大白菜橘红心类胡萝卜素组分及其基因分析

Analysis of the Carotenoids Pigments and Orange Inner Leaf Gene in Chinese Cabbage(Brassica rapa)

利用高效液相色谱法(HPLC),根据二极管阵列检测器和标准品的检测结果对橘红心和白心大白菜内叶类胡萝卜素组分进行鉴定;以橘红心大白菜自交系‘A21530’和白心大白菜自交系‘A21445’为亲本构建了一个269单株的F2分离群体,进行橘红心基因精细定位;根据大白菜基因组注释信息,筛选橘红心候选基因并克隆测序分析;基因内部开发标记,在F2群体进行候选基因验证。研究结果表明,橘红色是由于前番茄红素(7,9,7′,9′–四顺式–番茄红素)、9–顺式–β–胡萝卜素、前链孢红素(7,9,9′–三顺式–链孢红素)和其他胡萝卜素组分积累造成的。通过精细定位将橘红心基因定位于A09号染色体末端,其两侧紧密连锁的标记是SB13037和SB13049,这两个标记各有一个重组单株,与橘红色基因分别相距0.3和1.1 cM,物理距离为98.904 kb。根据大白菜基因组注释信息,分析定位区域的23个基因,筛选出1个编码类胡萝卜素异构酶的基因CRTISO,基因编号Bra031539。测序结果表明,Bra031539的编码区有53个SNP和6个碱基缺失,造成12个氨基酸突变和2个谷氨酸的缺失。根据缺失突变位点设计标记,在F2群体中进行验证,结果表明候选基因与橘红心性状共分离。

High-performance liquid chromatography （HPLC） , coupled with photodiode array detection and carotenoid standards was used to analyze caotenoid composition in different genotypes of Chinese cabbage. A F2 population constructed by homozygous breeding lines white inner leaf cabbage ＇A21445＇ and orange inner leaf cabbage ＇A21530＇was used for markers screening and fine mapping. The candidate or gene was screened and cloned according to Brassica rapa genome annotation information. An internal marker in the gene was developed and verified candidate gene in F2 population. Mapping of SSR and InDel markers on 269 individuals of F2 population, the or gene was located to a 1.4 cM interval with the physical distance of 98.904 kb on chromosome A09 of the B. rapa genome. HPLC analysis indicated that orange inner leaf of Chinese cabbage is due to the accumulation ofprolycopene （7Z, 9Z, 7＇Z, 9＇Z-tetra-cis-lycopene）, 9-cis-β-carotene, proneurosporene （7,9,9＇-tri-cis-neurosporene） and other carotenoids pigments. According to B. rapa genome annotation information, a candidate gene Bra031539, designated CRTISO, was predicted within the mapped or locus, A deletion of 6 bp and 53 SNPs were found in coding sequence of the orange type, corresponding to two glutamic acids （Glu） deletion and 12 amino acids mutations. The functional marker was cosegregated with or louts in F2 individuals.