Abstract：Salvia miltiorrhiza is a traditional Chinese herbal medicine, and its bioactive ingredients mainly include tanshinones and salvianolic acids. In order to explore the effects of multi-gene co-overexpression on the growth and accumulation of active substances in S. miltiorrhiza, in this study, the tissue expression and protein subcellular localization of cobacyl pyrophosphate synthetase gene 1 (SmCPS1) and cytochrome P450 monooxygenase gene (SmCYP76AH1) were analyzed, and pYLTAC380H-SmCPS1-SmCYP76AH1, an co-overexpression vector of SmCPS1 and SmCYP76AH1, was constructed using a multi-gene stacking expression system, and then the overexpression vector was transformed to S. miltiorrhiza clean seedlings by Agrobacterium tumefaciens-mediated method. Through callus induction culture, resistance and green fluorescence signal screening, genome integration identification and gene transcription level detection after transformation, double-gene co-overexpressed S. miltiorrhiza lines were obtained, and the content of tanshinone, photosynthetic and fluorescence characteristics of the double-gene co-overexpression lines were further determined. qRT-PCR showed that SmCPS1 and SmCYP76AH1 were expressed in the roots, stems and leaves of S. miltiorrhiza, both of which were highly expressed in the roots. Subcellular localization analysis showed that SmCPS1 was localized in chloroplasts and cytoplasm, and SmCYP76AH1 was localized in endoplasmic reticulum. The contents of tanshinone Ⅰ, tanshinone ⅡA and cryptotanshinone in roots of SmCPS1 and SmCYP76AH1 double-gene co-overexpression lines were extremely significantly increased (P≤0.01). Photosynthetic and fluorescence characteristics of leaves showed that the transpiration rate and stomatal conductance decreased significantly in SmCPS1 and SmCYP76AH1 co-overexpression lines, and the intercellular CO2 concentration (Ci) and net photosynthetic rate (Pn) varied with the expression level of SmCPS1 and SmCYP76AH1. The initial fluorescence (F0), maximum fluorescence (Fm) and non-photochemical quenching coefficient (NPQ) increased significantly in co-overexpressed lines, but the maximum photochemical efficiency (Fv/Fm) of photosystem Ⅱ (PSⅡ) remained unchanged. The multigene transformation method was established in this study and the double-gene co-overexpression lines were obtained which provides experimental materials and methods for further research on the regulation of tanshinone, and provide reference for exploring the function of of SmCPS1 and SmCYP76AH1 in the above ground part of S. miltiorrhiza.
[1] 白朕卿. 2018. 丹参酮合成关键基因SmCPS1与SmKSL1转录调控的研究[D]. 博士学位论文, 西北农林科技大学, 导师: 梁宗锁, pp.71-84. (Bai Z Q.2018. Study on the transcriptional regulation of SmCPS1 and SmKSL1 are involved in tanshinone biosynthesis[D]. Thesis for Ph. D., Northwest Agriculture and Forestry University, Supervisor: Z S. pp. 57(1): 71-84.) [2] 陈根云, 陈娟, 许大全. 2010. 关于净光合速率和胞间CO2浓度关系的思考[J]. 植物生理学通讯, 46(01): 64-66. (Chen G Y, Chen J, Xue D Q.2010. Thinking about the relationship between net photosynthetic rate and intercellular CO2 concentration[J]. Plant Physiology Communication, 46(01): 64-66.) [3] 华俊豪, 李光, 王义权. 2018. 文昌鱼中一个2A肽介导的多基因表达载体构建[J]. 