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本研究利用RACE技术从海南粗榧(Cephalataxus mannii)中克隆出紫杉烷13α-羟化酶基因。该基因含有一个1 488 bp长的开放阅读框(ORF),可编码495个氨基酸,该序列所编码氨基酸属于P450蛋白超家族系统,存在明显的疏水峰,含有线粒体靶向肽。进化树分析显示,该基因所编码的蛋白与紫杉醇合成途径中6种酰基转移酶来自同一个祖先,其中与紫杉烷13α-羟基化酶基因同源性最高,属于同一进化分支。实时荧光定量表达分析表明,紫杉烷13α-羟化酶基因在被茉莉酸甲酯(MeJA)诱导后的海南粗榧中表达量变化显著,处理后0~12 h目的基因表达量变化不明显,12~24 h目的基因表达量剧增,达到最大值,形成明显波峰。结果表明紫杉醇的合成可能与植物体抵御逆境胁迫存在重要关联。
In this study, taxane 13α-hydroxylase gene was cloned from Cephalataxus mannii by RACE technique. The gene contains an open reading frame (ORF) of 1 488 bp, which encodes 495 amino acids. The amino acids encoded by this sequence belong to the P450 superfamily system and show obvious hydrophobic peaks and contain mitochondrial targeting peptides. Phylogenetic tree analysis showed that the protein encoded by this gene came from the same ancestor with the six acylases in the taxol biosynthesis pathway, which shared the highest homology with the taxane 13α-hydroxylase gene and belong to the same evolutionary branch. The results of real-time fluorescence quantitative analysis showed that the expression level of taxane 13α-hydroxylase gene was significantly changed after being induced by methyl jasmonate (MeJA), but the expression of target gene did not change significantly from 0 to 12 h after treatment , 12 ~ 24 h target gene expression increased dramatically, reaching the maximum, the formation of significant peaks. The results showed that the synthesis of paclitaxel may be related to plant stress tolerance.