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Cloning and analyzing of chalcone isomerase gene (AaCHI) from Artemisia annua

  • Jiawei Ma
  • Xueqing Fu
  • Tingting Zhang
  • Hongmei Qian
  • Jingya Zhao
Original Article

Abstract

Artemisinin, isolated from Chinese medical herbal plant Artemisia annua L., was reported to be the main compound of anti-malaria drugs. However, the artemisinin content was very low in A. annua. Great efforts have been made to increase the artemisinin in A. annua. Chalcone isomerase (CHI) was a rate-limiting enzyme in flavonoids metabolic pathways. Terpenoids and flavonoids are from different biosynthetic pathways, but there is a synergistic action between them on a variety of biological processes in plants. Here, the full-length cDNA of CHI was isolated from A. annua. The AaCHI gene, contained a 690 bp open reading frame, which encoded a protein with 229-amino acids. An analysis of AaCHI transcript levels in multifarious tissues of A. annua showed that flower and bud had the highest transcription levels. Unexpectedly, the artemisinin biosynthetic genes and artemisinin content showed an increase in the AaCHI overexpression transgenic A. annua plants. The results indicated that overexpression of AaCHI gene was an effective method to improve the artemisinin content in A. annua. Taken together, our findings showed that AaCHI is involved in artemisinin production in A. annua and revealed a link between flavonoids and terpenoid production.

Keywords

Artemisinin content Flavonoids metabolic pathways Anti-malaria drugs 

Notes

Acknowledgements

This work was funded by the China National Transgenic Plant Research and Commercialization Project (Grant No. 2016ZX08002-001).

Authors’ contributions

JM and JZ conceived and designed the project. JM, XF, TZ and HQ performed the experiments. JM, FX and TZ analyzed the data. JM and FX wrote the manuscript. JZ revised the manuscript. All authors read and approved the final manuscript.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Human or animal rights

This article does not contain any studies with human or animal subjects performed by the any of the authors.

Supplementary material

11240_2018_1549_MOESM1_ESM.bmp (626 kb)
Fig. S1 PCR analysis of transgenic A. annua plants. “M” Marker, “+” Plasmid, “-” Wild type, “1-34” Transgenic A. annua. (BMP 625 KB)
11240_2018_1549_MOESM2_ESM.tif (79 kb)
Fig. S2 Southern blot of transgenic A. annua plants. Molecular size markers are given at left in kilobases. (TIF 78 KB)
11240_2018_1549_MOESM3_ESM.bmp (195 kb)
Fig. S3 The content of flavonoids in WT and transgenic A. annua plants. Error bars indicate SD (n = 3). Statistical significance was determined using Student’s t-test (*P < 0.05, **P < 0.01). (BMP 194 KB)

