Transcriptome-based Discovery of AP2/ERF Transcription Factors Related to Terpene Trilactones Synthesis in Ginkgo biloba
Keywords:AP2/ERF; Ginkgo biloba; MEP pathway; terpene trilactones; transcriptome sequencing
Ginkgo biloba is a unique tree in China with medicinally and phylogenetically important characteristics. Terpene trilactones (TTL) is a key active pharmaceutical ingredient in Ginkgo, so the content of TTL in Ginkgo has become one of the important indices for evaluating quality of the medicinal materials. By transcriptome sequencing on samples treated by chlormequat, ultraviolet (UV) and drought, totally 59820 contigs and 37564 unigenes were obtained. Furthermore, 18234 unigenes were annotated through COG, KEGG and GO analysis. There were 78 AP2/ERF transcription factors, 23 factors of up-regulation and 66 factors of down-regulation that were related with synthetic pathway of TTL in Ginkgo. Phylogenetic tree clustering analysis indicated that there were 42 AP2s could be clustered into ERF, DREB and RVA subfamilies. EMSA analysis demonstrated that GbERF13, GbERF25 and GbERF27 could bind with regulatory elements, such as E-box, in the upstream of GbMECPs promoter. Expression analysis showed that the expression level of GbERF25 was the highest in root, and GbERF25 and GbERF27 were expressed in relatively high transcription levels in leaf and other tissues. The results of qRT-PCR indicated that CCC treatment could significantly improve expression levels of ERF25 and ERF27, and UV and drought could induce transcription levels of ERF13 and ERF25, respectively. The results implied that ERF25 and ERF27 might involve in the induction and regulation of CCC treatment on synthesis of bilobalide in G. biloba. ERF13 might participate in the regulation of bilobalide synthesis induced by UV, and EFR25 might involve in the regulation of the synthesis induced by drought. During annual cycle of expression, the transcription levels of ERF13, ERF25 and ERF27 had significantly positive correlation with diterpene level with correlation coefficient 0.975. It implied that these transcription factors mainly acted on the MEP pathway that regulated synthesis of bilobalide. The aim of the research was to indicate the mechanism of environment or cultivation measure regulating target gene of TTL metabolic pathway by AP2/ERF, and establish metabolic network of AP2/ERF regulating TTL synthesis.
Chadwick M, Trewin H, Gawthrop F, Wagstaff C (2013). Sesquiterpenoids lactones: Benefits to plants and people. International Journal of Molecular Sciences 14(6):12780-12805.
Chao M, Hong X, Keli C (2005). Preliminary study on the ginkgolide b and bilobalide contents in different tissues and callus cultures of Ginkgo biloba. Natural Product Research & Development 17:603-605.
Cheng SY, Li LL, Yuan HH, Xu F, Cheng H (2015). Molecular cloning and characterization of GbMECT and GbMECP gene promoters from Ginkgo biloba. Genetics and Molecular Research 14(4):15112-15122.
Dai Y (2008). Molecular cloning and characterization of AP2-type transcription factors involved in isoprenoid biosynthetic pathway of Taxus cuspidata. PhD Thesis, Fudan University. Fudan University Publication.
Gao C, Li P, Song A, Wang H, Wang Y, Ren L, … Chen S (2015). Isolation and characterization of six AP2/ERF transcription factor genes in Chrysanthemum nankingense. International Journal of Molecular Sciences 16(1):2052-2065.
Han S, Wu Z, Jin Y, Yang W, Shi H (2015). RNA-Seq analysis for transcriptome assembly, gene identification, and SSR mining in ginkgo (Ginkgo biloba L.). Tree Genetics & Genomes 11(3):1-10.
Jie C, Renke T, Xiaojuan G, Zhengli F, Zheng W, Zhiyan Z, Xiaoli T (2015). The functional annotation of unigenes in COG, GO, KEGG, Swissprot and Nr database.
Kagaya Y, Ohmiya K, Hattori T (1999). RAV1, a novel DNA-binding protein, binds to bipartite recognition sequence through two distinct DNA. Nucleic Acids Research 27(2): 470-478.
Kim JH, Lee KI, Chang YJ, Kim SU (2012). Developmental pattern of Ginkgo biloba levopimaradiene synthase (GbLPS) as probed by promoter analysis in Arabidopsis thaliana. Plant Cell Reports 31(6):1119-1127.
Kim SM, Kuzuyama TChang YJ, Song KS, Kim SU (2006). Identification of class 2 1-deoxy-D-xylulose 5-phosphate synthase and 1-deoxy-D-xylulose 5-phosphate reductoisomerase genes from Ginkgo biloba and their transcription in embryo culture with respect to ginkgolide biosynthesis. Planta Medica 72(03):234-240.
Kumar S, Stecher G, Tamura K (2016). MEGA7: molecular evolutionary genetics analysis version 7.0 for bigger datasets. Molecular Biology & Evolution 33(7): 1870-1874.
Lange BM, Rujan T, Martin W, Croteau R (2000). Isoprenoid biosynthesis: the evolution of two ancient and distinct pathways across genomes. Proceedings of the National Academy of Sciences of the United States of America 97(24):13172-13177.
Leng P, Su S, Wang T, Jiang X, Wang S (2002). Effects of light intensity and light quality on photosynthesis, flavonol glycoside and terpene lactone contents of Ginkgo biloba L. seedlings. Journal of Plant Resources & Environment 11(1):1-4.