厦门大学学报(自然科学版), 57(1): 44-49. (Hua J H, Li G, Wang Y Q.2018. Construction of a 2A peptide-linked multicistronic expression vector for amphioxus[J]. Journal of Xiamen University (Natural Science), 57(1): 44-49.) [4] 刘昊天, 赵婧, 唐勋, 等. 2023. 马铃薯StbZIP10基因生物信息学及其锌胁迫下的表达分析[J]. 农业生物技术学报, 31(11): 2221-2230. (Liu H T, Zhao J, Tang X, et al.2023. Bioinformatics and expression analysis of StbZIP10 gene under zinc stress in potato[J]. Journal of Agricultural Biotechnology, 31(11): 2221-2230.) [5] 张换样, 李静, 朱永红, 等. 2021. 丹参叶柄遗传转化体系的建立及EDT1基因的导入[J]. 甘肃农业大学学报, 56(01): 66-71+84. (Zhang H Y, Li J, Zhu Y H, et al.2021. Establishment of genetic transformation system for petiole of Salvia miltiorrhiza and introduction of EDT1 gene[J]. Journal of Gansu Agricultural University, 56(01): 66-71+84.) [6] Bai Y, Zhou Y, Lei Q, et al.2023. Analysis of the HD-Zip Ⅰ transcription factor family in Salvia miltiorrhiza and functional research of SmHD-Zip12 in tanshinone synthesis[J]. PeerJ, 11: e15510. [7] Chappell J, Wolf F, Proulx J, et al.1995. Is the reaction catalyzed by 3-hydroxy-3-methylglutaryl coenzyme a reductase a rate-limiting step for isoprenoid biosynthesis in plants?[J]. Plant Physiology, 109(4): 1337-1343. [8] Cheng Q, Su P, Hu Y, et al.2014. RNA interference-mediated repression of SmCPS (copalyldiphosphate synthase) expression in hairy roots of Salvia miltiorrhiza causes a decrease of tanshinones and sheds light on the functional role of SmCPS[J]. Biotechnology Letters, 36(2): 363-369. [9] Contreras A, Leroy B, Mariage P A, et al.2019. Proteomic analysis reveals novel insights into tanshinones biosynthesis in Salvia miltiorrhiza hairy roots[J]. Scientific Reports, 9(1): 5768. [10] Cui G, Duan L, Jin B, et al.2015. Functional divergence of diterpene syntheses in the medicinal plant Salvia miltiorrhiza[J]. Plant Physiology, 169(3): 1607-1618. [11] Dai Z, Cui G, Zhou S F, et al.2011. Cloning and characterization of a novel 3-hydroxy 3-methylglutaryl coenzyme A reductase gene from Salvia miltiorrhiza involved in diterpenoid tanshinone accumulation[J]. Plant Physiology, 168(2): 148-157. [12] Gao W, Hillwig M L, Huang L, et al.2009. A functional genomics approach to tanshinone biosynthesis provides stereochemical insights[J]. Organic Letters, 11(22): 5170-5173. [13] Guo J, Ma X, Cai Y, et al.2016. Cytochrome P450 promiscuity leads to a bifurcating biosynthetic pathway for tanshinones[J]. New Phytologist, 210(2): 525-534. [14] Guo J, Zhou, Y J, Hillwig, M L, et al.2013. CYP76AH1 catalyzes turnover of miltiradiene in tanshinones biosynthesis and enables heterologous production of ferruginol in yeasts[J]. Proceedings of the National Academy of Sciences of the USA, 110(29): 12108-12113. [15] Hao G, Shi R, Tao R, et al.2013. Cloning, molecular characterization and functional analysis of 1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate reductase (HDR) gene for diterpenoid tanshinone biosynthesis in Salvia miltiorrhiza[J]. Plant Physiology and Biochemistry, 70(9): 21-32. [16] Kai G, Xu H, Zhou C, et al.2011. Metabolic engineering tanshinone biosynthetic pathway in Salvia miltiorrhiza hairy root cultures[J]. Metabolic Engineering, 13(3): 319-327. [17] Lange B M, Croteau R.1999. Isoprenoid biosynthesisvia a mevalonate-independent pathway in plants: Cloningand heterologous expression of 1-deoxy-D-xylulose-5-phosphate