Expression Vector Construction And Transformation Of Model Plants With Flavonoid Synthesis Related Genes In Herbaceous Peony(Paeonia Lactiflora Pall.)

The color of ornamental plants directly affects the value,which becomes the focus of flower breeding research.Herbaceous peony(Paeonia lactiflora Pall.)is a ornamental plant with high commercial value,which belongs to the Paeoniaceae family,but its color is mainly concentrated in pink,red,purple,white,wherever yellow cultivar is lack in the market,which limits the development to some degree.The new flower color breeding of P.lactiflora always receives much concern.However,the reports focus on cross breeding or genetic technology to achieve directly breeding are rarely reported,it could be poorly knowledge about molecular mechanism of flower color formation and regulatory mechanism of related genes in P.lactiflora.The key genes involved in flavo no id pathway have been isolated from many plants.Based on the previous transcriptome sequencing of P.lactiflora chimaera petals,10 candidate differentially expressed genes(DEGs)involved in flavonoid pathway that related to flower color were discovered,including CHI,ANS,DFR,FLS,PAL.This research cloned the five target genes,constructed the expression vector with these genes,and transformed into model plans by agrobacterium-mediated,in order to preliminarily explore the function to provide theoretical and practical support for new variety breeding of P.lactiflora.The main results were shown as follows:(1)Cloning of flavonoid pathway related genes in P.lactiflora.Total RNA was extracted from the petals of P.lactiflora 'Fen Yulou',and reverse transcribed to first-strand cDNA.Five key genes(CHI,ANS,DFR,FLS,PAL)were cloned with complete ORF according to the Race results.Analysis in the NCBI showed 99%,98%,99%,100%,100%similarity in their predicted amino acid sequences.(2)Constructing the over-expression vector with flavonoid pathway related genes in P.lactiflora.CHI,ANS,DFR and PAL were respectively digested by the double enzymes(Sma I and Sac I)while FLS was digested by the double enzymes(BamH I and Kpn I)after subcloning the ORFs with opposite restriction sites,and then ligated into the plasmid of pCAMBIA1301 vector.The completion of the construction of pB1301-CHI,pB1301-ANS,pB 1301-DFR pB1301-FLS,pB1301-PAL expression vectors were proved and sequenced,transferred into EHA105 by the freeze-thaw method finally,wihich can be transformation materials.(3)Gaining Arabidopsis homozygote(CHI,ANS,DFR,FLS,PAL).Transformation into Arabidopsis was conducted by floral-dip with recombinant agrobacterium transformants,finally,6 CHI homozygosis transgenic lines,2 ANS homozygosis transgenic lines,6 DFR homozygosis transgenic lines,3 FLS homozygosis transgenic lines,5 PAL homozygosis transgenic lines and 2 vector-control homozygosis transgenic lines were gained through a series of tests,including resistance screening of Ti generation,GUS staining,isolation screening of T2,T3 generation.The results of GUS staining,PCR confirmed the integration of the target genes into the genome of Arabidopsis,and qRT-PCR showed high level expression of target genes in transgenic lines compared to the control.(4)Preliminary phenotype analysis of Arabidopsis homozygote.There was no significant difference between CHI,FLS,PAL lines and the cotrol by preliminary observation.Although the flower of DFR,ANS lines had no significant difference,the back of rosette leaves in ANS lines showed variegated red,and a majority of the base of main veins showed slightly red,the color of rosette leaves in DFR lines was dark green,and the back of certain leaves turned red while the purple red especially showed in line 2.We will further research on the total flavonoids and anthocyanin content according to the phenotype afterwards.(5)Gaining transgenic tobacco paints(DFR,FLS).Transformation was conducted by infection of tobacco leaf discs with recombinant agrobacterium transformants,finally,43 DFR gene,66 FLS gene and 5 vector-control transgenic tobacco plants were obtained by screening with hygromycin.10 DFR lines and 12 FLS lines were determined by GUS staining and PCR,which showed that the DFR gene had been integrated into the genome of tobacco in 8 lines,and the FLS gene had been integrated into the genome of tobacco in 7 lines.Meanwile,the PCR-positive plants all successfully high expressed in transcription level compared to the control by qRT-PCR analysis.At present,these transgenic lins have not begin to bolt and flower due to the time and temperature,there was no significant difference in growth status and biological characteristics,we will further focus on the change of flower color and anthocyanin content afterwards.