Overexpression of Transcription Factor TwWRKY1 Gene Increased the Content of Wilforgine and Wilforine in Tripterygium wilfordii
YANG Li-Li1,*, XI Wen-Jing1,*, HE Ze-Dong1, CHENG Ling1, MA Zhi-Qing1,2, ZHANG Bin1,2, ZHU Chuan-Shu1,2,**
1 College of Plant Protection, Northwest A&F University, Yangling 712100, China; 2 Engineering and Technology Centers of Bio-pesticidein Shaanxi, Yangling 712100, China
Abstract：The methyl jasmonate (MeJA)-induced TwWRKY1 (GenBank No. GAVZ01022264.1) gene apparently regulated the biosynthesis of wilforgine in Tripterygium wilfordii. To elucidate the regulation mechanism and key target gene, the T. wilfordii sesquiterpene cyclase (Twses-CS) was selected for further exploring, the Twses-CS was screened as the key enzyme involved in the sesquiterpene biosynthesis pathway, the cis-acting element was analyzed in the Twses-CS promoter, and the expression of the TwWRKY1 and alkaloid content changes were detected. The results showed that the WRKY transcription factor might regulate the biosynthesis of sesquiterpene-pyridine alkaloids through regulating Twses-CS gene in T. wilfordii. Yeast one hybrid (Y1H) assays revealed that TwWRKY1 binded to the promoter of Twses-CS. Subcellular localization analysis indicated that the TwWRKY1 was located in the nucleus, consistent with its role as a typical transcription factor. Electrophoretic mobility shift assays (EMSA) further confirmed that TwWRKY1 formed a DNA-protein complex with a 30 bp probe containing 2 W-box sequences, indicating its binding ability to the W-box element. Overexpression of TwWRKY1 in hairy roots resulted in a significant increase in wilforgine and wilforine content about 1.87 and 1.90 times compared with the control, the content of wilforgine and wilforine was 249.53 and 638.25 μg/g DW, respectively. These results suggested that TwWRKY1 likely regulated the content of sesquiterpene-pyridine alkaloids in T. wilfordii by binding to the W-box element in the promoter of Twses-CS. This study provides basis for further exploration of alkaloid biosynthesis pathway and the transcription regulatory mechanism for sesquiterpene alkaloids, and provides a theoretical basis for using metabolic engineering and synthetic biology ways to solve the alkaloid shortage.
[1] 陈玲. 2021. 雷公藤TwWRKY1和TwWRKY5提高倍半萜生物碱含量的初步研究[D]. 硕士学位论文, 西北农林科技大学, 导师: 祝传书, pp. 1-28. (Chen L.2021. Preliminary study on increasing sesquiterpene alkaloid content by TwWRKY1 and TwWRKY5 in Tripterygium wilfordii[D]. Thesis for M.S., Northwest A&F University, Supervisor: Zhu C S, pp. 1-28.) [2] 张斌. 2020. 雷公藤萜类活性物质生物合成途径环化酶研究[D]. 博士学位论文, 西北农林科技大学, 导师: 张兴, pp. 23-37. (Zhang B.2020. Study on the cyclases involved in the biosynthesis pathway of the active terpenoids in Tripterygium wilfordii[D]. Thesis for Ph. D., Northwest A&F University, Supervisor: Zhang X, pp. 23-37.) [3] 祝传书, 刘艳, 陈蒙蒙, 等. 2018. 雷公藤转录因子TwWRKY1基因的克隆与表达分析[J]. 农业生物技术学报, 26(04): 595-605. (Zhu C S, Liu Y, Chen M M, et al., 2018. Cloning and expression analysis of transcription factor TwWRKY1 gene in Tripterygium wilfordii[J]. Journal of Agricultural Biotechnology, 26(04): 595-605.) [4] Beroza M.1952. Alkaloids from Tripterygium wilfordii Hook: Wilforgine and wilfortrine[J]. Journal of the American Chemical Society, 74(6): 1585-1588. [5] Brinker A M, Ma J, Lipsky P E, et al.2007. Medicinal chemistry and pharmacology of genus Tripterygium (Celastraceae)[J]. Phytochemistry, 68(6): 732-766. [6] Cao W, Wang Y, Shi M, et al.2018. Transcription factor SmWRKY1 positively promotes the biosynthesis of tanshinones in Salvia miltiorrhiza[J]. Frontiers in Plant Science, 9: 554. [7] Chen F, Tholl D, Bohlmann J, et al.2011. The family of terpene synthases in plants: A mid-size family of genes for specialized metabolism that is highly diversified throughout the kingdom[J]. Plant Journal, 66(1): 212-229. [8] Chen M, Yan T, Shen Q, et al.2017. GLANDULAR TRICHOME-SPECIFIC WRKY1 promotes artemisinin biosynthesis in Artemisia annua[J]. New Phytologist, 214(1): 304-316. [9] Hansen N L, Heskes A M, Hamberger B, et al.2017. The terpene synthase gene family in Tripterygium wilfordii harbors a labdane-type diterpene synthase among the monoterpene synthase TPS-b subfamily[J]. Plant Journal, 89(3): 429-441. [10] Hansen N L, Kjaerulff L, Heck Q K, et al.2022. Tripterygium wilfordii cytochrome P450s catalyze the methyl shift and epoxidations in the biosynthesis of triptonide[J]. Nature Communications, 13: 5011. [11] Hao X L, Xie C H, Ruan Q Y, et