|
|
Cloning and Expression Analysis of the GeCYP85A1 Gene in Gelsemium elegans |
ZHANG Yao1, CHEN Wen-Qiang1, PAN Li-Mei2, MU De-Tian1, ZHOU Yu1, LIN Xiao-Dong1, LIU Zhao-Ying3,*, TANG Qi1,* |
1 College of Horticulture/National Research Center of Engineering Technology for Utilization of Botanical Functional Ingredients, Hunan Agricultural University, Changsha 410128, China; 2 Guangxi Botanical Garden of Medicinal Plants, Nanning 510023, China; 3 College of Veterinary Medicine, Hunan Agricultural University, Changsha 410128, China |
|
|
Abstract Gelsemium elegans is a valuable traditional Chinese herb in China. The whole plant is highly toxic, but has anti-tumour and analgesic effects, as well as fattening effects on pigs and other livestock, and its main active constituents are terpenoid indole alkaloids (TIAs). Among them, the most representative ingredient is gelsenicine, which has the strongest toxicity and highest activity. In this study, 209 genes related to cytochrome P450 (CYP450) were screened based on the genomic and transcriptomic data of G.elegans, combined with metabolomic data, an important candidate gene GeCYP85A1 (GenBank No. OQ653843) related to the biosynthesis pathway of Oryza japonica was screened by "genome+expression profile+metabolome". The full-length cloning of this gene was performed by PCR, and its bioinformatics analysis was carried out. The expression pattern of the GeCYP85A1 gene in different tissues of G. elegans was analysed by qPCR. The results showed that the GeCYP85A1 gene was 1 392 bp long, encoding 463 amino acids, with a theoretical relative molecular mass of 53.390 06 kD and a theoretical isoelectric point of 9.07. It was a non-secretory protein with a subcellular localization in chloroplasts, a transmembrane region and no signal peptide. It was similar to Coffea arabica, Coffea eugenioides, Erythranthe guttata, Nicotiana tabacum, Olea europaea subsp. europaea, Phtheirospermum juttata, Salvia miltiorrhiza, Sesamum indicum, and Vitis riparia, and the amino acid sequences of the related CYP85A1 of the above 9 species were highly similar and belonged to a subfamily of the CYP450 family. By qPCR, the expression pattern of GeCYP85A1 in different parts of the plant was found to be consistent with the trend of the relative content of gelsenicine in different parts of the plant, which were root>flower>stem>leaf. This study provides a reference for further elucidation of the biosynthetic pathway for the TIAs-like alkaloids in G. elegans.
|
Received: 08 February 2023
|
|
Corresponding Authors:
* tangqi@hunau.edu.cn; liu_zhaoying@hunau.edu.cn
|
|
|
|
