二氢黄酮醇还原酶基因在红肉萝卜和白肉萝卜中的序列变异和表达差异  

Sequence Variation and Expression Profiles of Dihydroflavonol Reductase in Red Flesh and White Flesh Radish Germplasm

作　　者：孙玉燕[1] 张晓辉[1] 邱杨[1] 李锡香[1] 王海平[1] 沈镝[1] 宋江萍[1] 

机构地区：[1]中国农业科学院蔬菜花卉研究所/农业部蔬菜作物基因资源与种质创制北京科学观测实验站,北京100081

出　　处：《植物遗传资源学报》2014年第3期554-560,共7页Journal of Plant Genetic Resources

基　　金："十二五"国家科技支撑项目(2013BAD01B04-1;2012BAD02B01);国家高技术研究发展计划项目(2012AA021801-4);农业部园艺作物生物学与种质创制重点实验室

摘　　要：二氢黄酮醇还原酶是植物花青素合成的关键酶。本研究以中国特有红肉萝卜种质‘心里美’为试材,克隆和分析其二氢黄酮醇还原酶基因(DFR)。通过与大白菜DFR基因的完整CDS序列及白萝卜自交系‘36-2’全基因组序列(未公布)进行比对,获得单一的同源序列Rsa10008592。参照该序列设计5'和3'引物,以‘心里美’萝卜自交系‘HX12Q-49’的cDNA为模板,通过RT-PCR和TA克隆获得ORF为1164 bp(Genbank登录号KF280272)、编码387 aa(Genbank登录号AGU42192)的RsDFR基因的完整CDS序列。通过对白萝卜‘36-2’的Rsa10008592和‘心里美’萝卜‘HX12Q-49’的RsDFR序列比较分析,发现二者之间存在19个核苷酸和3个氨基酸的差异。氨基酸序列进化树显示,‘心里美’萝卜的RsDFR与大白菜和芥菜的DFR同源性较高。荧光定量PCR发现RsDFR在白萝卜和‘心里美’萝卜不同发育阶段的表达模式不同,在白萝卜中仅在肉质根发育的早期表达,而在‘心里美’萝卜的全生育期均有表达,且在破肚期的表达量最高。同时,对‘心里美’萝卜RsDFR基因编码的蛋白质进行了初步分析。Dihydroflavonol reductase is a key enzyme of anthocyanidin synthesis in plants. Cloning and analysis of dihydroflavonol reductase gene（ DFR） was carried out in China＇s peculiar radish germplasm Xinlimei with red flesh. By blasting the complete CDS sequence of Chinese cabbage DFR gene in the whole genome sequences database of white radish inbred line 36-2（ unpublished）,a homologous single gene Rsa10008592 was obtained. The complete CDS of RsDFR was amplified from the fleshy root of Xinlimei radish inbred line HX12Q-49 with the primers designed according to the Rsa10008592 sequence by RT-PCR. The ORF of RsDFR was 1164 bp in length（ Genbank accession number： KF280272）,and encoded a 387 aa of protein（ Genbank accession number： AGU42192）. The sequence difference of 19 nucloetide acids and 3 amino acids were found between Xinlimei radish and white radish through sequences comparison. Phylogenetic tree showed that RsDFR had the highest homology with the DFRs of Chinese cabbage and mustard. qRT-PCR result showed that the expression patterns of the RsDFRs in the fleshy roots of Xinlimei radish and white radish were different. RsDFR in white radish only expressed at early developmental stage,while expressed at all developmental stages and reached the peak at the splitting stage in Xinlimei radish. The protein from RsDFR was further analyzed.

关 键 词：'心里美’萝卜 二氢黄酮醇还原酶 克隆 序列分析 表达分析 

分 类 号：S631.1[农业科学—蔬菜学]