Liao Y, Xu F, Huang X, Zhang W, Cheng H, Wang X, … Shen Y (2015). Characterization and transcriptional profiling of Ginkgo biloba mevalonate diphosphate decarboxylase gene (GbMVD) promoter towards light and exogenous hormone treatments. Plant Molecular Biology Reporter 34(3):1-16.
Liao YL, Feng XU, Zhu J, Wang Y, Cheng SY (2008). Separation and determination of terpene trilactones by gas chromatography with wide bore capillary column. Acta Agriculturae Boreali-Occidentalis Sinica 17(1):146-149.
Lu X, Zhang L, Zhang F, Jiang W, Shen Q, Zhang L,… Tang K (2013). AaORA, a trichome-specific AP2/ERF transcription factor of Artemisia annua, is a positive regulator in the artemisinin biosynthetic pathway and in disease resistance to Botrytis cinerea. New Phytologist 198(4):1191-1202.
Magallón S, Hilu KW, Quandt D (2013). Land plant evolutionary timeline: Gene effects are secondary to fossil constraints in relaxed clock estimation of age and substitution rates. American Journal of Botany 100(3):556-573.
Mizoi J, Shinozaki K, Yamaguchi-Shinozaki K (2012). AP2/ERF family transcription factors in plant abiotic stress responses. Biochimica Et Biophysica Acta 1819(2):86-96.
Nakanishi K, Habaguchi K (1971). Biosynthesis of ginkgolide B, its diterpenoid nature, and origin of the tert-butyl group. Journal of the American Chemical Society 93(14):3546-3547.
Nakano T, Suzuki K, Fujimura T, Shinshi H (2006). Genome-wide analysis of the ERF gene family in Arabidopsis and rice. Plant Physiology 140(2):411-432.
Okamuro JK, Caster B, Villarroel R, Van MM, Jofuku KD (1997). The AP2 domain of APETALA2 defines a large new family of DNA binding proteins in Arabidopsis. Proceedings of the National Academy of Sciences of the United States of America 94(13):7076-7081.
Postel D, Vanlemmens P, Gode P, Ronco G, Villa P (2002). PlantCARE, a database of plant cis-acting regulatory elements and a portal to tools for in silico analysis of promoter sequences. Nucleic Acids Research 30(1):325-327.
Sabater-Jara AB, Souliman-Youssef S, Novo-Uzal E, Almagro L, Belchí-Navarro S, Pedreño MA (2013) Biotechnological approaches to enhance the biosynthesis of ginkgolides and bilobalide in Ginkgo biloba. Phytochemistry Reviews 12(1):191-205.
Sakuma Y, Qiang L, Dubouzet JG, Abe H, Shinozaki K, Yamaguchi-Shinozaki K (2002). DNA-binding specificity of the ERF/AP2 domain of Arabidopsis DREBs, transcription factors involved in dehydration- and cold-inducible gene expression. Biochemical & Biophysical Research Communications 290(3):998-1009.
Schmittgen TD, Livak KJ (2008). Analyzing real-time PCR data by the comparative CT method. Nature Protocols 3(6):1101-1108.
Selmar D (2013). Stress enhances the synthesis of secondary plant products: the impact of stress-related over-reduction on the accumulation of natural products. Plant & Cell Physiology 54(6):817-826.
Singh B, Kaur P, Gopichand, Singh RD, Ahuja PS (2008). Biology and chemistry of Ginkgo biloba. Fitoterapia 79(6):401-418.
Strømgaard K, Nakanishi K (2004). Cover picture: chemistry and biology of terpene trilactones from Ginkgo biloba. Angewandte Chemie 43(13):1640-1658.
Van dFL, Memelink J (2000). ORCA3, a jasmonate-responsive transcriptional regulator of plant primary and secondary metabolism. Science 289(5477):295-297.
Wang L, Lu Z, Li W, Xu J, Luo K, Lu W, Zhang L, Jin B (2016). Global comparative analysis of expressed genes in ovules and leaves of Ginkgo biloba L. Tree Genetics & Genomes 12(2):1-18.
Wang T, Zhang N, Du L (2005). Isolation of RNA of high quality and yield from Ginkgo biloba leaves. Biotechnology Letters 27(9):629-633.
Xu DL, Long H, Liang JJ, Zhang J, Chen X, Li JL, … Yu MQ (2012). De novo assembly and characterization of the root transcriptome of Aegilops variabilis during an interaction with the cereal cyst nematode. BMC Genomics 13(1):277-287.
Xu F, Zhang W, Sun N, Li L, Cheng S, Wang Y (2011). Effects of chlorocholine chloride on photosynthesis metabolism and terpene trilactones biosynthesis in the leaf of Ginkgo biloba. Acta Horticulturae Sinica 38(12):2253-2260.
Yu ZX, Li JX, Yang CQ, Hu WL, Wang LJ, Chen XY (2012). The jasmonate-responsive AP2/ERF transcription factors AaERF1 and AaERF2 positively regulate artemisinin biosynthesis in Artemisia annua L. Molecular Plant 5(2):353-365.
Zhu C, Cao F, Wang G, Geng G (2011). Effects of drought stress on annual dynamic changing pattern of the terpene lactones content in Ginkgo biloba leaves. China Forestry Science and Technology 25:15-20.
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