[1] 陈上, 王秀娟, 张逸风, 等. 2018. 雷公藤TwCYP88A1基因全长cDNA克隆与表达分析[J]. 中国实验方剂学杂志, 24(06): 32-36. (Chen S, Wang X J, Zhang Y F, et al.2018.Cloning and bioinformatics analysis of TwCYP88A1 gene in Tripteryguim wilfordii[J]. Chinese Journal of Experimental Traditional Medical Formulae, 24(06): 32-36.) [2] 陈炜, 赵建文, 张智俊, 等. 2019. 竹类植物中CYP85A1基因的克隆及表达分析[J]. 核农学报, 33(04): 653-663. (Chen W, Zhao J W, Zhang Z J, et al.2019. Cloning and expression analysis of CYP85A1 gene in bamboo plants[J]. Journal of Nuclear Agricultural Sciences, 33(04): 653-663.) [3] 陈越, 张青磊, 黄玉香, 等. 2016. 吲哚生物碱生物合成研究进展[J]. 世界科学技术-中医药现代化, 18(11): 1914-1920. (Chen Y, Zhang Q L, Huang Y X, et al.2016. A research progress on the biosynthetic pathways of indole alkaloids[J]. Modernization of Traditional Chinese Medicine and Materia Medica-World Science and Technology, 18(11): 1914-1920.) [4] 程海旭, 章国良. 2015. 甾体激素类药物与细胞色素P450代谢酶系相互作用的研究进展[J]. 中国临床药理学杂志, 31(13): 1328-1330. (Cheng H X, Zhang G L.2015.Progress in research of the interaction between the steroid hormone drugs and cytochrome P450 metabolic enzymes[J]. The Chinese Journal of Clinical Pharmacology, 31(13): 1328-1330.) [5] 韩博伦, 肖清铁, 文欢欢, 等. 2021. 紫苏分支酸合成酶PeCS基因克隆、生物信息学及原核表达分析[J]. 分子植物育种, 1-11. http://kns.cnki.net/kcms/detail/46.1068.S.20 210709.1531.027.html. (Han B L, Xiao Q T, Wen H H, et al.2021. Cloning, bioinformatics and prokaryotic express analysis of gene encoding chorismate synthase in Perilla frutescens[J]. Molecular Plant Breeding, 1-11. http://kns.cnki.net/kcms/detail/46.1068.S.20210709.153 1. 027.html [6] 何芳, 李国泽, 丁勇, 等. 2020. 樟叶越桔CYP450基因片段克隆与家族分析[J]. 分子植物育种, 18(02): 347-357. (He F, Li G Z, Ding Y, et al.2020. Cloning and family analysis of CYP450 gene fragment from Vaccinium dunalianum Wight[J]. Molecular Plant Breeding, 18(02): 347-357.) [7] 黄可馨, 罗丹, 王慧丰, 等. 2022. 基于细胞色素P450通路的前列腺癌差异关键基因的筛选与验证[J]. 广西医科大学学报, 39(01): 42-48. (Huang K X, Luo D, Wang H F, et al.2022. Screening and validation of differential key genes in prostate cancer based on cytochrome P450 pathway[J]. Journal of Guangxi Medical University, 39(01): 42-48.) [8] 李永康, 马雪祺, 冯婧娴, 等. 2021. 细胞色素P450酶在植物次生代谢产物生物合成中的研究进展[J]. 分子植物育种, 1-8. http://kns.cnki.net/kcms/detail/46.1068.S.20210707.1636.022.html. (Li Y K, Ma X Q, Feng J X, et al.2021. Advances of cytochrome P450s in the biosynthesis of plant secondary metabolites[J]. Molecular Plant Breeding, 1-8. http://kns.cnki.net/kcms/detail/46.1068.S.20210707.1636.022.html [9] 刘虹虹, 梁燕妮, 陈超杰. 2021. 对广西地区钩吻生物碱成分的含量检测分析研究[J]. 中国食品工业, (Z1): 116-118. (Liu H H, Liang Y N, Chen C J. 2021. Determination and analysis of alkaloids in Gelsemium elegans in Guangxi[J]. China Food Industry, (Z1): 116-118.) [10] 马小毛, 宁书菊, 叶齐, 等. 2020. 马蓝色氨酸合成酶基因的克隆及表达分析[J]. 中草药, 51(24): 6328-6336. (Ma X M, Ning S J, Ye Q, et al.2020. Cloning and expression analysis of tryptophan synthase gene BcTSB in Baphicacanthus cusia[J]. Chinese Traditional and Herbal Drugs, 51(24): 6328-6336.) [11] 穆德添, 万凌云, 韦树根, 等. 2022. 钩藤不同部位总RNA提取及UrSTR基因的克隆与表达分析[J]. 农业生物技术学报, 30(09): 1737-1746. (Mu D T, Wan L Y, Wei S G, et al.2022. Extraction of total RNA and cloning and expression analysis of UrSTR gene from Uncaria rhynchophylla[J]. Journal of Agricultural Biotechnology, 30(09): 1737-1746.) [12] 覃宏婷, 龚道勇, 李标. 2022. 石斛碱生物合成途径及萜类化合物CYP450酶的研究进展[J]. 