青脆枝中喜樹鹼生合成基因的表現與其拓樸異構酶I的胺基酸序列分析

Abstract

喜樹鹼（camptothecin, CPT）是一種單萜類吲哚生物鹼，並且是一種目前已廣泛應用在臨床上的抗癌物質，目前發現在喜樹（Camptotheca acuminata）、白花蛇根草（Ophiorrhiza pumila）、青脆枝（Nothapodytes nimmoniana）等植物都具有喜樹鹼，而在青脆枝根部樹皮則具有最高含量的喜樹鹼，但青脆枝中喜樹鹼的生合成卻仍未被完全解析，故本研究從青脆枝當中選殖出四個喜樹鹼生合成相關基因，anthranilate synthase （NnASA）、tryptophan decarboxylase （NnTDC）、cytochrome P450 reductase （NnCPR）、secologanin synthase（NnSLS）及一個拓樸異構酶I（topoisomerase I，NnTop1）。利用離葉系統進行不同的處理研究，當以25 oC以上的高溫或施加1 μM BA的處理，皆可使四個喜樹鹼生合成基因表現量提高；當施加200 μM甲基茉莉酸（MeJA）處理，NnASA、NnTDC及NnCPR表現量會受到誘導，但NnSLS則不受其誘導；而高濃度的氯化鈉溶液處理則會抑制NnASA、NnTDC及NnCPR基因的表現，亦會造成喜樹鹼的累積銳減。 本研究同時發現青脆枝的拓樸異構酶I在喜樹鹼的作用區域中出現兩個胺基酸位置突變（Asn421Lys, Leu530Ile），其可能與青脆枝的抵抗喜樹鹼機制有關。 本研究結果除了可提供青脆枝生產喜樹鹼的最適生長條件之外，亦可作為日後研究喜樹鹼與拓樸異構酶I交互作用的參考依據。

Camptothecin (CPT), a monoterpene indole alkaloid, is an important anticancer compound that has been obtained in several plants including Camptotheca acuminata, Ophiorrhiza pumila and Nothapodytes nimmoniana. Current report revealed that the highest level of CPT was detected in root bark of N. nimmoniana, but the CPT biosynthesis in N. nimmoniana is still unclear. In this study, four CPT biosynthetic related genes, including anthranilate synthase (NnASA), tryptophan decarboxylase (NnTDC)，cytochrome P450 reductase (NnCPR), secologanin synthase (NnSLS) and a topoisomerase I were cloned from N. nimmoniana. The expression profiling of CPT-related genes were monitored under several treatments for disclosing the pivotal regulatory network. Four CPT - related genes were all induced when plant grew under the warmer ambient temperature condition (> 25 oC). 6-benzyladenine (BA), a kind of cytokinin, is active plant growth regulator to elevate the expression level of all CPT - related genes. NnASA、NnTDC and NnCPR but not NnSLS were induced by 200 μM methyl jasmonate but reduced by high concentration of sodium chloride (salt). Moreover, the CPT amount in leaf treated with high concentration of salt also decreased significantly. Two mutation sites (Asn421Lys, Leu530Ile) existing in topoisomerase I of N. nimmoniana were identified. This result suggested that the CPT-resistant mechanism in N. nimmoniana. Our results revealed the CPT-biosynthesis and resistance through genetic and molecular network and provided a perspective for cultivating N. nimmoniana.