药学学报, 2021, 56(2): 630-638
丁宁, 海燕, 王晓晖, 屠鹏飞, 高博闻, 史社坡. 白木香查尔酮异构酶基因的克隆鉴定与表达分析[J]. 药学学报, 2021, 56(2): 630-638.
DING Ning, HAI Yan, WANG Xiao-hui, TU Peng-fei, GAO Bo-wen, SHI She-po. Cloning and expression analysis of chalcone isomerase from Aquilaria sinensis [J]. Acta Pharmaceutica Sinica, 2021, 56(2): 630-638.

丁宁1, 海燕1, 王晓晖1, 屠鹏飞1, 高博闻2, 史社坡1
1. 北京中医药大学中药学院中药现代研究中心, 北京 100029;
2. 包头医学院, 内蒙古 包头 014060
查尔酮异构酶(chalcone isomerase,CHI)是黄酮类成分生物合成途径中的关键酶之一,在植物防御反应中发挥重要作用。本研究根据白木香转录组测序结果并结合RT-PCR技术首次从白木香愈伤组织中克隆得到1个CHI基因,命名为AsCHI1。白木香AsCHI1基因的开放阅读框(ORF)长654 bp,编码蛋白含217个氨基酸,其蛋白分子质量为23.11 kDa。AsCHI1蛋白具有查尔酮异构酶保守的活性位点,系统进化树显示AsCHI1蛋白为I型CHI蛋白,与棉花(Gossypium hirsutum) CHI蛋白亲缘关系较近。构建原核表达载体pET28a-AsCHI1并在E.coli BL21(DE3)菌株中成功表达AsCHI1,利用Ni2+亲和色谱纯化得到可溶性AsCHI1重组蛋白。体外酶活性分析证明重组蛋白AsCHI1可以催化柚皮素查尔酮转化为柚皮素。实时荧光定量PCR检测结果表明白木香愈伤组织经盐、甘露醇、低温以及重金属胁迫诱导后,AsCHI1基因的表达量明显上升;植物激素脱落酸、赤霉素和水杨酸均能够诱导愈伤组织中AsCHI1基因表达,说明AsCHI1在白木香自我防御反应中发挥作用。本研究结果为进一步探讨白木香中黄酮类成分的生物合成及其在白木香防御反应中的作用提供参考。
关键词:    白木香      沉香      查尔酮异构酶      克隆鉴定      表达分析     
Cloning and expression analysis of chalcone isomerase from Aquilaria sinensis
DING Ning1, HAI Yan1, WANG Xiao-hui1, TU Peng-fei1, GAO Bo-wen2, SHI She-po1
1. Modern Research Center for Traditional Chinese Medicine, School of Chinese Materia Medica, Beijing University of Chinese Medicine, Beijing 100029, China;
2. Baotou Medical College, Baotou 014060, China
Chalcone isomerases (CHIs) play an essential role in the biosynthesis of flavonoids important in plant self-defense. Based on the transcriptome data of Aquilaria sinensis Calli, a full-length cDNA sequence of CHI1 (termed as AsCHI1) was cloned by reverse transcription PCR. AsCHI1 contains a complete open frame (ORF) of 654 bp. The deduced protein is composed of 217 amino acids, with a predicted molecular weight of 23.11 kDa. The sequence alignment and phylogenetic analysis revealed that AsCHI1 has conserved most of the active site residues in type I CHIs, indicating a close relationship with the CHI from Gossypium hirsutum. The recombinant AsCHI1 protein was obtained by heterologous expression of AsCHI1 in E. coli BL21(DE3). The purified AsCHI1 protein exhibited CHI activity by catalyzing the production of naringenin from naringenin chalcone. Remarkably, AsCHI1 expression in A. sinensis Calli treated with various abiotic stresses including salt, mannitol, cold, and heavy metals could be markedly increased, and plant hormones such as abscisic acid (ABA), gibberellin (GA3), and salicylic acid (SA) could also increase the expression of AsCHI1, suggesting that AsCHI1 might play an important role in plant self-defense. The results expand our understanding of the biosynthesis of flavonoids in A. sinensis and give further insight into the defensive responses of A. sinensis to abiotic and biotic stresses.
Key words:    Aquilaria sinensis    agarwood    chalcone isomerase    gene cloning and function determination    expression analysis   
收稿日期: 2020-10-09
DOI: 10.16438/j.0513-4870.2020-1588
基金项目: 国家自然科学基金资助项目(81773842).
