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杨俊, 姜正旺, 王彦昌. 红肉猕猴桃DFR基因的克隆及表达分析[J]. 植物科学学报, 2010, 28(6): 673-681.
引用本文: 杨俊, 姜正旺, 王彦昌. 红肉猕猴桃DFR基因的克隆及表达分析[J]. 植物科学学报, 2010, 28(6): 673-681.
YANG Jun, JIANG Zheng-Wang, WANG Yan-Chang. Cloning and Expression of Dihydroflavonol 4-Reductase in Actinidia chinensis var. rufopulpa[J]. Plant Science Journal, 2010, 28(6): 673-681.
Citation: YANG Jun, JIANG Zheng-Wang, WANG Yan-Chang. Cloning and Expression of Dihydroflavonol 4-Reductase in Actinidia chinensis var. rufopulpa[J]. Plant Science Journal, 2010, 28(6): 673-681.

红肉猕猴桃DFR基因的克隆及表达分析

Cloning and Expression of Dihydroflavonol 4-Reductase in Actinidia chinensis var. rufopulpa

  • 摘要: 利用葡萄(Vitis vinifera)果实的DFR-cDNA序列搜索猕猴桃(Actinidia Lindl.)EST数据库,拼接所有与该cDNA相似片段成contig并借此设计引物,分离出红肉猕猴桃(A.chinensis var.rufopulpa)中DFR的两个克隆(AcDFR1AcDFR2)的片段。在此基础上利用5′RACE和3′RACE技术,分别克隆到具有完整阅读框的AcDFR1(1264 bp)和靠近3′端的部分AcDFR2序列(880 bp)。AcDFR1与茶DFR-cDNA(Camellia sinensis)相似达84%,且AcDFR1与非洲菊变种(Gerbera hybrida var.regina)的氨基酸序列相似达80%。据DFR聚类分析,AcDFR1AcDFR2蛋白类型上差异显著。实时定量PCR分析表明,AcDFR1在‘金魁’(A.chinensis var.deliciosa‘Jinkui’)中表达很高,AcDFR1AcDFR2在‘金农’(A.chinensis var.chinensis‘Jinnong’)与‘红阳’(A.chinensis var.chinensis‘Hongyang’)中较低,且在果实发育后期(大约花后90~120 d)均有所升高,二者可能参与了红肉猕猴桃中花青素的积累,而在绿肉猕猴桃‘金魁’与黄肉猕猴桃‘金农’中,AcDFR1可能还参与了类黄酮代谢过程中的上游分支途径。

     

    Abstract: Two DFR cDNA sequences similar to full-length DFR cDNA in Vitis vinifera were reconstructed by overlapping ESTs from Actinidia dbEST.Based on these two sequences,a full-length AcDFR1(1264 bp) and partial sequence of AcDFR2 cDNA near 3’ end(880 bp) was obtained using 3’RACE and 5’RACE in A. chinensis var.rufopulpa.AcDFR1,whose nucleotide sequence exhibited 84% identity with DFR cDNA of Camellia sinensis,showed 80% identity with Gerbera hybrida var.regina at the amino acid level.Alignment analysis of DFR showed that AcDFR1 differed considerably from AcDFR2.To understand the diversity of expression among green,yellow,and red-fleshed cultivars,real-time PCR was conducted.AcDFR1 expression was extremely high in green-flesh kiwifruit(Jinkui) but relatively low in the yellow-flesh fruit(Jinnong) and red-flesh fruit(Hongyang).There was an increasingly higher expression of AcDFR1 and AcDFR2 in Jinnong and Hongyang fruit after development,especially between 90 to 120 days after flowering.This indicated that the two transcriptions both catalyzed the accumulation of anthocyanins during fruit growing development.Results also suggested that AcDFR1 and AcDFR2 both participated in the anthocyanin pathway.In addition,AcDFR1 may have additional functions related to other flavonol metallization referred to upper-stream pathways of Jinnong and Jinkui fruit.

     

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