Molecular Cloning And Functional Analysis Of Dihydroflavonol 4-reductas (DFR) Gene And Screening Of Lignin Genes In Paeonia Ostii

Paeonia ostii is the traditional Chinese ornamental plant and medicinal plant,belonging to Paeonia.In recent years,Paeonia ostii is choosed as oil crop because of its high oil yield and oil quality and development potential.However,the direct use of seed oil squeeze will appear black foreign body in the production process.So it needs to remove the testa of seeds,which will reduce the oil yield.The main ingredients in the seed coat have been proved are flavonoids and lignins in the oil peony through pigment analysis.Therefore,cloning and analysis of related genes not only help to understand the mechanism of black pigments and lignin synthesis,but also help to change the testa composition through genetic engineering.In the experiment,dihydroflavonol 4-reductase(DFR)gene of P.ostii,an important gene for flavonoid synthesis,was cloned and the function was analysized.The results showed that PoDFR contained an open reading frame(ORF)of 1092 bp in length,encoding 364 amino acids.The relative molecular weight of PoDFR is 40796.17 Da,and the theoretical isoelectric point(PI)is 5.76.The expression level of PoDFR gene in the four developmental stages of P.ostii seed was analyzed by real-time quantitative PCR method.The results showed that the expression level of PoDFR gene was increased to the highest level at S2 stage and then decreased.We get the PoDFR protein through prokaryotic expression method.And this protein can catalyze dihydroquercetin synthesis to leucocyanidin and synthesis cyanidin with PoANS protein.A total of 110,151 gene sequences were obtained from the testa and kernel of P.ostii by RNA-seq.The average length of the gene was 1010 bp,and the rate of gene annotation was 50.44%.A total of 19,530 differentially expressed genes were obtained by transcript expression level.Eight genes related to lignin synthesis pathway were screened out,including caffeic acid / 5-hydroxy ferulic acid-O-methyltransferase(COMT),Caffeoyl CoA O-methyltransferase(CCoAOMT),cinnamoyl-CoA reductase(CCR),cinnamyl alcohol dehydrogenase(CAD),coumaric acid-3-hydrogenase(C3H),ferulic acid-5-hydroxy(F5H),4-coumaric acid coenzyme A ligase(4CL)and coumaryl coenzyme A: shikimic acid / quinine carnosyltransferase(HCT).The expression patterns of these candidate genes in the development of the seed coat were obtained by real-time quantitative PCR,and it was proved that these genes were closely related to the accumulation of lignin.In summary,the above data laid the foundation for separating,identificating of related genes and exploring the synthesis mechanism of lignin.