药学学报, 2022, 57(5): 1526-1536
引用本文:
周浩, 路星星, 敖雯雯, 廖海民, 张明生, 强玮*. 钩藤STR基因及其启动子的克隆与分析[J]. 药学学报, 2022, 57(5): 1526-1536.
ZHOU Hao, LU Xing-xing, AO Wen-wen, LIAO Hai-min, ZHANG Ming-sheng, QIANG Wei*. Cloning and analysis of STR gene and its promoter from Uncaria[J]. Acta Pharmaceutica Sinica, 2022, 57(5): 1526-1536.

钩藤STR基因及其启动子的克隆与分析
周浩, 路星星, 敖雯雯, 廖海民, 张明生, 强玮*
贵州大学生命科学学院/农业生物工程研究院, 山地植物资源保护与保护种质创新教育部重点实验室, 山地生态与农业生物工程协同创新中心, 贵州 贵阳 550025
摘要:
根据钩藤转录组中STR基因核心序列设计特异性引物,并进行RACE扩增,克隆到UrSTR基因(GeneBank:OL310251)的cDNA全长1 541 bp,编码345个氨基酸;采用染色体步移克隆到UrSTR基因启动子(GeneBank:OL310252)序列1 179 bp。系统进化发育树分析显示,钩藤UrSTR蛋白与同为茜草科的美丽帽柱木和短小蛇根草的STR蛋白聚为一类,其亲缘关系最近;亚细胞共定位实验表明UrSTR蛋白定位于液泡膜上;SDS-PAGE结果表明pET-28a-UrSTR重组蛋白成功表达且大小与预期相符;启动子顺式作用元件分析表明其含有光响应、胁迫响应和激素响应有关的多种调控元件;启动子活性分析UrSTR基因启动子具有转录活性。成功克隆了UrSTR基因及其启动子序列并对其进行生物信息学分析和启动子活性分析;后期需要优化UrSTR原核表达体系,为进一步纯化UrSTR蛋白研究其结构和功能奠定基础。
关键词:    钩藤      异胡豆苷合成酶      启动子      亚细胞定位      原核表达     
Cloning and analysis of STR gene and its promoter from Uncaria
ZHOU Hao, LU Xing-xing, AO Wen-wen, LIAO Hai-min, ZHANG Ming-sheng, QIANG Wei*
Key Laboratory of Plant Resource Conservation and Germplasm Innovation in Mountainous Region (Ministry of Education), Collaborative Innovation Center for Mountain Ecology & Agro-Bioengineering (CICMEAB), College of Life Sciences/Institute of Agro-Bioengineering, Guizhou University, Guiyang 550025, China
Abstract:
On the basis of the Uncaria transcriptome, specific primers were designed for UrSTR. The full-length cDNA of UrSTR (GeneBank:OL310251) was 1 541 bp, encoding 345 amino acid residues, and the promoter region sequence of UrSTR (GeneBank:OL310252) was 1 179 bp. Phylogenetic tree is revealed that UrSTR had a closest relationship with STR from Ophiorrhiza pumila and Ophiorrhiza japonica. Localization of UrSTR protein is revealed located in the vacuole membrane. Plant-care analysis indicated that the promoter region sequence of UrSTR, covering multiple light, stress and hormone-response cis-regulatory elements, and verified transcriptional activity. The results of SDS-PAGE show that pET-28a-UrSTR recombinant protein was successfully expressed, and the size was anticipated. The UrSTR prokaryotic expression system needs to be optimized in the later stage. The research lays the foundation for further purification to study its structure and functional characterization of the UrSTR protein.
Key words:    Uncaria rhynchophylla    strictosidine synthase    promoter    subcellular localization    prokaryotic expression   
收稿日期: 2021-11-01
DOI: 10.16438/j.0513-4870.2021-1700
基金项目: 贵州省基础研究计划(黔科合基础-ZK[2022]一般096);贵州大学培育项目(贵大培育[2019]51号);贵州大学引进人才科研项目(贵大人基合字[2017]58);贵州省中药材现代产业技术体系建设项目(GZCYTX-02);国家喀斯特石漠化防治工程技术研究中心建设(2012FU125X13);贵州省生物学一流学科建设项目(GNYL[2017]009).
通讯作者: 强玮,Tel:13678505784,E-mail:wayneqiang@126.com
Email: wayneqiang@126.com
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参考文献:
[1] Chinese Pharmacopoeia Commission. Pharmacopoeia of the People's Republic of China (中华人民共和国药典)[S]. Vol 1. Beijing:China Medical Science Press, 2015:257.