References

  1. Abdin MZ, Israr M, Rehman RU et al (2003) Artemisinin, a novel antimalarial drug: biochemical and molecular approaches for enhanced production. Planta Med 69(4):289–299.  https://doi.org/10.1055/s-2003-38871 Google Scholar
  2. Brown GD, Sy LK (2004) In vivo transformations of dihydroartemisinic acid in Artemisia annua plants. Tetrahedron 60(5):1139–1159.  https://doi.org/10.1016/j.tet.2003.11.070 Google Scholar
  3. Burbulis IE, Winkelshirley B (1999) Interactions among enzymes of the Arabidopsis flavonoid biosynthetic pathway. Proc Natl Acad Sci USA 96(22):12929–12934.  https://doi.org/10.1073/pnas.96.22.12929 Google Scholar
  4. Chen DH, Ronald PC (1999) A rapid DNA minipreparation method suitable for AFLP and other PCR applications. Plant Mol Biol Rep 17(1):53–57.  https://doi.org/10.1023/A:1007585532036 Google Scholar
  5. Cheng H, Li L, Cheng S et al (2011) Molecular cloning and function assay of a chalcone isomerase gene (GbCHI) from Ginkgo biloba. Plant Cell Rep 30(1):49–62.  https://doi.org/10.1007/s00299-010-0943-4 Google Scholar
  6. Chung HS, Chang LC, Lee SK et al (1999) Flavonoid constituents of Chorizanthe diffusa with potential cancer chemopreventive activity. J Agric Food Chem 47(1):36–41.  https://doi.org/10.1021/jf980784o Google Scholar
  7. Dastmalchi M, Dhaubhadel S (2015) Soybean chalcone isomerase: evolution of the fold, and the differential expression and localization of the gene family. Planta 241(2):507–523.  https://doi.org/10.1007/s00425-014-2200-5 Google Scholar
  8. Gould KS (2002) Do anthocyanins function as antioxidants in leaves? Imaging of H2O2 in red and green leaves after mechanical injury. Plant Cell Environ 25(10):1261–1269.  https://doi.org/10.1046/j.1365-3040.2002.00905.x Google Scholar
  9. Grotewold E (2006) The Science of Flavonoids. Springer, New York, pp 71–97Google Scholar
  10. He Q, Fu X, Shi P et al (2017) Glandular trichome-specific expression of alcohol dehydrogenase 1 (ADH1) using a promoter-GUS fusion in Artemisia annua L. Plant Cell Tissue Organ Culture 130(2):1–12.  https://doi.org/10.1007/s11240-017-1204-9 Google Scholar
  11. Hirsch AM, Bauer WD, Bird DM et al (2003) Molecular signals and receptors: controlling rhizosphere interactions between plants and other organisms. Ecology 84(4):858–868Google Scholar
  12. Ho WE, Peh HY, Chan TK et al (2014) Artemisinins: pharmacological actions beyond anti-malarial. Pharmacol Therapeut 142(1):126–139.  https://doi.org/10.1016/j.pharmthera.2013.12.001 Google Scholar
  13. Jez JM, Bowman ME, Dixon RA et al (2000) Structure and mechanism of the evolutionarily unique plant enzyme chalcone isomerase. Nat Struct Mol Biol 7(9):786–791.  https://doi.org/10.1038/79025 Google Scholar
  14. Kang JH, Mcroberts J, Shi F et al (2014) The flavonoid biosynthetic enzyme chalcone isomerase modulates terpenoid production in glandular trichomes of tomato. Plant Physiol 164(3):1161–1174.  https://doi.org/10.1104/pp.113.233395 Google Scholar
  15. Koes RE, Quattrocchio F, Mol JNM (1994) The flavonoid biosynthetic pathway in plants: function and evolution. Bioessays 16(2):123–132.  https://doi.org/10.1002/bies.950160209 Google Scholar
  16. Kumar R, Vashisth D, Misra A et al (2016) RNAi down-regulation of cinnamate-4-hydroxylase increases artemisinin biosynthesis in Artemisia annua. Sci Rep 6:26458.  https://doi.org/10.1038/srep26458 Google Scholar
  17. Lan X, Quan H, Xia X et al (2016) Molecular cloning and transgenic characterization of the genes encoding chalcone synthase and chalcone isomerase from the Tibetan herbal plant Mirabilis himalaica. Biotechnol Appl Biochem 63(3):419–426.  https://doi.org/10.1002/bab.1376 Google Scholar
  18. Laughlin JC (1995) The influence of distribution of anti-malarial constituents in Artemisia annua L. on time and method of harvest. Acta Hort 390(390):67–74.  https://doi.org/10.17660/ActaHortic.1995.390.8 Google Scholar
  19. Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2–∆∆CT method. Methods 25(4):402–408.  https://doi.org/10.1006/meth.2001.1262 Google Scholar
  20. Lu X, Zhang L, Zhang F et al (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 Phytol 198(4):1191–1202.  https://doi.org/10.1111/nph.12207 Google Scholar
  21. Mercke P, Bengtsson M, Bouwmeester HJ et al (2000) Molecular cloning, expression, and characterization of amorpha-4,11-diene synthase, a key enzyme of artemisinin biosynthesis in Artemisia annua L. Archiv Biochem Biophys 381(2):173–180.  https://doi.org/10.1006/abbi.2000.1962 Google Scholar
  22. Morales MR, Charles DJ, Simon JE (1993) Seasonal accumulation of artemisinin in Artemisia annua L. Acta Hort 344(344):416–420.  https://doi.org/10.17660/ActaHortic.1993.344.48 Google Scholar
  23. Murashige T, Skoog F (1962) A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiol Plantarum 15(3):473–497.  https://doi.org/10.1111/j.1399-3054.1962.tb08052.x Google Scholar
