千里光橙花叔醇合酶SsNES的功能鉴定
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摘要
通过对千里光转录组数据筛选得到一个较短的萜类合酶基因。对其进行系统进化树和序列比对分析,初步确定为一个橙花叔醇合酶,命名为SsNES (GenBank登录号MH518312)。通过蛋白同源建模表明SsNES虽然序列较短,但具有完整的保守功能域,并且能正确折叠。通过对该基因的克隆,原核表达载体的构建,成功在大肠杆菌中表达出可溶性蛋白;用大肠杆菌代谢工程鉴定SsNES的体外催化功能,结果表明SsNES可以催化反式法尼烯焦磷酸(FPP)生成反式橙花叔醇。在千里光茎叶的提取物中也检测到反式橙花叔醇,表明该基因作为橙花叔醇合酶在千里光中行使功能;通过RT-PCR表达模式的分析,SsNES在茎中表达最高,其次是花、叶,在根中不表达;在水杨酸(SA)、茉莉酸甲酯(MeJA)、丙甲菌素(Ala)的处理后该基因均能在6 h时就被诱导表达,表明该基因可能参与到千里光应对胁迫响应的防御过程中。SsNES生化功能的鉴定不仅为倍半萜合酶的研究提供多样性,也解析了千里光中橙花叔醇的生物合成,为千里光萜类化合物介导的病虫害防御提供理论基础。
A short terpene synthase gene was obtained by screening the transcriptome data of Senecio scandens. The phylogenetic tree and sequence alignment putatively identified this gene as a nerolidol synthase gene, named SsNES(GenBank MH518312). Protein homology modeling indicated that SsNES contained a complete conserved domain and folded correctly. SsNES was cloned and successfully expressed in Escherichia coli as soluble protein. The biochemical function of SsNES was characterized by E. coli metabolic engineering, which showed that SsNES catalyzed formation of trans-nerolidol with(E, E)-farnesyl diphosphate as the substrate. Nerolidol was also detected in stems and leaves of S. scandens, indicating that SsNES might act as the nerolidol synthase in plant. RT-PCR analysis indicated that SsNES was mainly expressed in stem, flowers and leaves, and no expression was observed in roots. After the treatment of SA, MeJA or Ala, SsNES was induced significantly at 6 h, indicating involvement in the defense response of S. scandens. The identification of SsNES not only clarified biosynthesis of nerolidol in S. scandens, but also provided diversity of sesquiterpene synthase, as well as theoretical basis for disease and pest defense mediated by the terpene metabolites.
A short terpene synthase gene was obtained by screening the transcriptome data of Senecio scandens. The phylogenetic tree and sequence alignment putatively identified this gene as a nerolidol synthase gene, named SsNES(GenBank MH518312). Protein homology modeling indicated that SsNES contained a complete conserved domain and folded correctly. SsNES was cloned and successfully expressed in Escherichia coli as soluble protein. The biochemical function of SsNES was characterized by E. coli metabolic engineering, which showed that SsNES catalyzed formation of trans-nerolidol with(E, E)-farnesyl diphosphate as the substrate. Nerolidol was also detected in stems and leaves of S. scandens, indicating that SsNES might act as the nerolidol synthase in plant. RT-PCR analysis indicated that SsNES was mainly expressed in stem, flowers and leaves, and no expression was observed in roots. After the treatment of SA, MeJA or Ala, SsNES was induced significantly at 6 h, indicating involvement in the defense response of S. scandens. The identification of SsNES not only clarified biosynthesis of nerolidol in S. scandens, but also provided diversity of sesquiterpene synthase, as well as theoretical basis for disease and pest defense mediated by the terpene metabolites.
引文
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[15] Starks C M, Back K, Chappell J, et al. Structural basis for cyclic terpene biosynthesis by tobacco 5-epi-aristolochene synthase[J]. Science, 1997, 277: 1815.
[16] Ke Zhou, Yang Gao, Hoy J A, et al. Insights into diterpene cyclization from structure of bifunctional abietadiene synthase from Abies grandis[J]. J Biol Chem, 2012, 287(9): 6840.
[17] Hillwig M L, Xu M, Toyomasu T, et al. Domain loss has independently occurred multiple times in plant terpene synthase evolution[J]. Plant J, 2011, 68(6): 1051.
[18] Cane D E, Sohng J K, Lamberson C R, et al. Pentalenene synthase. Purification, molecular cloning, sequencing, and high-level expression in Escherichia coli of a terpenoid cyclase from Streptomyces UC5319[J]. Biochemistry,1994, 33(19): 5846.