[2] Wei GY, Xu CY. Research progress on antihypertensive effects of rhynchophylline and isorynchophylline[J]. China Med Pharm (中国医药科学), 2020, 10:32-36.
[3] Yu X, Zhu LL, Liu J, et al. Research progress on monoterpenoid indole alkaloids in Uncariae Ramulus Cum Uncis and their pharmacological activities[J]. Chin Tradit Herb Drugs (中草药), 2021, 52:6052-6065.
[4] Qin N, Lu X, Liu YJ, et al. Recent research progress of Uncaria spp. based on alkaloids:phytochemistry, pharmacology and structural chemistry[J]. Eur J Med Chem, 2021, 210:112960.
[5] Stöckigt J, Antonchick AP, Wu F, et al. The pictet-spengler reaction in nature and in organic chemistry[J]. Angew Chem Int Ed, 2011, 50:8538-8564.
[6] Wu SW, Yang MQ, Xiao YL. Synthetic biology studies of monoterpene indole alkaloids[J]. Chin J Org Chem (有机化学), 2018, 38:2243-2258.
[7] O'Connor SE, Maresh JJ. Chemistry and biology of monoterpene indole alkaloid biosynthesis[J]. Nat Prod Rep, 2006, 23:532-547.
[8] Kutchan TM. Expression of enzymatically active cloned strictosidine synthase from the higher plant Rauvolfia serpentina in Escherichia coli[J]. FEBS Lett, 1989, 257:127-130.
[9] Treimer J, Zenk M. Purification and properties of strictosidine synthase, the key enzyme in indole alkaloid formation[J]. Eur J Biochem, 1979, 101:225-233.
[10] Yasuyo Y, Hiroshi S, Mami Y, et al. Camptothecin biosynthetic genes in hairy roots of Ophiorrhiza pumila:cloning, characterization and differential expression in tissues and by stress compounds[J]. Plant Cell Physiol, 2003, 44:395-403.
[11] Lu Y, Wang HS, Wang W, et al. Molecular characterization and expression analysis of a new cDNA encoding strictosidine synthase from Ophiorrhiza japonica[J]. Mol Biol Rep, 2009, 36:1845-1852.
[12] Singh S, Kamble SN, Satdive RK, et al. Heterologous overexpression of Nothapodytes foetida strictosidine synthase enhances levels of anti-cancer compound camptothecin in Ophiorrhiza rugosa[J]. Plant Cell Tissue Organ Cult, 2020, 141:67-76.
[13] Zhu YZ, Fan HH, Li DH, et al. Molecular cloning, bioinformation analysis and expression of the strictosidine synthase in Dendrobium officinale[J]. Acta Sci Pol Hort Cult, 2020, 19:111-124.
[14] Canel C, LopesCardoso MI, Whitmer S, et al. Effects of over-expression of strictosidine synthase and tryptophan decarboxylase on alkaloid production by cell cultures of Catharanthus roseus[J]. Planta, 1998, 205:414-419.
[15] Sharma A, Verma P, Mathur A, et al. Overexpression of tryptophan decarboxylase and strictosidine synthase enhanced terpenoid indole alkaloid pathway activity and antineoplastic vinblastine biosynthesis in Catharanthus roseus[J]. Protoplasma, 2018, 255:1281-1294.
[16] Cui LJ, Ni XL, Ji Q, et al. Co-overexpression of geraniol-10-hydroxylase and strictosidine synthase improves anti-cancer drug camptothecin accumulation in Ophiorrhiza pumila[J]. Sci Rep, 2015, 5:8227.
[17] Zhang JG, Chen JJ, Geng CA. Advances in indole alkaloids from traditional Chinese medicine of Uncariae Ramulus Cum Uncis documented in Chinese Pharmacopoeia[J]. China J Chin Mater Med (中国中药杂志), 2019, 44:685-695.
[18] Zhou H, Qiang W, Ao WW, et al. Effect of exogenous abscisic acid on biosynthesis of alkaloids in Uncaria[J]. Mol Plant Breeding (分子植物育种), 2021.
[19] Wang Z, Ye SF, Li JJ, et al. Fusion primer and nested integrated PCR (FPNI-PCR):a new high-efficiency strategy for rapid chromosome walking or flanking sequence cloning[J]. BMC Biotechnol, 2011, 11:109.
