Study On The Formation Of Flower Color From Ornamental Sunflower (Helianthus Annuus)

The cultivated sunflower(Helianthus annuus)that known as ornamental includes a number of varieties.They differ in flower color of ray floret and tubular floret.During recent years the importance of the sunflower as an ornamental plant has increased. They are used to a compulsory part of cottage gardens and perennial borders in Europe and American countries.Recently they are also popular in China as cut flowers.Flavonoids represent a large group of secondary metabolites widespread among plants and involved in many processes.Anthocyanins are the most conspicuous class,which are important to many diverse functions within plants. Anthocyanins biosynthesis has been studied extensively from many plant species. Most structural genes and regulatory genes of this pathway have been isolated.To date,however,researches of pigment biosynthesis are limited to ornamental sunflower.Also little has been reported about genes expression along the anthocyanins biosynthetic pathway and regulation of floral color change in ornamental sunflower.The major research contents of this dissertation included:(1)Ornamental sunflower with different petal color was used in order to analyze growth characters in different sowing seasons.(2)The flower color pigments of ornamental sunflower were detected by using specific color reactions and UV-visible spectra,et al.(3)Based on genes involved in anthocyanins synthesis from others plants,6 structural genes in ornamental sunflower were isolated with RT-PCR.Transcription and expression analysis will also be done with semi-quantitative RT-PCR.(4)Based on EST (expressed sequence tag)databases,osDFR gene was isolated from ornamental sunflower with technology of in silico cloning.Southern blotting,expression in Escherichia coli BL21 and PCR-mediated modification of osDFR gene were also done.The main experimental results were as follows:(1)6 cultivars of ornamental sunflower with different petal color were used to study the growth characters in different sowing seasons.The results showed that there were very significant differences of correlation coefficients in growth period characters and biological characters in different sowing seasons.The results of canonical correlation analysis indicated that there were significant correlation of canonical coefficients between growth period characters and biological characters.GP was related to NRF in spring-sowing season.DSM was related to NF in summer-sowing season.GP was related to WH in autumn-sowing season.And DSM was related to NRF in winter-sowing season.The growth curves and logistic model showed that the growth of plant height was significantly different among 3 cultivars of ornamental sunflower. The plant types of ornamental sunflower in different sowing seasons were also very significantly different.(2)Specific color reactions indicated that the flower color pigments of ornamental sunflower were flavonoids,and probably including flavones,flananones,flavanonols and so on.The pigments of red and purple flower from 'Joker' and 'Ring of Fire' might contain anthocyanins.The results of UV-visible spectra showed that the flower color pigments from four cultivars of ornamental sunflower probably included flavanonols.The high pressure liquid chromatography-mass spectrometry(HPLC-MS) was used to isolate pigments and to determinate the molecular weight.The results showed that the flower color pigments might conclude pelargonidin with diglucoside in 'Ring of Fire',malvidin-3-rutinoside and cyaniding-3-glucoside in 'Joker',and taxfolin-3-glucoside in 4 cultivars.The research had been made on stability of the anthocyanins in ornamental sunflower.Conclusion was drawn that the indoors nature light and low temperature were good for the stability of the anthocyanins.(3)Based on the conserved motifs of cloned genes involved in anthocyanin synthesis, primers were designed and used to amplify the 6 structural genes from ornamental sunflower cultivar Ring of Fire.6 structural genes(PAL,CHS,CHI,F3'H,DFR,ANS) were obtained and submitted to GenBank excluded CHS gene.The accession numbers were EF566469,EU366166,EU366167,EF492066,DQ884194,respectively.Sequence analysis showed that these genes shared high similarity with those from other plants.The results of phylogenetic analysis met that described in plant taxonomy. And the evolution rates of PAL and CHS gene were quicker than F3'H,DFR and ANS. The expressions of these genes were detected in different organs of ornamental sunflower by using semi-quantitative RT-PCR.The transcripts of CHS gene was not detected in tubular floret,and other five genes were all expressed in ray floret,tubular floret,bud,leaf,and bark of ornamental sunflower.Expression detection of flower development indicated that expression levels of all most structural genes in first florescence and full bloom stage were higher and then decreased after bloom.The expressions of these genes were related to the ray floret color.Transcripts of CHS gene were higher in chocolate petal than in other color petals.Transcripts of F3'H gene was higher in purple petal(such as 'Ring of Fire' and 'Mouling Rouge')than in chocolate petals(such as'Joker' and 'Autumn Beauty').Expressions of DFR and ANS genes were higher in petal with deep color('Mouling Rough' and 'Autumn Beauty') than those with multicolour('Ring of Fire' and 'Joker').(4)By using in silico elongation based on ESTs database,cDNA sequence osDFR had been successfully isolated from Helianthus annuus cultivar 'Ring of Fire'.The data revealed that the cDNA sequence contained a 1079 bp open reading frame,encoding a protein of 354 amino acid residues.Sequence analysis indicated that osDFR gene is a potentially biofunctional gene.Southern blotting analysis showed that osDFR was present as a small gene family or DFR-like genes in four cultivars 'Valentine', 'Ikarus','Ring of Fire',and 'Joker'.Phylogenetic analysis of DFRs showed that DFRs of monocots and eudicots were clearly classified into different branches. Eudicot DFRs further diverged into two groups.This phylogenetic tree indicates that Asn-type DFRs were widely distributed in plants,whereas Asp-type DFRs were only found in limited plant species that were scattered throughout the eudicots.And DFRs other than Asn-or Asp-type were present in a few plant species.DFRs of Compsitae constituted a monophyletic group,and all were Asn-type DFRs.By using PCR, substrate binding region of DFR from Helianthus annuus was knocked out,and conserved amino acid residue asparagine was mutated into aspartate or leucine. KODFR,N134D and N134L mutated were successfully amplified.Plant expression vector pCAM-KODFR,pCAM-N134D and pCAM-N134L were also successfully constructed,which will be used for gene transformation.