Analysis Of Flowering Transcriptome Of Paeonia Lactiflora And Functional Verification Of PlFT Gene

Chinese herbaceous peony(Paeonia lactiflora Pall.)is a new high-grade cut flower in recent years.It has beautiful flowers,rich colors and high ornamental value.However,the florescence of single species of Paeonia lactiflora is short,and the florescence of group species is concentrated,which not only affects the ornamental period of Paeonia lactiflora,but also leads to the labor shortage when cutting Paeonia lactiflora flowers.The florescence of Paeonia lactiflora can be controlled by protected cultivation,but the production cost is high.In recent years,many studies on the molecular mechanism of flowering regulation have provided technical support for the use of molecular technology to regulate the florescence of plants.In this study,we used Paeonia lactiflora variety 'Dafugui' as the test material and collected the potential flower buds that are in the critical period of flower bud differentiation,the flower buds after the completion of flower bud morphology and the flowers in the full-bloom stage of Paeonia lactiflora to sequence the transcriptome According to the transcriptome analysis results,we screened the genes related to the regulation of Paeonia lactiflora flowering;then,we isolated and cloned the gene related to flowering and constructed the expression vector of it for genetic transformation.The aim is to provide theoretical basis for exploring the molecular mechanism of flowering regulation of Paeonia lactiflora and clarifying the key genes regulating Paeonia lactiflora flowering The main results are as follows(1)Using Illumina HiSeq platform to analyze RNA-Seq comparative transcriptome of the Paeonia lactiflora 'Dafugui' samples.9 cDNA libraries(3 biological repeats)were constructed.92.53 Gb data and 81788 Unigenes were obtained.The sequence length of Unigenes is mainly between 200-3000 nt.Seven functional databases(KEGG,GO,NR,NT,SwissProt,Pfam and KOG)were annotated to the selected Unigenes.19,281(23.57%)Unigenes were annotated together,and 56,599(69.20%)Unigenes were annotated to any database.It was found that the similarity between the Unigenes and Vitis vinifera in NR database was the highest(21.41%).The number of Unigenes annotated by the KOG database to the category of 'Cell cycle control,cell division,chromosome part' is 670.We screened the intergroup differential expression genes(DEGs).The GO function classification of DEGs showed that the number of DEGs annotated to GO function in T1(critical stage of flower bud differentiation),T2(after flower bud morphogenesis)and T3(full-bloom stage)increased compared with the previous period,and the two most annotated secondary categories are the same in the GO function classification.KEGG pathway analysis were carried out for DEGs and the significant enrichment metabolic pathways are circadian rhythm pathway and isoflavone biosynthesis pathway.Among them,plant circadian rhythm pathway is related to flowering regulation.We selected 19 DEGs related to flowering regulation of Paeonia lactiflora from plant circadian rhythm pathway,and randomly analyzed 9 genes(TCP21,CHE?CO?PRR5?PAP1,MYB75?HY5?ELF3?GI?FT?CRY1)by qRT-PCR.The expression of the samples in the three periods was consistent with the result of transcriptome sequencing(2)PlFT related to Paeonia lactiflora flowering was isolated and cloned.The total RNA was extracted from the leaves of Paeonia lactiflora 'Dafugui' and the first strand of cDNA was synthesized by reverse transcription.According to the full-length sequence of DEGs FT(gene ID CL8783.Contig2_All)in transcriptome data,PlFT gene was cloned and analyzed by bioinformatics.The total length of PlFT gene is 592 bp,which has a complete open reading frame 522 bp and encodes 173 amino acids.The gene login number is MT249229.The pCAMBIA1301-PlFT over-expression vector was successfully constructed After sequencing and bacterial solution PCR verification,the recombinant plasmid was transformed into Agrobacterium EHA105 by freeze-thaw method,and the positive transformation factors were obtained.(3)The mutant ft-10 Arabidopsis was genetically transformed(functional replenishment experiment).Through Agrobacterium transformation,pCAMBIA1301-PlFT was introduced into mutant Arabidopsis ft-10,and the seeds were screened for hyg resistance.Finally,35 PlFT transgenic lines were obtained,and some of them were identified by GUS tissue staining.The transcription level of PlFT in 10 transgenic ft-10 Arabidopsis lines was analyzed by qRT-PCR.The results showed that PlFT could be expressed successfully in transgenic Arabidopsis lines,and the expression was significantly higher than that in the control group.The phenotypes such as flowering time and leaf number of transgenic mutant Arabidopsis ft-10 of PlFT gene,wild type Arabidopsis Col-0 and mutant Arabidopsis ft-10 were observed and analyzed.All lines of transgenic Arabidopsis with PIFT gene flowered earlier than those of the control group,and the transgenic Arabidopsis lines had the least number of rosette leaves in first blooming.The results of phenotypic and qRT-PCR analysis showed that PlFT could promote flower formation.