通讯作者: 高博闻,E-mail:shishepo@163.com;史社坡,E-mail:gaobw001@163.com
Email: shishepo@163.com;gaobw001@163.com
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海燕  在本刊中的所有文章
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屠鹏飞  在本刊中的所有文章
高博闻  在本刊中的所有文章
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[1] Chinese Pharmacopoeia Commission. Pharmacopoeia of the People's Republic of China (中华人民共和国药典:一部)[S]. Beijing:China Medical Science Press, 2015.
[2] Zhang Z, Yang Y, Wei JH, et al. Advances in studies on mechanism of agarwood formation in Aquilaria sinensis and its hypothesis of agarwood formation induced by defense response[J]. Chin Tradit Herb Drugs (中草药), 2010, 41:156-159.
[3] Li L, Zhang GQ, Shi XF. Advances in pharmacological research and clinical application of agarwood[J]. Asia-Pac Tradit Med (亚太传统医药), 2019, 15:199-204.
[4] Zheng SZ, Zheng PJ, Yang RH, et al. Historical and cultural accumulation of agarwood[J]. Chaoshan Businessman (潮商), 2010, 2:66-71.
[5] Persoon GA, Van Beek HH. Growing' the Wood of the Gods':agarwood production in southeast Asia[M]//Snelder DJ, Lasco RD, editors. Smallholder Tree Growing for Rural Development and Environmental Service:Lessons from Asia. Netherlands:Springer, 2008, 5:245-262.
[6] CITES. Amendments to Appendix I and II of CITES[C]. Bangkok:Proceedings of Thirteenth Meeting of the Conference of the Parties, 2004:2-14.
[7] Wei JH, Yang Y, Zhang Z, et al. Artificial liquid transfusion technology in inducing agarwood formation in the whole body of cultivated Aquilaria sinensis (Lour.) Gilg trees:CN, 201010104119.5[P]. 2010-06-30.
[8] Zhang XL, Liu YY, Wei JH, et al. Production of high-quality agarwood in Aquilaria sinensis trees via whole-tree agarwood induction technology[J]. Chin Chem Lett, 2012, 23:727-730.
[9] Kumeta Y, Ito M. Characterization of δ-guaiene synthases from cultured cells of Aquilaria, responsible for the formation of the sesquiterpenes in agarwood[J]. Plant Physiol, 2010, 154:1998-2007.
[10] Ito M, Okimoto KI, Yagura T, et al. Induction of sesquiterpenoid production by methyl jasmonate in Aquilaria sinensis cell suspension culture[J]. J Essent Oil Res, 2005, 17:175-180.
[11] Naef R. The volatile and semi-volatile constituents of agarwood, the infected heartwood of Aquilaria species:a review[J]. Flavour Fragr J, 2011, 26:73-89.
[12] Xu YH, Liao YC, Zhang Z, et al. Jasmonic acid is a crucial signal transducer in heat shock induced sesquiterpene formation in Aquilaria sinensis[J]. Sci Rep, 2016, 6:21843.
[13] Zhang Z, Zhang XL, Yang Y, et al. Hydrogen peroxide induces vessel occlusions and stimulates sesquiterpenes accumulation in stems of Aquilaria sinensis[J]. Plant Growth Regul, 2014, 72:81-87.
[14] Ye W, He X, Wu HQ, et al. Identification and characterization of a novel sesquiterpene synthase from Aquilaria sinensis:an important gene for agarwood formation[J]. Int J Biol Macromol, 2018, 108:884-892.
[15] Xu YH, Zhang Z, Wang MX, et al. Identification of genes related to agarwood formation:transcriptome analysis of healthy and wounded tissues of Aquilaria sinensis[J]. BMC Genomics, 2013, 14:227.
[16] Wang XH, Gao BW, Liu X, et al. Salinity stress induces the production of 2-(2-phenylethyl)chromones and regulates novel classes of responsive genes involved in signal transduction in Aquilaria sinensis calli[J]. BMC Plant Biol, 2016, 16:119.
[17] Ye W, Wu HQ, He X, et al. Transcriptome sequencing of chemically induced Aquilaria sinensis to identify genes related to agarwood formation[J]. PLoS One, 2016, 11:e0155505.
[18] Yang MX. Flavonoids from Wild Aquilaria sinensis Leaves, Their Anti-cancer Activities and the Preliminary Studies on Chemical Intervention Effect in Agarwood Formation Induced by Wounding (野生白木香叶中的黄酮类化合物, 抗癌活性及对伤害诱导结香化学干预作用初步研究)[D]. Jinan:Jinan University, 2016.
