Identification of a biosynthetic gene cluster in rice for momilactones
Identification of a biosynthetic gene cluster in rice for momilactones
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DOI：
10.1074/jbc.m703344200
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发表时间：
2007-11-23
影响因子：
4.8
通讯作者：
Yamane, Hisakazu
中科院分区：
文献类型：
--
作者：
Shimura, Kazuhiro;Okada, Atsushi;Yamane, Hisakazu
水稻二萜类植物抗毒素，例如莫米内酯和植物卡桑，是在用几丁质寡糖引发剂处理的悬浮培养的水稻细胞中以及在用紫外线照射的稻叶中产生的。共同底物香叶基香叶基二磷酸通过两步连续环化转化为二萜烃前体，然后通过几个氧化步骤转化为生物活性植物抗毒素。有人提出，微粒体细胞色素 P-450 单加氧酶 (P-450s) 参与二萜烃的下游氧化，产生植物抗毒素，并且脱氢酶参与莫米内酯的生物合成。然而，尚未鉴定出参与二萜烃下游氧化的酶。在这项研究中，我们发现一个假定的脱氢酶基因 (AK103462) 和两个功能未知的 P-450 基因 (CYP99A2 和 CYP99A3) 与参与莫米内酯生物合成的二萜环化酶基因 OsKS4 和 OsCyc1 一起形成几丁质寡糖引发剂和紫外线诱导基因簇。通过在大肠杆菌中进行异源表达和随后的酶测定进行的功能分析表明，AK103462 蛋白可催化 3 β-羟基-9 β H-pimara-7,15-dien-19,6 β-olide 转化为 momilactone A。CYP99A2 和 CYP99A3 的双重敲低特异性抑制了激发子诱导的产生 莫米内酯，强烈表明 CYP99A2、CYP99A3 或两者都参与莫米内酯生物合成。这些结果为 4 号染色体上存在参与莫米内酯生物合成的基因簇提供了有力的证据。
Rice diterpenoid phytoalexins such as momilactones and phytocassanes are produced in suspension-cultured rice cells treated with a chitin oligosaccharide elicitor and in rice leaves irradiated with UV light. The common substrate geranylgeranyl diphosphate is converted into diterpene hydrocarbon precursors via a two-step sequential cyclization and then into the bioactive phytoalexins via several oxidation steps. It has been suggested that microsomal cytochrome P-450 monooxygenases (P-450s) are involved in the downstream oxidation of the diterpene hydrocarbons leading to the phytoalexins and that a dehydrogenase is involved in momilactone biosynthesis. However, none of the enzymes involved in the downstream oxidation of the diterpene hydrocarbons have been identified. In this study, we found that a putative dehydrogenase gene (AK103462) and two functionally unknown P-450 genes (CYP99A2 and CYP99A3) form a chitin oligosaccharide elicitor- and UV-inducible gene cluster, together with OsKS4 and OsCyc1, the diterpene cyclase genes involved in momilactone biosynthesis. Functional analysis by heterologous expression in Escherichia coli followed by enzyme assays demonstrated that the AK103462 protein catalyzes the conversion of 3 beta-hydroxy- 9 beta H-pimara-7,15-dien-19,6 beta-olide into momilactone A. The double knockdown of CYP99A2 and CYP99A3 specifically suppressed the elicitor-inducible production of momilactones, strongly suggesting that CYP99A2, CYP99A3, or both are involved in momilactone biosynthesis. These results provide strong evidence for the presence on chromosome 4 of a gene cluster involved in momilactone biosynthesis.