoding 647 amino acids with protein molecular weight（Mw）70.835k D and isoelectric point（p I）5.20.Amino acid sequence alignment and phylogenetic analysis showed that DcHsp70 was cytoplasmic Hsp70 contained three highly conserved labels of Hsp70 family.q RT-PCR analysis showed the expression of DcHsp70 was induced under heat stress condition.Arabidopsis overexpressed DcHsp70showed improved thermotolerance.Lower accumulation of reactive oxygen species（ROS）,higher activities of anti-oxidant enzymes,and lower MDA content and relative electrolyte leakage value in Arabidopsis overexpressed DcHsp70 were observed than WT under heat stress condition.The expression level of At HSP101,At HSP90,At HSP18.2,At HSFA2 and At APX gene were significantly changed in transgenic Arabidopsis before and after heat treatment.In addition,yeast two-hybrid analysis demonstrated that DcHsf A1d could interact with DcHsf70.4.DcHsp90 was obtained via homology-based cloning combined with Tail-PCR technology,which contained a full-length of g DNA（ATG-TAA）of 3046bp,an open reading frame（ORF）of 2094bp and encoding 697 amino acids with protein molecular weight（Mw）79.980k D and isoelectric point（p I）4.94.Amino acid sequence aligment and phylogenetic analysis showed that DcHsp90 was cytoplasmic Hsp90.q RT-PCR analysis showed the expression of DcHsp90 was induced under heat stress condition.Arabidopsis overexpressed DcHsp90 showed improved thermotolerance,whereas Arabidopsis transformed with antisense DcHsf A1d had impaired thermotolerance.Lower accumulation of O₂^-and equal level of H₂O₂were detected in Arabidopsis overexpressed DcHsp90.Higher activities of POD and SOD,lower MDA content and relative electrolyte leakage value in Arabidopsis overexpressed DcHsp90 were observed than WT under heat stress condition.The expression level of 7 stress-related genes were significantly changed in transgenic Arabidopsis before and after heat treatment.5.With transcriptome data for reference,five different DcHsf genes were cloned.NCBI Blast and phylogenetic analysis showed that three genes were DcHsf A2,DcHsf A4a and DcHsf A6,respectively,the other two were DcHsf B1.Molecular weights and isoelectric points were varies in these five DcHsf genes.Characteristics analysis of amino acid sequence showed that these five DcHsf genes contained a conserved Hsf DNA-binding domain（DBD）at the N-terminus having a central helix-turn-helix structure.Besides,we cloned eight different Dcs Hsp genes,NCBI Blast,molecular weight and phylogenetic analysis showed that these genes were DcHsp18.5-C I,DcHsp18.5-C II,DcHsp22.6-C IV,DcHsp16.1-C V,DcHsp22.2-C VI,DcHsp21.4-CP,DcHsp23.7-MT and DcHsp15.8-PX,respectively.These eight genes characterized with a conservedα-crystallin domain（ACD）of s Hsps.These results laid the foundation for further analysis of gene functions.6.At least 19 Hsf genes were identified from P.mume genome.Their chromosomal locations,gene structures,conserve motifs,phylogenetic relationships and gene expression level under heat stress condition were analyzed.These 19 Pm Hsf members were divided into three categories（A,B and C）according to the functional domain features and conserve motifs.q RT-PCR analysis showed that Pm Hsf A2,Pm Hsf A3 and Pm Hsf A6 became predominant transcripts in response to heat stress.7.At least 28 s Hsp genes were identified from P.mume genome.Their chromosomal locations,gene structures,conserve motifs and phylogenetic relationship were analyzed.24 members belonged to six different cytoplasmic/nuclear（Classes CI,CII,CIII,CIV,CV and CVI）subclasses and 2 each belonged to chloroplast and mitochondria subfamily.q RT-PCR analysis showed that most of the Pms Hsps were induced by heat stress.In addition,Pms Hsp19 expression was also induced by salt,dehydrate,oxidative stress and ABA treatment.Arabidopsis overexpressed Pms Hsp19showed improved thermotolerance,lower accumulation of reactive oxygen species（ROS）,higher activities of anti-oxidant enzymes,and lower MDA content and relative electrolyte leakage under heat stress condition.The expression level of At HSP101,At P5CS,At HSFA2 and At APX were significantly changed in transgenic Arabidopsis before and after heat treatment.