|
|
Effect of Over-expressing TwHMGR and TwDXR on the Biosynthesis of Terpenoids in Tripterygium wilfordii |
, , , , , , |
|
|
Abstract Abstract 3-Hydroxy-3-methylglutaryl-CoA reductase (HMGR) and 1-deoxy-D-xylulose 5-phosphate reductoisomerase (DXR) catalyse the reaction of 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) to mevalonic acid (MVA) which is the first committed enzyme in the MVA pathway in the cytosol, and the reaction of 1-deoxy-D-xylulose 5-phosphate (DXP) to 2-C-methyl-D-erythritol 4-phosphate (MEP) which is the first committed enzyme in the MEP pathway in the plastid, respectively. For the study of the effect of over-expressing TwHMGR(KU246037.1) and TwDXR(KJ174341.1) genes on the production of secondary metabolites in Tripterygium wilfordfii, the expressing vector of TwHMGR and TwDXR was constructed and integrated into the genome of T. wilfordfii via Agrobacterium-mediated transformation methods. Three transgenic hairy roots were screened and confirmed respectively. The results of qRT-PCR showed that the expressing level of target genes in transgenic hairy roots were significantly higher than those in non-transgenic control, with highest levels at 4.73 times and 5.81 times, respectively. According to the results of high-performance liquid chromatography (HPLC), the contents of wilforgine, wilforine and triptolide of transgenic hairy roots significantly higher than controls. The results showed that the increase of TwHMGR gene and TwDXR gene expression could improve the yield of terpenoids secondary metabolites of T. wilfordii. This study provides the basis for the further use of metabolic engineering methods to improve the content of secondary metabolites and to elucidate of terpenoid biosynthetic pathway in T. wilfordii.
|
Received: 30 October 2017
Published: 21 May 2018
|
|
|
|
1.Alam P, Abdin MZ, 2011. Over-expression of HMG-CoA reductase and amorpha-4,11-diene synthase genes in Artemisia annua L. and its influence on artemisinin content [J]. Plant Cell Report, 30:1919-1928.2.Brinker AM, Ma J, Lipsky P E, et al. 2007. Medicinal chemistry and pharmacology of genus Tripterygium (Celastraceae) [J]. Phytochemistry, 68(6):732-766.3.Brinker AM, Raskin I. 2005 Determination of triptolide in root extracts of Tripterygium wilfordii by solid-phase extraction and reverse phase high-performance liquid chromatography [J]. Journal of Chromatography A, 1070(1-2):65-70.4.Chappell J, Wolf F, Proulx J, et al. 1995. Is the reaction catalysed by 3-hydroxy-3-methylglutaryl coenzyme A reductase a rate limiting step for isoprenoid biosynthesis in plants [J]? Plant Physiology, 109: 1337-1343.5.Dai Z, Cui G, Zhou SF 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]. Journal of Plant Physiology, 168:148-157.6.Dong N, Ponciano G, McMahan CM, et al. 2013. Overexpression of 3-hydroxy-3-methylglutaryl coenzyme A reductase in Parthenium argentatum (guayule) [J]. Industrial Crops and Products, 46:15-24.7.Han M, Heppel SC, Su T, et al. 2013. Enzyme Inhibitor Studies Reveal Complex Control of Methyl-D-Erythritol 4-Phosphate (MEP) Pathway Enzyme Expression in Catharanthus roseus [J]. PLoS ONE 8(5): e62467.8.Harker M, Holmberg N, Clayton JC, et al. 2003. Enhancement of seed phytosterol levels by expression of an N terminal Hevea brasiliensis (rubber tree) 3-hydroxy-3-methylglutaryl-CoA reductase [J]. Plant Biotechnology Journal, 1:113-121.9.Hasunuma T, Takeno S, Hayashi S, et al. 2008. Overexpression of 1-Deoxy-D-Xylulose-5-Phosphate Reductoisomerase Gene in Chloroplast Contributes to Increment of Isoprenoid Production [J]. JOURNAL OF BIOSCIENCE AND BIOENGINEERING, 105(5):518-526.10.Kim YJ, Lee OR, Oh JY, et al. 2014. Functional analysis of 3-hydroxy-3-methylglutaryl coenzyme A reductase encoding genes in triterpene saponin-producing ginseng [J]. Plant Physiology, 165:373-387.11.Kim YK, Kim JK, Kim YB, et al. 2013. Enhanced Accumulation of Phytosterol and Triterpene in Hairy Root Cultures of Platycodon grandiflorum by Overexpression of Panax ginseng 3?Hydroxy-3-methylglutaryl-coenzyme A Reductase [J]. Journal of Agricultural and Food Chemitry, 61: 1928-1934.12.Lange I, Poirier BC, Herron BK, et al. 2015. Comprehensive