[20] Liu YG, Chen YL. High-efficiency thermal asymmetric interlaced PCR for amplification of unknown flanking sequences[J]. Biotechniques, 2007, 43:651-656.
[21] Yu XS, Li X, Wang XH, et al. Screening and stability evaluation of reference genes in Uncaria rhynchophylla qRT-PCR analysis[J]. J Agric Biotechnol (农业生物技术学报), 2021, 29:587-597.
[22] Prashanth S, Christina DS. Engineering a microbial biosynthesis platform for de novo production of tropane alkaloids[J]. Nat Commun, 2020, 585:614-639.
[23] Paddon CJ, Westfall PJ, Pitera DJ, et al. High-level semi-synthetic production of the potent antimalarial artemisinin[J]. Nature, 2013, 496:528-532.
[24] Liu XN, Cheng J, Zhang GH, et al. Engineering yeast for the production of breviscapine by genomic analysis and synthetic biology approaches[J]. Nat Commun, 2018, 9:448.
[25] Xu K, Zhao YJ, Nadeem A, et al. O-Glycosyltransferases from Homo sapiens contributes to the biosynthesis of glycyrrhetic acid 3-O-mono-β-D-glucuronide and glycyrrhizin in Saccharomyces cerevisiae[J]. Synth Syst Biotechnol, 2021, 6:173-179.
[26] Reshma SV, Nitish S, Nagendra HG. Characterization of a hypothetical protein YVRE from Bacillus subtilis indicates its key role as glucono-lactonase in pentose phosphate pathway and glucose metabolism[J]. Bioinformation, 2017, 13:430-438.
[27] Cao N, Wang CH. Strictosidine synthase, an indispensable enzyme involved in the biosynthesis of terpenoid indole and β-carboline alkaloids[J]. China J Nat Med, 2021, 19:591-607.
[28] McCoy E, Galan MC, O'Connor SE. Substrate specificity of strictosidine synthase[J]. Bioorg Med Chem Lett, 2006, 16:2475-2478.
[29] Zou T, Li SC, Li MX, et al. An atypical strictosidine synthase, OsSTRL2, plays key roles in anther development and pollen wall formation in rice[J]. Sci Rep, 2017, 7:6863.
[30] Gray CH, McGarry LC, Spence HJ, et al. Novel beta-propeller of the BTB-Kelch protein Krp1 provides a binding site for Lasp-1 that is necessary for pseudopodial extension[J]. J Biol Chem, 2009, 284:30498-30507.
[31] Luca DV, Cutler AJ. Subcellular localization of enzymes involved in indole alkaloid biosynthesis in Catharanthus roseus[J]. Plant Physiol, 1987, 85:1099-1102.
[32] Miettinen K, Dong L, Navrot N, et al. The seco-iridoid pathway from Catharanthus roseus[J]. Nat Commun, 2014, 5:3606.
[33] Thomas DM, Dnaiel RB, Ronald JB, et al. Expression of enzymatically active and correctly targeted strictosidine synthase in transgenic tobacco plants[J]. Planta, 1991, 185:148-152.
[34] Lu XX, Qiang W, Fu W, et al. Effects of methyl jasmonate on alkaloids biosynthesis in Uncaria rhynchophylla[J]. Mol Plant Breeding (分子植物育种), 2021. https://kns.cnki.net/kcms/detail/46.1068.s.20210603.1049.004.html
[35] Yasuyo Y, Akiko U, Hiroshi S, et al. Metabolite profiling of alkaloids and strictosidine synthase activity in camptothecin producing plants[J]. Phytochemistry, 2003, 62:461-470.
[36] Chang CH, Liu ZW, Wang YY, et al. A bZIP transcription factor, CaLMF, mediated light-regulated camptothecin biosynthesis in Camptotheca acuminata.[J]. Tree Physiol, 2019, 39:372-380.
[37] Bernonville TD, Carqueijeiro I, Lanoue A, et al. Flivory elicits a strong defense reaction in Catharanthus roseus:metabolomic and transcriptomic analyses reveal distinct local and systemic responses[J]. Sci Rep, 2017, 7:40453.
[38] Lu S, Xu R, Jia JW, et al. Cloning and functional characterization of a β-pinene synthase from Artemisia annua that shows a circadian pattern of expression[J]. Plant Physiol, 2002, 130:477-486.
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