Screening Of Candidate Genes Regulating The Anemone Flower Type And Study Of Expression And Regulation Mechanism In Herbaceous Peony(Paeonia Lactiflora Pall.)

Herbaceous peony(Paeonia lactiflora Pall.)as well as Paeonia suffruticosa is a famous flower in China.A number of cultivars with rich variety of color and flower pattern have been cultivated by artificial selection for a long time,and its flower shape is directly related to the ornamental and commercial merit.Previous studies have shown that,the floral pattern change of paeonia lactiflora mainly comes from the petaling of female,stamen and the natural increase of petal number.However,the molecular mechanisms of petals type in P.lactiflora remain unclear.Therefore,in this study,we selected inner-and outer-petals from P.lactiflora`Zi Fengyu' with anemone type as study object,and screening out the related genes regulating petals type through the conjoint analysis of comparative transcriptome.On this basis,this study performed the function verification of RACE cloning,bioinformatics analysis,gene expression characteristics and promoter methylation regulation,which provide theoretical guidance for in-depth study of P.lactiflora PlAP2 gene function and molecular breeding of genetic improvement.Besides,the codon usage patterns of PIAP2 gene and P.lactiflora genome were analyzed based on the sequencing results by multivariate statistical analysis.The main research results are as follows:1.Comparative RNA-seq transcriptome sequencing of inner-and outer-petals from P.lactiflora 'Zi Fengyu'RNA-seq transcriptome sequencing of inner-and outer-petals from P.lactiflora 'Zi Fengyu' obtained 394.46 Mb clean reads and 43,704 annotated Unigenes.979 differential expression genes(DEGs)were detected between inner-and outer-petals,of which 408 DEGs were upregulated in outer-petals.GO function analysis showed that DEGs were mainly enriched in Metabolic process,Single-organism process,Organelle,membrane,Catalytic activity and Binding.KEGG pathway analysis showed that DEGs were mainly enriched in Signal transduction,Carbohydrate metabolism,Biosynthesis of other secondary metabolites,Metabolism of terpenoids and polyketides,etc.Besides,some important DEGs such as PISTILLATA(PI)?APETALA2(AP2),Agamous-like MADS-box(AGL6)?AGAMOUS(AG)were identified.2.Comparative iTRAQ proteome of inner-and outer-petals from P.lactiflora 'Zi Fengyu' and screening of candidate genes(1)iTRAQ proteome of inner-and outer-petals from P.'Zi Fengyu' showed that 266 differential expression proteins(DEPs)were detected between inner-and outer-petals,of which 115 DEGs were upregulated in outer-petals.GO function analysis showed that DEPs were mainly enriched in metabolic process(131 DEPs),single-organism process(102 DEPs),extracellular region(12 DEPs),vacuole(12 DEPs),catalytic activity(137 DEPs).KEGG pathway analysis showed that DEPs were mainly enriched in 10 signaling pathways,including Biological oxidations,Glycolysis/Gluconeogenesis,etc.Besides,some important DEPs such as PDC2?ALDH3F1?ALDH2B7?APETALA2 were identified.(2)Based on the analysis results of transcriptome and proteome,using Venny software,39 common DEGs/DEPs between inner-and outer-petals from P.lactiflora 'Zi Fengyu' were selected as candidate gene regulating flower type,which mainly participate in biological oxidations,glycolysis/gluconeogenesis?carbon fixation in photo synthetic organisms,pyruvate metabolism,etc.qPCR validation showed that the gene expressions of APETALA2?ALDH3Fl?PDC2?NLTP6?At1g22430 were significantly different between inner-and outer-petals(P<0.01).Further western blot analysis showed that the protein expression of APETALA2 was obviously higher in outer-petals than that in inner-petals.Therefore,we speculate that APETALA2(AP1)was identified as an important candidate gene regulating P.lactiflora flower type.3.Cloning and expression pattern analysis of APETALA2(PlAP2)in P.lactiflora(1)RACE analysis showed that the full-length cDNA of PIAP2 gene consists of 1935 bp and contains a 1578 bp open reading frame(ORF)encoding 525 amino acid proteins.Analysis of protein structure and function showed that PlAP2 protein was unstable and hydrophilic,meanwhile no signal peptide and transmembrane structure indicated P1AP2 protein was non-secretory.Nuclear localization signal located between 139th and 147th amino acid(KKSRRGPRS).The secondary structure of P1AP2 protein contained 24%?-helices,19%?-sheet,28%?-turn and 28%random coil.PlAP2 protein had eight glycosylation sites and Sixty-four phosphorylation sites.P1AP2 protein had two same conservative domains:AP2/Ethylene-Responsive factors(ERF),which was located in the region between 151 and 213,between 243 and 306,respectively.Prediction of subcellular localization showed that PlAP2 located mostly in cytoplasm(45.0%),rarely in microbody,mitochondrial matrix and lysosome.Analysis of evolutionary tree showed that the AP2 of Paeonia lactiflora were highly homologous and close to Paeonia suffruticosa.