al.2021. The transcription factor OpWRKY2 positively regulates the biosynthesis of the anticancer drug camptothecin in Ophiorrhiza pumila[J]. Horticulture Research, 8: 7. [12] Huang X, Zhang W, Liao Y, et al.2024. Contemporary understanding of transcription factor regulation of terpenoid biosynthesis in plants[J]. Planta, 259(1): 2. [13] Kato N, Dubouzet E, Kokabu Y, et al.2007. Identification of a WRKY protein as a transcriptional regulator of benzylisoquinoline alkaloid biosynthesis in Coptis japonica[J]. Plant Cell Physiology, 48(1): 8-18. [14] Lange B M, Fischedick J T, Lange M F, et al.2017. Integrative approaches for the identification and localization of specialized metabolites in Tripterygium roots[J]. Plant Physiology, 173(1): 456-469. [15] Li J, Huang H C, Zuo Y Q, et al.2024. PatWRKY71 transcription factor regulates patchoulol biosynthesis and plant defense response[J]. BMC Plant Biology, 24: 8. [16] Li S, Zhang P, Zhang M, et al.2013. Functional analysis of a WRKY transcription factor involved in transcriptional activation of the DBAT gene in Taxus chinensis[J]. Plant Biology (Stuttg), 15(1): 19-26. [17] Lião L M.2003. SESQUITERPENE PYRIDINE ALKALOIDS, The Alkaloids: Chemistry and Biology[M]. Academic Press, Pittsburgh, 60: 287-343. [18] Lv H, Jiang L, Zhu M, et al.2019. The genus Tripterygium: A phytochemistry and pharmacological review[J]. Fitoterapia, 137: 104190. [19] Ma D, Pu G, Lei C, et al.2009. Isolation and characterization of AaWRKY1, an Artemisia annua transcription factor that regulates the amorpha-4,11-diene synthase gene, a key gene of artemisinin biosynthesis[J]. Plant Cell Physiology, 50(12): 2146-2161. [20] Ma Z Q, Li YJ, Wu L P, et al.2014. Isolation and insecticidal activity of sesquiterpenes alkaloids from Tripterygium wilfordii Hook f.[J]. Industrial Crops and Products, 52:642-648. [21] Miao G P, Li W, Zhang B, et al.2015. Identification of genes involved in the biosynthesis of Tripterygium wilfordii Hook. f. secondary metabolites by suppression subtractive hybridization[J]. Plant Molecular Biology Reporter, 33(4): 756-769. [22] Schluttenhofer C, Yuan L.2015. Regulation of specialized metabolism by WRKY transcription factors[J]. Plant Physiology, 167(2): 295-306. [23] Shi M, Zhang S, Zheng Z, et al.2024. Molecular regulation of the key specialized metabolism pathways in medicinal plants[J]. Journal of Integrative Plant Biology, 66(3): 510-531. [24] Song H, Cao Y P, Zhao L G, et al.2023. Review: WRKY transcription factors: Understanding the functional divergence[J]. Plant Science, 334: 111770. [25] Suttipanta N, Pattanaik S, Kulshrestha M, et al.2011. The transcription factor CrWRKY1 positively regulates the terpenoid indole alkaloid biosynthesis in Catharanthus roseus[J]. Plant Physiology, 157(4): 2081-2093. [26] Tong Y R, Su P, Guan H Y, et al.2018. Eudesmane-type sesquiterpene diols directly synthesized by a sesquiterpene cyclase in Tripterygium wilfordii[J]. Biochemical Journal, 475(17): 2713-2725. [27] Tu L, Su P, Zhang Z, et al.2020. Genome of Tripterygium wilfordii and identification of cytochrome P450 involved in triptolide biosynthesis[J]. Nature Communications, 11: 971. [28] Wang C, Hao XL, Wang Y, et al.2022. Identification of WRKY transcription factors involved in regulating the biosynthesis of the anti-cancer drug camptothecin in Ophiorrhiza pumila[J]. Horticulture Research, 9(1): uhac099. [29] Xu Y H,Wang J W,Wang S, et al.2004. Characterization of GaWRKY1, a cotton transcription factor that regulates the sesquiterpene synthase gene (+)-delta-cadinene synthase-A[J]. Plant Physiology, 135(1): 507-515. [30] Yu H, Guo W, Yang D, et al.2018. Transcriptional profiles of SmWRKY family genes and their putative roles in the biosynthesis of tanshinone and phenolic acids in Salvia miltiorrhiza[J]. International Journal of Molecular Science, 19(6): 1593. [31] Zerbe P, Hamberger B, Yuen M M, et al.2013. Gene discovery of modular diterpene metabolism in nonmodel systems[J]. Plant Physiology, 162(2): 1073-1091. [32] Zhang Y, Gao J, Ma L, et al.2023. Tandemly duplicated CYP82Ds catalyze 14-hydroxylation in triptolide biosynthesis and precursor production in Saccharomyces cerevisiae[J]. Nature Communications, 14: 875. [33] Zhu C S, Miao G P, Guo J, et al.2014. Establishment of Tripterygium wilfordii Hook.f. hairy root culture and optimization of its culture conditions for the production of triptolide and wilforine[J]. Journal of Microbiology and Biotechnology, 24(6): 823-834.
