| Flower color is an important index to measure the ornamental value of flowers,and also one of the main characters of ornamental plant variety innovation.Double-color variety is a large group among nine major color families of Paeonia lactiflora Pall.and the double-colored P.lactiflora ’Hebao Jinlian’ owns yellow inner petals and red outer petals.Understanding the molecular mechanism of the formation on the difference between inner and outer petals is of great significance for the genetic regulation of flower color in the future.In this study,the changes of morphology,color and anatomical structure of inner and outer petals of P.lactiflora ’Hebao Jinlian’ were observed during flower blooming,and the changes of flavonoid components and content of inner and outer petals were analyzed by liquid chromatography-mass spectrometry;The transcriptomics of inner and outer petals were studied by full-length transcriptome and next-generation sequencing technology to analyse the expression patterns of flower color formation related differentially expressed genes（DEGs）,and the related candidate genes involved in flower color formation were excavated;On this basis,the ATP citrate lyase（ACL）gene was cloned,and the gene function was identified by overexpression on tobacco and transient silencing on P.lactiflora,so as to clarify the function of PlACL2 gene on flower color formation.The main results are as follows:（1）With the P.lactiflora flowers gradually opening,the length and width of the petals increase progressively;The values of the outer petals were significantly larger than those of the inner petals in the same period,and the red outer petals and the yellow inner petals became lighter;Red and colorless cells were observed by the anatomical structure in the red outer petals,while light yellow and colorless cells were observed in the yellow inner petals.Colored cells were mainly distributed in the epidermis,while colorless cells were mainly distributed in the spongy and palisade tissues,and the colored cells were long-oval.（2）One anthocyanin detected in the outer petals was cyanidin-3,5-O-diglucoside,which was not identified in the inner petals.The HPLC peaks of anthoxanthins were same in the inner and outer petals.A total of five anthoxanthins were identified,including quercetin-7-O-glucoside,kaempferol-3-O-galloyl glucoside,isorhamnetin-3-O-glucoside,kaempferol-7-O-glucoside,kaempferol-3-O-glucoside.The content of anthoxanthins in the outer petals was significantly higher than that in the inner petals all along.（3）A total of 684,251 polymerase reads and 22,144,925 subreads sequences were obtained by a mixed sample of full-length transcriptome,and a total of 74,968 transcripts with good overall assembly results were obtained after redundancy removal.Based on the reference gene bank,24 independent samples were analyzed by the next-generation sequencing.A total of 32.33 GB clean reads were obtained after filtration.Most transcripts were completely covered,and the density range was relatively concentrated,and the number of detected genes tended to be saturated;5,061 DEGs belonged to 57 transcription factor families,and a total of 6,315 DEGs were differentially expressed at different developmental stages.The expression levels of DEGs verified by quantitative real-time polymerase chain reaction（qRT-PCR）were basically consistent with the transcriptome sequencing data.（4）KEGG enrichment analysis of DEGs showed that phenylpropanoid biosynthesis and flavonoid biosynthesis pathways were significantly enriched.One chalcone synthase（CHS）,one chalcone isomerase（CHI）,two flavonol synthase（FLS）and one anthocyanindin synthase（ANS）expressed higher in the outer petals and decreased gradually with developmental stages.In addition,one ACL was screened from DEGs,which encoded the enzyme that can regulate the malonyl-CoA to produce substrate for the flavonoid synthesis.It also expressed highly in the outer petals,and gradually decreased with the development stages.（5）The candidate PIACL2 gene was cloned by RACE cloning technology,and the cDNA full length sequence was 2,147 bp and the open reading frame was 1,824 bp,608 amino acids encoded,nucleotide and amino acids sequence had high homology with other plants ACL2.The molecular weight of P.lactiflora PlACL2 protein was 178,042.78 Da,and the protein was hydrophobic.Based on the construction of overexpression vector pCAMBIA2300-PlACL2 containing P.lactiflora PlACL2 gene sequence,it was found located in the cytoplasm by subcellular localization observation of rice protoplast.（6）The P.lactiflora PlACL2 gene overexpressed in tobacco by leaf disk method for Agrobacterium mediated transformation and the positive transgenic plants were detected by PCR and qRT-PCR;Compared with wild type（WT）tobacco,the expression level of P.lactiflora PIACL2 gene was significantly improved,and a*value of petals became obviously higher.The P.lactiflora PIACL2 gene was transiently silenced in the outer petals of P.lactiflora,and the positive transgenic plants were detected by PCR and qRT-PCR;Compared with WT and TRV empty vector,the expression level of P.lactiflora PlACL2 gene significantly expressed lower,and a*value decreased significantly. |