重庆工商大学学报(自然科学版), 39(06): 1-13. (Qin H T, Gong D Y, Li B.2022. Research progress on dendrobine biosynthetic pathway and terpenoid CYP450 enzymes[J]. Journal of Chongqing Technology and Business University (Natural Science Edition), 39(06): 1-13.) [13] 宋恩峰, 张彩碟. 2017. 钩吻现代功效研究进展[J]. 现代中药研究与实践, 31(05): 74-77. (Song E F, Zhang C D.2017. Research progress of Gelsemium elegans[J]. Research and Practice on Chinese Medicines, 31(05): 74-77.) [14] 孙铭学, 徐庆强, 孟文琪, 等. 2020. 钩吻药理及毒理机制研究进展[J]. 毒理学杂志, 34(04): 336-341. (Sun M X, Xu Q Q, Meng W Q, et al.2020. Research progress on pharmacology and toxicological mechanism of Gelsemium elegans[J]. Journal of Toxicology, 34(4): 336-341.) [15] 滕林佐, 陈丽莉, 何含杰, 等. 2020. 四季桂香叶醇合成酶基因GES的克隆及表达模式分析[J]. 分子植物育种, 18(06): 1825-1831. (Teng L Z, Chen L L, He H J, et al.2020. Cloning and analysis of expression pattern of geranyllinalool synthases (GES) gene in Osmanthus fragans var. semperflorens[J]. Molecular Plant Breeding, 18(06): 1825-1831.) [16] 王昭玉, 封润霞,苏智, 等. 2020. 沙棘HrCYP85A1基因生物信息及表达分析[J]. 分子植物育种, 18(15): 4952-4957. (Wang Z Y, Feng R X, Su Z, et al.2020.Bioinformatics and expression analysis of HrCYP85A1 gene in Seabuckthorn[J]. Molecular Plant Breeding, 18(15): 4952-4957.) [17] 杨成佳, 成旭, 胡冰, 等. 2023. P450酶在植物三萜化合物生物合成中的催化与调控[J]. 生物加工过程, 21(01): 39-49. (Yang C J, Cheng X, Hu B, et al.2023. The catalysis and regulation of cytochrome P450 enzymes in plant triterpenoids biosynthesis[J]. Chinese Journal of Bioprocess Engineering, 21(01): 39-49.) [18] 杨然, 方磊, 李佳, 等. 2018. 环烯醚萜苷类生物合成途径及相关酶的研究进展[J]. 中草药, 49(10): 2482-2488. (Yang R, Fang L, Li J, et al.2018.Research progress on biosynthetic pathways and related enzymes of iridoid glycosides[J]. Chinese Traditional and Herbal Drugs, 49(10): 2482-2488.) [19] 袁志航, 邬静, 孙志良. 2015. 钩吻研究进展[J]. 中兽医医药杂志, 34(03): 26-28. (Yuan Z H, Wu J, Sun Z H.2015.Research and development on Gelsemium elegans Benth.[J]. Journal of Traditional Chinese Veterinary Medicine, 34(03): 26-28.) [20] 张翼, 邵冬南, 薛飞, 等. 2021. 陆地棉细胞色素P450超家族基因GhCYP85A2-1的克隆与功能分析[J]. 新疆农业科学, 58(02): 197-205. (Zhang Y, Shao D N, Xue F, et al.2021. Gossypium hirsutum cytochrome P450 superfamily gene cloning and functional analysis of GhCYP85A2-1[J]. Xinjiang Agricultural Sciences, 58(02): 197-205.) [21] 章瑶, 穆德添, 周宇, 等. 2022. 钩吻看家基因筛选及生物碱合成相关酶基因的表达分析[J]. 生物工程学报, 39(1): 286-303. (Zhang Y, Mu D T, Zhou Y, et al.2022. Screening of housekeeping genes in Gelsemium elegans and expression patterns of genes involved in its alkaloid biosynthesis[J]. Chinese Journal of Biotechnology, 39(1): 286-303.) [22] 赵雅婷, 武淑鹏, 胡春丽, 等. 2019. 钩吻的化学成分及药理作用研究进展[J]. 中国实验方剂学杂志, 25(03): 200-210. (Zhao Y T, Wu S P, Hu C L, et al.2019. Reviews on chemical compositions and pharmacological effect of Gelsemium elegans[J]. Chinese Journal of Experimental Traditional Medical Formulae, 25(3): 200-210.) [23] Allison M H, Tamil C M S, Berin A B, et al.2018. Biosynthesis of bioactive diterpenoids in the medicinal plant Vitex agnus-castus[J]. The Plant Journal, 93(5): 943-958. [24] Forman V, Luo D, Geu F F, et al.2022. A gene cluster in Ginkgo biloba encodes unique multifunctional cytochrome P450s that initiate