reductoisomerase from peppermint[J]. Archives of Biochemistry and Biophysics, 365(1): 170-174. [18] Li C X, Zhang J G, Ren Z Y, et al.2021. Development of 'multiresistance rice' by an assembly of herbicide, insect and disease resistance genes with a transgene stacking system[J]. Pest Management Science, 77(3): 1536-1547. [19] Li Q, Fang X, Zhao Y, et al.2023. The SmMYB36-SmERF6/SmERF115 module regulates the biosynthesis of tanshinones and phenolic acids in Salvia miltiorrhiza hairy roots[J]. Horticulture Research, 10(1): 238. [20] Ma Y, Ma X H, Meng F Y, et al.2016. RNA interference targeting CYP76AH1 in hairy roots of Salvia miltiorrhiza reveals its key role in the biosynthetic pathway of tanshinones[J]. Biochemical and Biophysical Research Communications, 477(2): 155-160. [21] Newman J D, Chappell J.1999. Isoprenoid biosynthesis in plants: Carbon partitioning within the cytoplasmic pathway[J]. Critical Reviews in Biochemistry and Molecular Biology, 34(2): 95-106. [22] Pulido P, Perello C, Rodriguez-Concepcion M.2012. New insights into plant isoprenoid metabolism[J]. Molecular Plant, 5(3): 964-967. [23] Rohmer M, Knani M, Simonin P, et al.1993. Isoprenoid biosynthesis in bacteria: A novelpathway for the early steps leading to isopentenyl diphosphate[J]. The Biochemical Journal, 295(2): 517-524. [24] Shi M, Luo X, Ju G, et al.2014. Increased accumulation of the cardio-cerebrovascular disease treatment drug tanshinone in Salvia miltiorrhiza hairy roots by the enzymes 3-hydroxy-3-methylglutaryl CoA reductase and 1-deoxy-dxylulose 5-phosphate reductoisomerase[J]. Functional and Integrative Genomics, 14(3): 603-615. [25] Shi M, Luo X, Ju G, et al.2016. Enhanced diterpene tanshinone accumulation and bioactivity of transgenic Salvia miltiorrhiza hairy roots by pathway engineering[J]. Journal of Agricultural and Food Chemistry, 64(12): 2523-2530. [26] Xu H, Song J, Luo H, et al.2016. Analysis of the genome sequence of the medicinal plant Salvia miltiorrhiza[J]. Molecular Plant, 9(6): 949-952. [27] Xue Y F, Fu C, Chai C Y, et al.2023. Engineering the staple oil crop brassica napus enriched with α-linolenic acid using the Perilla FAD2-FAD3 fusion gene[J]. Journal of Agricultural and Food Chemistry, 71(19): 7324-7333. [28] Yu N, Signorile L, Basu S, et al.2016. Isolation of functional tubulin dimers and of tubulin-associated proteins from mammalian cells[J]. Current Biology, 26(13): 1728-1736. [29] Zhao S J, Zhang J J, Tan R H, et al.2015. Enhancing diterpenoid concentration in Salvia miltiorrhiza hairy roots through pathway engineering with maize C1 transcription factor[J]. Journal of Experimental Botany, 66(22): 7211-7226. [30] Zhou W, Wang S, Shen Y, et al.2022. Overexpression of SmSCR1 promotes tanshinone accumulation and hairy root growth in Salvia miltiorrhiza[J]. Frontiers in Plant Science, 13: 860033. [31] Zhou Y., Feng J., Li Q., et al.2020. SmMYC2b enhances tanshinone accumulation in Salvia miltiorrhiza by activating pathway genes and promotinglateral root development[J]. Frontiers in Plant Science, 11: 559438. [32] Zhu Q, Yu S, Zeng. D.et al.2017. Development of "purple endosperm rice" by engineering anthocyanin biosynthesis in the endosperm with a high-efficiency transgene stacking system[J]. Molecular Plant, 10(7): 918-929.