(2)Using qPCR technology,we detected and analyzed the differential expression analysis of PlAP2 gene between inner-petal and outer-petal,at four developmental stages(flower-bud stage,S1;initiating bloom stage,S2;bloom stage,S3;wither stage,S4),among different Paeonia lactiflora cultivars('Zi Fengyu','Tongyun Jinyan','Honglou Jinju').Differential expressions between inner-petal and outer-petal showed that the expression level of PlAP2 in outer-petal was apparently higher than that in inner-petals from 'Zi Fengyu' cultivar.PlAP2 gene expression was higher at S3 stage than that at S1,S2,S4 stages.Differential expression among different cultivars showed,the expression level of PIAP2 gene was significantly or extremely significantly higher in 'Tongyun Jinyan' than that in 'Honglou Jinju' and 'Zi Fengyu'.Besides,there were significant differences in the morphological characteristics of petals among different stages and cultivars from‘Zi Fengyu',‘Tongyun Jinyan','Honglou Jinju'.Comprehensive results indicated that PIAP2 gene expression was closely related to Paeonia lactiflora petals.4.Promoter methylation analysis of candidate gene PlAP2 regulating flower type in P.lactifloraUsing genome walking,we obtained PlAP2 5' upstream promoter sequence with a length of 2000 bp,containing a CpG island(-665 bp?-872 bp),where NF-kappaB,GATA-1,Sp1,C/EBP were located.Methylation sequencing by BSP+Miseq showed that 7 CpG sites were detected in the CpG island of PIAP2 promoter,such as CpG-1,CpG-2,CpG-3,CpG-4,CpG-5,CpG-6,CpG-7,and the methylation levelof CpG sites ranged from 19.73%to 65.78%.Difference significance analysis of methylation level between inner-petal and outer-petal tissues showed that the methylation level in inner-petal(45.37%)was significantly higher than that in outer-petal(35.85%)at CpG-3 site(P<0.01).Difference significance analysis of methylation level among different developmental stages showed that the methylation level of CpG-2 site at S4 stage(57.01%)was significantly higher than that at S3 stage(40.62%)(P<0.01),and significantly higher than that at S1 stage(45.49%),S2 stage(45.85%)(P<0.05).The methylation level of CpG-3 site at S3 stage(32.36%)was significantly lower than that at S1 stage(42.99%),S2 stage(43.71%)and S4 stage(43.37%)(P<0.05).Besides,the methylation level at CpG-3,CpG-6 sites showed significantly negative correlations with AP2 expression(P<0.01),and the CpG-3 is located in the binding site of transcription factor Spl.5.Codon usage pattern analysis of P.lactiflora transcriptome and the candidate gene PlAP2 regulating flower type(1)In this study,43,704 differential genes were screened out by transcriptome sequencing of P.lactiflora 'Zi Fengyu' with anemone type,followed by the further filtering analysis according to the principle of CDS sequence characteristics and greater than 300 bp,we finally obtained 24,216 genes as research object.Mobyle software was used to calculate different parameters for the codon usage,such as GC content,average GC content of the first and second positions(GC12),GC content of the third position(GC3s),effective number of codon(ENC),codon adaptation index(CAI),relative synonymous codon usage(RSCU),Neutrality plot was analyzed based on GC12 value as the ordinate,GC3s value as the abscissa.ENC-GC3s plot was analyzed based on ENC value as the ordinate,GC3s value as the abscissa.Parity Rule 2(PR2)plot was analyzed by ENC value as the ordinate,GC3s value as the abscissa.Besides,we probed into the influence of mutational pressure and translational selection by the multivariate statistical analysis.Finally,we took 5%CAI value as high-expression and low-expression sample groups,then calculated the RSCU value,and analyzed the significant difference to determine the optimal codons by chi-square test.Results showed,GC content at the third position of codons was 46.37%.GC content of most genes were mainly distributed between 35%and 45%.Neutrality analysis showed there was significant negative correlation(r=-0.03,P<0.01)between GC3s and GC12 value.ENC-plot showed most of genes on or close to the expected curve,but also some points with low-EVC values were below it.The(ENCexp-ENCobs)/ENCexp ratio of most genes ranged from-0.1 to 0.3.Parity Rule 2-plot showed the frequency of.T nucleotide at the third position was higher than A,G was higher than C,suggesting that the use frequencies of four nucleotide were not balanced.Correspondence analysis showed that the first axis showed 20.55%variation,while the other three axes showed 13.99%,12.24%,11.45%,respectively,suggesting that the first axis was main index evaluating the codon usage bias of Paeonia lactiflora transcriptome.Mutation pressure and selection analysis showed there were significant positive correlations(r=0.758,P<0.01;r=0.293,P<0.01)between the first axis and GC3s,CAI value,respectively.Using the delta RSCU and significant chi-square test methods,we defined 22 codons as the major preference codons in the Paeonia lactiflora transcriptome,most of preference codons ending with A or U.(2)In this study,eight AP2 genes coding sequences from different plant species were selected using the GenBank database.Their nucleotide composition(GC content),genetic index,relative synonymous codon usage(RSCU)and relative codon usage bias(RCBS)were calculated with R software to compare codon bias and base composition dynamics of AP2 gene codon usage patterns in different plant species.Results showed,the usage of AP2 gene codons from different plant species were influened by GC bias,especially GC3s.Overall,base composition analysis indicated that the usage frequency of codon AT in the gene coding sequence was higher than GC among AP2 gene coding sequences from different plants.Furthermore,most AP2 gene coding sequences ended with A/T;AG A,GCU and UGU had relatively high RSCU values as the most dominant codon;the usage characteristic of the AP2 gene codon in Paeonia lactiflora was similar to Paeonia suffruticosa.Genetic selection pressure analysis showed that there was a low preference in the usage of AP2 gene codon with relatively low optimal codon frequency(Fop)values,Relative codon usage bias(RCBS)and high effective number of codons(ENC)value among different plant species.