ginkgolide biosynthesis[J]. Nature Communications, 13(01): 5143-5143. [25] Guengerich F P.2019. Cytochrome P450 research and the journal of biological chemistry[J]. The Journal of Biological Chemistry, 294(5): 1671-1680. [26] Huang C Y, Yang K, Cao J J, et al.2021. Integration of metabolomics and transcriptomicsto comprehensively evaluate the metabolic effects of Gelsemium elegans on pigs[J]. Animals (Basel), 11(5): 1192. [27] Huang S J, Zuo M T, Qi X J, et al.2021. Phosphoproteomics reveals NMDA receptor-mediated excitotoxicity as a key signaling pathway in the toxicity of gelsenicine[J]. Food and Chemical Toxicology, 156: 112507. [28] Lin H L, Qiu H Q, Cheng Y, et al.2021. Gelsemium elegans Benth: Chemical components, pharmacological effects, and toxicity mechanisms[J]. Molecules, 26(23): 7145. [29] Liu M, Shen J, Liu H, et al.2011. Gelsenicine from Gelsemium elegans attenuates neuropathic and inflammatory pain in mice[J]. Biological and Pharmaceutical Bulletin, 34(12): 1877-1880. [30] Liu Y S, Tang Q, Cheng P, et al.2020. Whole-genome sequencing and analysis of the Chinese herbal plant Gelsemium elegans[J]. Acta Pharmaceutica Sinica B, 10(2): 374-382. [31] Negar G, Ayman O S E.2004. Expression of cytochrome P450 in lung tumor[J]. Current Drug Metabolism, 5(2): 203-210. [32] Pang X Y, Tang C Z, Guo R C, et al.2021.Non-cytochrome P450 enzymes involved in the oxidative metabolism of xenobiotics: Focus on the regulation of gene expression and enzyme activity[J]. Pharmacology & Therapeutics, 233: 108020. [33] Pérez-España V H, Sánchez-León N, Vielle-Calzada J.2011. CYP85A1 is required for the initiation of female gametogenesis in Arabidopsis thaliana[J]. Plant Signaling & Behavior, 6(3): 321-326. [34] Prema S K, Puja D, J Bennett A, et al.2019. Functional characterization of the cytochrome P450 monooxygenase CYP71AU87 indicates a role in marrubiin biosynthesis in the medicinal plant Marrubium vulgare[J]. BMC Plant Biology, 19(1): 114. [35] Rodríguez Arcas M J, García-Jiménez E, Martínez-Martínez F., et al.2010. Role of CYP450 in pharmacokinetics and pharmacogenetics of antihypertensive drugs[J]. Farmacia Hospitalaria, 35(02): 84-92. [36] Sonam T, Gaurav A, Punita L, et al.2014. Significant role of CYP450 genetic variants in cyclophosphamide based breast cancer treatment outcomes: A multi-analytical strategy[J]. Clinica Chimica Acta, 434: 21-28. [37] Tang Q, Ma X J, Mo C M, et al.2011. An eficient approach to finding Siraitia grosvenorii triterpene biosynthetic genes by RNA-seq and digital gene expression analysis[J]. BMC Genomics, 12: 343. [38] Wang J F, Liu Y L, Cai Y F, et al.2010. Cloning and functional analysis of geraniol 10-hydroxylase, a cytochrome P450 from Swertia mussotii Franch[J]. Bioscience, Biotechnology, and Biochemistry, 74(8): 1583-1590. [39] Zhang J S, Dai L H, Yang J G, et al.2016. Oxidation of cucurbitadienol catalyzed by CYP87D18 in the biosynthesis of mogrosides from Siraitia grosvenorii[J]. Plant Cell Physiology, 57(5): 1000-1007. |
|
|
|