青脆枝細胞色素P450及還原酶基因表達分析及喜樹鹼代謝工程之研究

Abstract

由青脆枝篩選得到的3個細胞色素P450還原酶cDNA基因，分別命名為NfCPR1、NfCPR2及NfCPR3。其演繹之胺基酸序列，具有典型細胞色素P450還原酶的特徵，包括保守flavin adenine dinucleotide (FAD)結合區、flavin mononucleotide (FMN)結合區、nicotinamide adenine dinucleotide phosphate (NADPH)結合區和細胞色素P450結合區。序列親緣性演化樹分析顯示NfCPR1屬於第一群，而NfCPR2和NfCPR3屬於第二群。NfCPR1轉錄層次分布在各個器官，種子有較高基因表現，而NfCPR2轉錄層次也分布在各個器官，於葉子有較高基因表現，NfCPR3以花苞和種子有較高基因表現。NfCPR1基因表現受到NAA、 6-BA、GA3、ABA、SA和MeJA抑制，NfCPR2基因表現也受NAA、6-BA和MeJA抑制，但受2,4-D誘導，而NfCPR3基因表現輕微受到SA和MeJA抑制，卻受GA3和ABA促進增加基因表現，NfCPR1基因表現不受創傷影響，NfCPR2/3基因表現受創傷影響。 將青脆枝細胞色素P450基因，參與生物鹼代謝的Nf3-12、Nf5-14、Nf5-30、基因和參與類黃酮代謝的Nf1-25、Nf1-26、Nf1-39、Nf1-44、Nf3-76的基因及日日春之geraniol 10 hydroxylase (CrG10H) 基因，分別搭配青脆枝細胞色素P450還原酶基因NfCPR2和阿拉伯芥還原酶基因AtCPR，經由桿狀病毒表現系統，異源表現於昆蟲細胞。高效液相層析儀和質譜儀分析，證明CrG10H具有利用narigenin和geraniol為基質之雙重催化能力，進一步將CrG10H和AtCPR共同表達，則可增加對narigenin之催化能力，酵素動力參數kcat / Km比值指出，催化narigenin效率約為催化geraniol的1/10，顯示CrG10H基因除了是萜類生合成關鍵酵素，也參與苯基丙酸類的生物合成。另將NfCPR2與CrG10H共同表現，可輔助CrG10H催化narigenin生成eriodictyol，在含有0.5 mM NADPH的條件下反應，其酵素活性可達17.49 nmol/h/mg，若不添加NADPH，則無法偵測產物的活性，顯示NfCPR2為需NADPH型的細胞色素P450還原酶。 喜樹鹼(camptothecin, CPT)是很重要的抗癌藥物，商業生產喜樹鹼可由青脆枝提煉，青脆枝中喜樹鹼含量以根較高(0.864 mg/g 乾物重)，10-羥基喜樹鹼(10-hydroxycamptothecin, HCPT)含量以種子較高(0.096 mg/g乾物重)，不同發育階段果實，喜樹鹼含量最高的時期是成熟果中種殼，葉片中含量較多的成分為CPT和9-甲氧基喜樹鹼(9-Methoxycamptothecin, MCPT)，已知喜樹鹼生合成路徑需要細胞色素P450和細胞色素P450還原酶參與，將青脆枝之細胞色素P450基因Nf2-16、Nf3-12、Nf4-15、Nf5-30和細胞色素P450還原酶NfCPR，以基因槍法轉殖於喜樹葉片進行暫時性表現，結果僅轉殖細胞色素P450基因即可提升喜樹葉片中CPT含量，表示青脆枝之Nf2-16、Nf3-12、Nf4-15和Nf5-30可能參與喜樹鹼代謝；喜樹葉片和癒傷組織轉殖，轉殖條件農桿菌濃度OD600值為0.5、acetosyringone濃度100 μM、Pluronic acid F68濃度0.02 %、共培養3天有較佳轉殖率。使用含細胞色素P450基因Nf2-16質體之農桿菌品系A281感染轉殖喜樹無菌苗葉片，轉殖後2個月挑選均質的轉殖細胞系測定CPT和HCPT，轉殖細胞系CPT含量皆高於未轉殖細胞，最高可增加5倍(0.58 μg/g乾重)，顯示青脆枝細胞色素Nf2-16在喜樹鹼生合成途徑扮演重要角色。

