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期 刊 ：植物遗传资源学报 2014年 15卷 3期

关键词：心里美萝卜；二氢黄酮醇还原酶；克隆；序列分析；

Keywords:‘Xinlimei’ radish, anthocyanidin, dihydroflavonol reductase, cloning, sequencing analysis,

摘 要 ：二氢黄酮醇还原酶是植物花青素合成的关键酶。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 序列与白萝卜自交系‘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 384 aa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，发现它们之间存在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 的同源性较高。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。

Abstract:Dihydroflavonol reductase is a key enzyme in anthocyanidin synthesis of 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the complete CDS sequence of Chinese cabbage DFR gene with white radish inbred line ‘36-2’ whole genome sequences (unpublished), a homologous single gene Rsa10008592 was obtained.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 contained 1,164 bp (Genbank accession number: KF280272), which encoded the protein of 384 aa. 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 RsDFR had the highest homology with the DFRs of Chinese cabbage and mustard. qRT-PCR result showed 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 it expressed at all developmental stages and reached the peak at the splitting stage in ‘Xinlimei’ radish. The protein from RsDFR was further analyzed.

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