Cloning And Overexpression Vector Construction Of DFR,a Related Gene To Pink Leaf In Ornamental Kale

Ornamental kale（Brassica oleracea L.var.acephala DC）is a variant of Brassica oleracea,biennial herb.It occupies an important place in the landscape in our country because of it’s changeable leaf type and color,long ornamental period,easy to cultivative and strong cold resistance.Leaf color is an important horticultural character of kale,it determines the commercial value and ornamental value of kale cultivars.This study measured the degree of the pigment content of kale cultivars with different colour,Cloned the related gene to pink leaf,analyzed the protein sequence which it encodes,and constructed a plant expression vector.The aim of the study is to reveal the molecular mechanism of the kale’s pink leaves.principal results are as follows:1.The degree of the pigment content of five different colour kale cultivars was measured by pH differential method.Anthocyanins content in the overall level on a scale of0-0.780^-11 mg·g FW.The redder cultivars are the higher total anthocyanin content is,considering the previous analysis results of anthocyanin composition:There are a total of 8kinds of cyanine are indentified in kale,they are all cyanidin-3-O-glucoside with different acylations.These results suggest that red leaves are mainly affected by anthocyanin content.In each cultivar,the total content of anthocyanins in inner leaf is higher than that in outor leaf;the total content of chlorophyll in inner leaf is higher than that in outor leaf.These results suggest that the formation and accumulation of anthocyanin and chlorophyll in kale is different between different parts of the plant.2.The material are pink-leaved inbred lines’P02-9,white-leaved inbred lines’W02-7,and the F₂ population built by these two inbred lines.A series of molecular markers were dsigned according to the Brassica oleracea sequencing database,these molecular markers were tested between parents,pools and F₂ populations,finally a co-segregated marker was got.A gene which encodes dihydroflavonol 4-reductase（DFR）named Bo9g058630 in number nine chromosome of kale was identified as the candidate gene relate to pink leaf.3.The DFR gene of kale was cloned and sequenced,The full-length cDNA of DFR is1158bp,encodes a protein of 385 amino acids.Bioinformatics analysis showed that the protein belongs to the short-chain dehydrogenase/reductase family（SDR）.It is a stable hydrophilic protein,the molecular weight is 42886.04,contains two transmembrane regions,two conservative structural domains.Protein secondary structure prediction show that the protein is consisted mainly of alpha helix,beta turn,extend strand,and random coil,These structures distribute throughout the whole protein.4.The cDNA of DFR was fused with GUS reporter gene to construct a plant expression vector named pCambia3301-DFR by In-Fusion technology.