Three unique NADPH:cytochrome P450 reductase (CPR) cDNAs, whose corresponding genes designed as NfCPR1~3 from Nothapodytes nimmoniana have been characterized. The encoded proteins contained the conserved CPR functional domains including the flavin adenine dinucleotide (FAD)-, flavin mononucleotide (FMN)-, nicotinamide adenine dinucleotide phosphate (NADPH)-, and P450-binding motifs. Phylogenetic analysis showed that NfCPR1 is a class I isoform, whereas NfCPR2 and NfCPR3 are class II isoforms. NfCPR1 transcripts were detected in all examined organs, the highest level being in the seeds. NfCPR2 transcripts were also present in all examined organs but predominantly in leaves. In contrast, NfCPR3 transcripts were only detected in flower buds and seeds at almost equal expression levels. NfCPR1 gene expression was repressed by NAA, 6-BA, GA3, ABA, SA, and MeJA. NfCPR2 gene expression was also repressed by NAA, 6-BA, and MeJA but was induced by 2,4-D. NfCPR3 gene expression was slightly inhibited by SA, MeJA, and 2,4-D and was induced by GA3 and ABA. Moreover, NfCPR1 expression did not change during wounding treatment, whereas NfCPR2 and NfCPR3 were induced in response to wounding. The cytochrome P450 gene CrG10H from Catharanthus roseus and cytochrome P450 reductase NfCPR2 from Nothapodytes nimmoniana, AtCPR from Arabidopsis thaliana were cloned and heterologously expressed in baculovirus-infected insect cells. As reported in a previous study, CrG10H hydroxylated the monoterpenoid geraniol at the C-10 position to generate 10-hydroxygeraniol. Interestingly, CrG10H also catalyzed 3’-hydroxylation of naringenin to produce eriodictyol. Co-expression of an Arabidopsis NADPH P450 reductase substantially increased the ability of CrG10H to hydroxylate naringenin. The catalytic activity of CrG10H was approximately 1/10 times efficient with naringenin than with geraniol, judged by the kcat/Km values. Thus, G10H also plays an important role in the biosynthetic pathway of flavonoids, in addition to its previously described role in the metabolism of terpenoids. when the recombinant NfCPR2 protein was coexpressed with CrG10H and NADPH was added into the reaction, the production of eriodictyol from naringein increased and CrG10H activity reached 17.49 nmol/h/mg. However, no activity of CrG10H was detectable if NADPH was not added. This result indicates that the NfCPR2 efficiently interacts with cytochrome P450 to transfer electrons from NADPH. Camptothecin (CPT) derivatives are clinically used anti-neoplastic alkaloids and are extracted from Nothapodytes nimmoniana for commercial production. The highest content of CPT and 10-hydroxycamptothecin (HCPT) in different tissues of Nothapodytes nimmoniana was 0.864 mg/g dry weight in the roots and 0.096 mg/g dry weight in seeds, respectively. During fruit development, the highest aboundance of CPT was detected in seed coat from a mature fruit. The major camptothecin-related alkaloids accumulated in leaf of Nothapodytes nimmoniana were CPT and MCPT. Since cytochrome P450 monooxygenases were known to have function in association with cytochrome P450 reductases (CPR), overexpression vectors containing Nothapodytes nimmoniana cytochrome P450 gene, Nf2-16, Nf3-12, Nf4-15, Nf5-30 with or without CPR were transferred into C. acuminata by particle bombardment on leaf discs as transient expression analysis for CPT and HCPT content. The most abundant accumulation of CPT was detected in all leaf discs transformed with overexpression vectors without NfCPR. The transgenic callus containing Nf2-16 genes via Agrobacterium method were confirmed by polymerase chain reaction analysis. High-performance liquid chromatography analysis revealed that the CPT and HCPT contents were higher after transformation. The transformed calli contain 1.24~2.19 μg CPT /g dry weight, nearly 2~5 folds increase over the non-transformed calli (0.44~0.58 μg/g dry weight). It indicates that Nf2-16 might play an important role in camptothecin biosythesis.