Assessment of Transcriptional Regulation Facilitates Metabolic Engineering of Isoprenoid Accumulation in Arabidopsis [J], Plant Physiology, 169:1595-1606.13.Liao P, Hemmerlin A, Bach TJ, et al. 2016. The potential of the mevalonate pathway for enhanced isoprenoid production [J]. Biotechnology Advances, 34 (5):697-713.14.Liao P, Zhou W, Zhang L, et al. 2009. Molecular cloning, characterization and expression analysis of a new gene encoding 3-hydroxy-3-methylglutaryl coenzyme A reductase from Salvia miltiorrhiza [J]. Acta Physiologiae Plantarum, 31:565-572.15.LIU J, XU YH, LIANG L, et al. 2015.Molecular cloning, characterization and expression analysis of the gene encoding 1-deoxy-D-xylulose 5-phosphate reductoisomerase from Aquilaria sinensis (Lour.) Gilg [J]. Journal of Genetics, 94(2):239-24916.Ma ZQ, Li YJ, Wu LP, et al. 2014. Isolation and insecticidal activity of sesquiterpenes alkaloids from Tripterygium wilfordii Hook f [J]. Industrial Crops and Products, 52: 642-648.17.Mahmoud SS, Croteau RB, 2001. Metabolic engineering of essential oil yield and composition in mint by altering expression of deoxyxylulose phosphate reductoiso-merase and menthofuran synthase [J]. Proceedings of the National Academy of Sciences of the United States of America, 98:8915-8920.18.Schaller H, Grausem B, Benveniste P, et al. 1995. Expression of the Hevea brasiliensis Mull. Arg. 3-hydroxy-3-methylglutaryl-coenzyme A reductase 1 in tobacco results in sterol overproduction [J]. Plant Physiology, 109:761-770.19.Song AAL, Abdullah JO, Abdullah MP, et al. 2012. Overexpressing 3-Hydroxy-3-Methylglutaryl Coenzyme A Reductase (HMGR) in the Lactococcal Mevalonate Pathway for Heterologous Plant Sesquiterpene Production [J]. PLoS ONE, 7(12): e52444. 20.Vaccaro M, Malafronte N, Alfieri M, et al. 2014. Enhanced biosynthesis of bioactive abietane diterpenes by overexpressing AtDXS or AtDXR genes in Salvia sclarea hairy roots [J]. Plant Cell Tissue and Organ Culture, 119:65-77.21.van Deenen N, Bachmann AL, Schmidt T, et al. 2012. Molecular cloning of mevalonate pathway genes from Taraxacum brevicorniculatum and functional characterisation of the key enzyme 3-hydroxy-3-methylglutarylcoenzyme A reductase [J]. Molecular Biology Reports, 39:4337-4349.22.Vranová E, Coman D, and Gruissem W, et al. 2013.Network Analysis of the MVA and MEP Pathways for Isoprenoid Synthesis [J], Annual Review of Plant Biology, 64:665-700.23.Wang H, Nagegowda DA, Rawat R, et al. 2012. Overexpression of Brassica juncea wild-type and mutant HMG-CoA synthase 1 in Arabidopsis up-regulates genes in sterol biosynthesis and enhances sterol production and stress tolerance [J]. Plant Biotechnology Journal, 10:31-42.24.Wang X, Tian W, Li Y. 2008. Development of an efficient protocol of RNA isolation from recalcitrant tree tissues [J]. Molecular Biotechnology, 38(1): 57-64.25.Wu SJ, Shi M, Wu JY, 2009. Cloning and characterization of the 1-deoxy-D-xylulose 5-phosphate reductoisomerase gene for diterpenoid tanshinone biosynthesis in Salvia miltiorrhiza (Chinese sage) hairy roots [J]. Biotechnology and Applied Biochemitry, 52:89-95.26.Zhang J, Huo YB, LiuY, et al. 2016. Differential expressed analysis of Tripterygium wilfordii unigenes involved in terpenoid backbone biosynthesis [J], Journal of Asian Natural Products Research,21:1-10.27.Zhao LS, Chang WC, Xiao YL, et al. 2013. Methylerythritol Phosphate Pathway of Isoprenoid Biosynthesis [J], Annual Review of Biochemitry, 82:497-530.28.Zhu CS, Miao GP, 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 Microbiology Biotechnology, 24(6):823-834.29.武莹, 李威, 祝传书等. 2012. 雷公藤HMGR基因克隆、序列分析及诱导表达 [J]. 西北农林科技大学学报(自然科学版), 40(11):103-111. (Wu Y, Li W, Zhu CS, et al. 2012. Characterization and expression of gene encoding HMGR from Tripterygium wilfordii [J], Journal of North-west A&F University (Nat. Sci. Ed.), 40(11):103-111. )祝传书, 陈欣, 郭嘉等. 2014. 雷公藤1-脱氧-D-木酮糖-5-磷酸还原酶基因(TwDXR)的克隆与表达分析 [J]. 农业生物技术学报, 22(3):298-308. (Zhu CS, Chen X, Guo J, et al. 2014. Cloning and Expression Analysis of 1-deoxy-D-xylulose 5-phosphate Reductoisomerase Gene (DXR) in Tripterygiun wilfordii [J], Journal of Agricultural Biotechnology, 22(03): 298-308.) |
|
|
|