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[20] Li G, Kollner T G, Yin Y, et al. Nonseed plant Selaginella moellendorffi has both seed plant and microbial types of terpene synthases[J]. Proc Natl Acad Sci USA, 2012, 109: 14711.
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[22] Tamogami S, Takahashi Y, Abe M, et al. Conversion of airborne nerolidol to DMNT emission requires additional signals in Achyranthes bidentata[J]. FEBS Lett, 2011, 585(12): 1807.
[2] 赵宏. 千里光属及其近缘属倍半萜类化合物研究[D]. 济南:山东大学, 2013.
[3] 王锋鹏. 现代天然产物化学[M]. 北京:科学出版社,2009: 402.
[4] 王凌健, 方欣, 杨长青, 等. 植物萜类次生代谢及其调控[J]. 中国科学, 2013, 43(12): 1030.
[5] Vranova E, Coman D, Gruissem W. Network analysis of the MVA and MEP pathways for isoprenoid synthesis[J]. Annu Rev Plant Biol, 2013, 64: 665.
[6] Wendt K U, Schulz G E. Isoprenoid biosynthesis: manifold chemistry catalyzed by similar enzymes [J]. Structure, 1998, 6(2): 127.
[7] Peters R J, Carter O A, Zhang Y, et al. Bifunctional abietadiene synthase: mutual structural dependence of the active sites for protonation-initiated and ionization-initiated cyclizations[J]. Biochemistry, 2003, 42(9): 2700.
[8] Koksal M, Hu H, Coates R M, et al. Structure and mechanism of the diterpene cyclase ent-copalyl diphosphate synthase[J]. Nat Chem Biol, 2011, 7(7): 431.
[9] Zhou K, Peters R J. Investigating the conservation pattern of a putative second terpene synthase divalent metal binding motif in plants[J]. Phytochemistry, 2009, 70(3): 366.
[10] Zi J, Mafu S, Peters R J. To gibberellins and beyond! Surveying the evolution of (di)terpenoid metabolism[J]. Ann Rev Plant Biol, 2014, 65: 259.
[11] Martin V J, Pitera D J, Withers S T, et al. Engineering a mevalonate pathway in Escherichia coli for production of terpenoids[J]. Nat Biotechnol, 2003, 21: 796.
[12] Liang J, Liu J, Wang Q, et al. Direct production of dihydroxylated sesquiterpenoids by a maize terpene synthase [J]. Plant J, 2018, 94: 847.
[13] Lee S, Badieyan S, Bevan D R, et al. Herbivore-induced and floral homoterpene volatiles are biosynthesized by a single P450 enzyme (CYP82G1) in Arabidopsis[J]. Proc Natl Acad Sci USA, 2010, 107(49): 21205.
[14] 梁瑾, 王强. 大豆橙花叔醇合酶基因克隆及功能鉴定[J]. 中国油料作物学报, 2015(6): 744.
[15] Starks C M, Back K, Chappell J, et al. Structural basis for cyclic terpene biosynthesis by tobacco 5-epi-aristolochene synthase[J]. Science, 1997, 277: 1815.
[16] Ke Zhou, Yang Gao, Hoy J A, et al. Insights into diterpene cyclization from structure of bifunctional abietadiene synthase from Abies grandis[J]. J Biol Chem, 2012, 287(9): 6840.
[17] Hillwig M L, Xu M, Toyomasu T, et al. Domain loss has independently occurred multiple times in plant terpene synthase evolution[J]. Plant J, 2011, 68(6): 1051.
[18] Cane D E, Sohng J K, Lamberson C R, et al. Pentalenene synthase. Purification, molecular cloning, sequencing, and high-level expression in Escherichia coli of a terpenoid cyclase from Streptomyces UC5319[J]. Biochemistry,1994, 33(19): 5846.
[19] Proctor R H, Hohn T M. Aristolochene synthase. Isolation, characterization, and bacterial expression of a sesquiterpenoid biosynthetic gene (Ari1) from Penicillium roqueforti[J]. J Biol Chem, 1993, 268(6): 4543.
[20] Li G, Kollner T G, Yin Y, et al. Nonseed plant Selaginella moellendorffi has both seed plant and microbial types of terpene synthases[J]. Proc Natl Acad Sci USA, 2012, 109: 14711.
[21] Tholl D, Sohrabi R, Huh J H, et al.The biochemistry of homoterpenes-common constituents of floral and herbivore-induced plant volatile bouquets[J]. Phytochemistry, 2011, 72(13): 1635.
[22] Tamogami S, Takahashi Y, Abe M, et al. Conversion of airborne nerolidol to DMNT emission requires additional signals in Achyranthes bidentata[J]. FEBS Lett, 2011, 585(12): 1807.