青脆枝中拓樸異構酶I基因的選殖及分子特性分析

本研究以青脆枝（Nothapodytes nimmoniana）為試驗材料，其為一台灣原生植物，全株皆具喜樹鹼（camptothecin，CPT），喜樹鹼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，目前除在青脆枝中發現外，在喜樹（Camptotheca acuminata）、白花蛇根草（Ophiorrhiza pumila）等植物都具有喜樹鹼，其之所以具有抗癌活性是因它會和拓樸異構酶I（topoisomerase I，Top1）及去氧核醣核酸分子形成複合體，此複合體可干擾DNA拓樸異構酶之作用機制，以達到抗癌效果。不論是人類、酵母菌或是植物體中，皆具有拓樸異構酶I，而喜樹鹼可干擾人體癌細胞的拓樸異構酶I之活性，但是卻不會干擾具喜樹鹼植物自己本身的拓樸異構酶I，故本研究從青脆枝當中選殖出一個拓樸異構酶I，簡稱NnTopl，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變異（Asn421Lys，Leu530Ile），其可能與青脆枝的抵抗喜樹鹼機制有關。並利用南方墨點法確認NnTopl於青脆枝基因體中至少有三個拷貝數；喜樹鹼主要累積在根、種子及樹皮中，而利用即時聚合酶連鎖反應（Real time PCR，qPCR）技術進行組織表現量的分析結果顯示樹皮中有最多NnTopl mRNA累積。

關鍵字

青脆枝；喜樹鹼；拓樸異構酶I

並列摘要

Nothapodytes nimmoniana, a native plant in Taiwan containing Camptothecin (CPT) was used in this study. 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. The antitumor activity of CPT is ascribed to their ability to stabilize the covalent binding of topoisomerase I (Top 1) to DNA (the cleavable complex) forming a reversible ternary complex. Top1 sequences have been identifying in all living organisms. Intriguingly, the enzymatic activity of Top 1 in CPT-producing plants would not be altered by the self-produced CPT. In this study, a topoisomerase I cDNA was cloned from N. nimmoniana. Two shift sites (Asn421Lys, Leu530Ile) existing in topoisomerase I of N. nimmoniana were identified. Southern blot analysis indicated that there are at least 3 copies of NnTop1 in the N. nimmoniana genome. Gene expression analysis of NnTop1 in different tissues in N nimmoniana revealed that NnTop1 is highly expressed in bark tissues. Analysis of CPT content showed that CPT is mainly accumulated in root, seed and bark of N. nimmoniana.

並列關鍵字

Nothapodytes nimmoniana ； camptothecin ； topoisomerase I

國際替代計量

青脆枝中拓樸異構酶I基因的選殖及分子特性分析

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