  24. Paddon CJ, Westfall PJ, Pitera DJ et al (2013) High-level semi-synthetic production of the potent antimalarial artemisinin. Nature 496(7446):528–532.  https://doi.org/10.1038/nature12051 Google Scholar
  25. Patra N, Srivastava AK (2014) Enhanced production of artemisinin by hairy root cultivation of Artemisia annua in a modified stirred tank reactor. Appl Biochem Biotechnol 174(6):2209.  https://doi.org/10.1007/s12010-014-1176-8 Google Scholar
  26. Peer WA, Murphy AS (2007) Flavonoids and auxin transport: modulators or regulators? Trends Plant Sci 12(12):556–563.  https://doi.org/10.1016/j.tplants.2007.10.003 Google Scholar
  27. Peplow M (2016) Synthetic biology’s first malaria drug meets market resistance. Nature 530(7591):389–390.  https://doi.org/10.1038/530390a Google Scholar
  28. Pourcel L, Irani NG, Koo AJ et al (2013) A chemical complementation approach reveals genes and interactions of flavonoids with other pathways. Plant J 74(3):383–397.  https://doi.org/10.1111/tpj.12129 Google Scholar
  29. Ro DK, Paradise EM, Ouellet M et al (2006) Production of the antimalarial drug precursor artemisinic acid in engineered yeast. Nature 440(7086):940–943.  https://doi.org/10.1038/nature04640 Google Scholar
  30. Shen Q, Lu X, Yan TX et al (2016) The jasmonate-responsive AaMYC2 transcription factor positively regulates artemisinin biosynthesis in Artemisia annua. New Phytol 210(4):1269–1281.  https://doi.org/10.1111/nph.13874 Google Scholar
  31. Teoh KH, Polichuk DR, Reed DW et al (2006) Artemisia annua L. (Asteraceae) trichome-specific cDNAs reveal CYP71AV1, a cytochrome P450 with a key role in the biosynthesis of the antimalarial sesquiterpene lactone artemisinin. FEBS Lett 580(5):1411–1416.  https://doi.org/10.1016/j.febslet.2006.01.065 Google Scholar
  32. Teoh KH, Polichuk DR, Reed DW et al (2009) Molecular cloning of an aldehyde dehydrogenase implicated in artemisinin biosynthesis in Artemisia annua. Botany 87(6):635–642.  https://doi.org/10.1139/B09-032 Google Scholar
  33. Tu Y (2011) The discovery of artemisinin (qinghaosu) and gifts from Chinese medicine. Nat Med 17(10):1217–1220.  https://doi.org/10.1038/nm.2471 Google Scholar
  34. Wallaart TE, Pras N, Beekman AC et al (2000) Seasonal variation of artemisinin and its biosynthetic precursors in plants of Artemisia annua of different geographical origin: proof for the existence of chemotypes. Planta Med 66(01):57–62.  https://doi.org/10.1055/s-2000-11115 Google Scholar
  35. Wang BS, Pinder D, Wu SC et al (2010a) Effects of the aqueous extract of sugarcane leaves on antimutation and nitric oxide generation. Food Chem 124(2):495–500.  https://doi.org/10.1016/j.foodchem.2010.06.060 Google Scholar
  36. Wang H, Ma C, Li Z et al (2010b) Effects of exogenous methyl jasmonate on artemisinin biosynthesis and secondary metabolites in Artemisia annua L. Ind Crops Prod 31(2):214–218.  https://doi.org/10.1016/j.indcrop.2009.10.008 Google Scholar
  37. Wang LY, Zhang TT, Fu XQ et al (2017) Molecular cloning, characterization, and promoter analysis of the isochorismate synthase gene from Artemisia annua. J Zhejiang Univ 18(8):662–673.  https://doi.org/10.1631/jzus.B1600223 Google Scholar
  38. Zhang Y, Teoh KH, Reed DW et al (2008) The molecular cloning of artemisinic aldehyde Delta11(13) reductase and its role in glandular trichome-dependent biosynthesis of artemisinin in Artemisia annua. J Biol Chem 283(31):21501–21508.  https://doi.org/10.1074/jbc.M803090200 Google Scholar
  39. Zhang L, Jing FY, Li FP et al (2009) Development of transgenic Artemisia annua (Chinese wormwood) plants with an enhanced content of artemisinin, an effective anti-malarial drug, by hairpin-RNA-mediated gene silencing. Biotechnol Appl Biochem 52(3):199–207.  https://doi.org/10.1042/BA20080068 Google Scholar
  40. Zhang Y, Fu XQ, Hao XL et al (2016) Molecular cloning and promoter analysis of the specific salicylic acid biosynthetic pathway gene phenylalanine ammonia-lyase (AaPAL1) from Artemisia annua. Biotechnol Appl Biochem 63(4):514–524.  https://doi.org/10.1002/bab.1403 Google Scholar
  41. Zhang D, Sun W, Shi Y et al (2018a) Red and blue light promote the accumulation of artemisinin in Artemisia annua L. Molecules 23(6):1329.  https://doi.org/10.3390/molecules23061329 Google Scholar
  42. Zhang TT, Ma JW, Wang LY et al (2018b) Improving artemisinin content of Artemisia annua L. Through overexpression of flavanone 3-hydroxylase gene (AaF3H). Curr Biotechnol 8(1): 55–62 (in Chinese).  https://doi.org/10.19586/j.20952341.2017.0073 Google Scholar

Copyright information

© Springer Nature B.V. 2019

Authors and Affiliations

  • Jiawei Ma
    • 1
  • Xueqing Fu
    • 1
  • Tingting Zhang
    • 1
  • Hongmei Qian
    • 1
  • Jingya Zhao
    • 1
  1. 1.Plant Biotechnology Research Center, Fudan-SJTU-Nottingham Plant Biotechnology R&D Center, School of Agriculture and BiologyShanghai Jiao Tong UniversityShanghaiPeople’s Republic of China

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