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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 in plant anthocyanin synthesis. In this study, we obtained the dihydroflavonol reductase gene (DFR) from Chinese red sweet radish germplasm ¡° Mei Li Mei¡ ±. A single homologous sequence, Rsa10008592, was obtained by comparing the complete CDS sequence of Chinese cabbage DFR gene and the whole genome sequence of ’36 -2 ’(unpublished data) of Chinese white radish inbred line. The ORF of 1164 bp (GenBank accession number: KF280272) was obtained by RT-PCR and TA cloning with the cDNA of ’Xinyimei’ radish inbred line ’HX12Q-49’ 387 aa (Genbank accession number AGU42192). By comparing the RsDFR sequences of Rsa10008592 of ’36 -2 ’and’ HX12Q-49 ’of’ Meixi ’with white radish, we found that there were 19 nucleotides and 3 amino acids difference between the two. The amino acid sequence phylogenetic tree showed that the RsDFR of ’Meili Mei’ radish was highly homologous to the DFR of Chinese cabbage and mustard. Fluorescent quantitative PCR revealed that the expression pattern of RsDFR was different at different developmental stages of Radish and Radix et Rhizoma Radish and was expressed only in the early stage of development of Radix Forsythia and in whole growth period of radish in ’ And the highest expression in the broken period. At the same time, a preliminary analysis of the proteins encoded by RsDFR gene from Radix et Rhizoma Rhei was carried out.