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以富含花青素的紫山药‘蓣引紫006’为材料,克隆得到花青素生物合成途径中关键酶基因DaF3H(KF561995),其序列全长为1 325 bp,最大开放阅读框为1 089 bp,编码362个氨基酸。DaF3H蛋白属于水溶性的胞质不稳定蛋白,无跨膜区和信号肽结构,为α–酮戊二酸和二价铁离子依赖型的加氧酶家族[2OG-Fe(Ⅱ)Oxygenase superfamily]。氨基酸序列与胡桃(ACR47976)、琴叶拟南芥(CAC26921)相似性最高,达到80%。DaF3H在山药幼叶中表达量最高,在成熟叶片和茎中几乎不表达。茎叶和块茎中花青素积累量增速与DaF3H表达密切相关。运用接头PCR法对DaF3H的启动子序列进行了克隆,元件预测显示该序列中存在GT-1光调控元件以及生长素、金属离子、MYB-bZIP-MYC等元件的结合位点,叶片遮光处理试验表明,DaF3H受光调节。
The key enzyme gene DaF3H (KF561995) of anthocyanin biosynthetic pathway was cloned from purple yam “Qianyanzi 006” rich in anthocyanin, with a full length of 1 325 bp and a maximum open reading frame of 1 089 bp, encoding 362 amino acids. DaF3H protein belongs to water-soluble cytoplasmic unstable protein and has no transmembrane domain and signal peptide structure, and is a family of α-ketoglutarate and ferrous iron-dependent oxygenases [2OG-Fe (Ⅱ) Oxygenase superfamily] . The amino acid sequence has the highest similarity to walnuts (ACR47976) and thaliana (CAC26921), reaching 80%. DaF3H was the highest expressed in young leaves of Yam and almost not expressed in mature leaves and stems. The accumulation of anthocyanin in stems, leaves and tubers was closely related to the expression of DaF3H. The promoter sequence of DaF3H was cloned by the linker PCR method. The predicted results showed that GT-1 light regulatory elements and the binding sites of auxin, metal ions, MYB-bZIP-MYC, It shows that DaF3H is regulated by light.