[19] Jez JM, Bowman ME, Dixon RA, et al. Structure and mechanism of the evolutionarily unique plant enzyme chalcone isomerase[J]. Nat Struct Biol, 2000, 7:786-791.
[20] Cheng AX, Zhang XB, Han XJ, et al. Identification of chalcone isomerase in the basal land plants reveals an ancient evolution of enzymatic cyclization activity for synthesis of flavonoids[J]. New Phytol, 2018, 217:909-924.
[21] Sun MJ, Wang HJ, Ding FJ, et al. Qualitative analysis of flavonoids in Tetrastigma hemsleyanum based on UPLC-Quadrupole/Exactive Orbitrap mass spectrometry and mass defect filter method[J]. J Chin Mass Spectr Soc, 2020, 41:359-367.
[22] Ribeiro IA, Ribeiro MHL. Naringin and naringenin determination and control in grapefruit juice by a validated HPLC method[J]. Food Control, 2008, 19:432-438.
[23] Tunen AJ, Koes RE, Spelt CE, et al. Cloning of the two chalcone flavanone isomerase genes from Petunia hybrida:coordinate, light-regulated and differential expression of flavonoid genes[J]. EMBO J, 1988, 7:1257-1263.
[24] Kimura Y, Aoki T, Ayabe S. Chalcone isomerase isozymes with different substrate specificities towards 6'-hydroxyand 6'-deoxychalcones in cultured cells of Glycyrrhiza echinata, a leguminous plant producing 5-deoxyflavonoids[J]. Plant Cell Physiol, 2001, 42:1169-1173.
[25] Shimada N, Aoki T, Sato S, et al. A cluster of genes encodes the two types of chalcone isomerase involved in the biosynthesis of general flavonoids and legume-specific 5-deoxy(iso) flavonoid in Lotus Japonicus[J]. Plant Physiol, 2003, 131:941-951.
[26] Zhu H, Hu HY, Lu CY, et al. Progresses on flavonoid metabolism in plants and its regulation[J]. J Xiamen Univ (厦门大学学报), 2007, 46:136-143.
[27] Xu WY, Gao WW, He CN. The influences of environmental factors on flavonoid biosynthesis[J]. World Sci Technol/Mod Tradit Chin Med Mater Med (世界科学技术一中医药现代化), 2006, 8:68-72.
[28] Chen D, Bi D, Song YL, et al. Flavanoids from the stems of Aquilaria sinensis[J]. Chin J Nat Med, 2012, 10:287-291.
[29] Peng K, Mei WL, Wu J. Flavones from the stem of Aquilaria sinensis[J]. J Trop Subtrop Bot (热带亚热带植物学报), 2010, 18:97-100.
[30] Lin HZ, Li HN, Mei QX, et al. Research progress of agarwood leaves[J]. Pharm Today (今日药学), 2011, 21:547-549.
[31] Zhang HC, Liu JM, Lu HY, et al. Enhanced flavonoid production in hairy root cultures of Glycyrrhiza uralensis Fisch by combining the over-expression of chalcone isomerase gene with the elicitation treatment[J]. Plant Cell Rep, 2009, 28:1205-1213.
[32] Park NI, Xu H, Li XH, et al. Enhancement of flavone levels through overexpression of chalcone isomerase in hairy root cultures of Scutellaria baicalensis[J]. Funct Integr Genomics, 2011, 11:491-496.
[33] Christie PJ, Alfenito MR, Walbot V. Impact of low-temperature stress on general phenylpropanoid and anthocyanin pathways:enhancement of transcript abundance and anthocyanin pigmentation in maize seedlings[J]. Planta (Heidelberg), 1994, 194:541-549.
[34] Wang H, Hu TJ, Huang JZ, et al. The expression of Millettia pinnata chalcone isomerase in Saccharomyces cerevisiae salt-sensitive mutants enhances salt-tolerance[J]. Int J Mol Sci, 2013, 14:8775-8786.
[35] Chen HQ, Wei JH, Yang JS, et al. Chemical constituents of agarwood originating from the endemic genus Aquilaria plants[J]. Chem Biodivers, 2012, 9:236-250.
[36] Yagura T, Ito M, Kiuchi F, et al. Four new 2-(2phenylethyl) chromone derivatives from withered wood of Aquilaria sinensis[J]. Chem Pharm Bull, 2003, 51:560-564.
[37] Ibrahim SR, Mohamed GA. Natural occurring 2-(2-phenylethyl) chromones, structure elucidation and biological activities[J]. Nat Prod Res, 2015, 29:1489-1520.
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