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根据物种水平上蛋白保守序列设计特异引物,扩增获得‘红阳’猕猴桃花青素合成途径中查尔酮合成酶(chalconesynthase,CHS)和无色花青素双加氧酶(leucoanthoc yanidin dioxygenase,LDOX)基因的特异片段,用RACE(rapid-amplification of cDNAends)技术克隆出这两个基因的cDNA全长,长度分别为1501bp(AcCHS)和1381bp(AcLDOX),分别编码389个和355个氨基酸。通过比对发现AcCHS与棉花(Gossypium hirsutum)、山茶(Camellia japonica)和黄蜀魁(Abelmoschus manihot)的CHS序列相似性较高,达到95%,与葡萄(Vitis vinifera)和苹果(Malus×domestica)的相似性分别为94%和93%;AcLDOX与山葡萄(Vitis amurensis)和葡葡的相似性分别高达94%和93%。用实时荧光定量PCR分析AcCHS和AcLDOX在‘红阳’(红肉)、‘金魁’(绿肉)和‘金农’(黄肉)3种不同果肉颜色的猕猴桃内果皮中的表达,发现AcCHS的表达量在‘红阳’果实转色期(花后65d)较高,而在‘金农’开花后表达量呈持续下降趋势;AcLDOX在‘红阳’果实发育早期呈上升趋势,花后65d后迅速下降,在‘金魁’果实发育后期呈明显上升趋势,在‘金农’开花后呈持续下降趋势。
According to the conserved sequence of the protein at the species level, specific primers were designed to amplify chalconesynthase (CHS) and leucoanthoc yanidin dioxygenase (AChE) in the anthocyanin biosynthesis pathway of ’Hongyang’ LDOX) genes were cloned. The full-length cDNAs of these two genes were cloned by rapid amplification of cDNA ends (RACE) with lengths of 1501 bp (AcCHS) and 1381 bp (AcLDOX), encoding 389 and 355 amino acids, respectively. By comparison, we found that the similarity of CHS sequences between AcCHS and Gossypium hirsutum, Camellia japonica and Abelmoschus manihot was 95%, similar to that of Vitis vinifera and Malus × domestica 94% and 93% respectively. The similarities between AcLDOX and Vitis amurensis and grapevine were 94% and 93% respectively. The expression of AcCHS and AcLDOX in kiwifruit of three different flesh colors of ’Hongyang’ (red meat), ’Jinkui’ (green meat) and ’Jinnong’ (yellow meat) were analyzed by real-time fluorescence quantitative PCR and found that The expression level of AcCHS in ’Hongyang’ fruits was higher during turning-color (65 days after anthesis) and continued to decrease after flowering in ’Jinnong’; AcLDOX showed an upward trend in the early development of ’Hongyang’ After 65 days, it dropped rapidly and showed a clear upward trend in the late stage of ’Jinkui’ fruit development, and continued to decline after the flowering of ’Jinnong’.