[논문]환경스트레스 처리에 의한 개똥쑥 artemisinin 생합성 증진

김경운; 황철호

doi:10.5010/jpb.2022.49.4.307

[국내논문] 환경스트레스 처리에 의한 개똥쑥 artemisinin 생합성 증진
Enhanced biosynthesis of artemisinin by environmental stresses in Artemisia annua 원문보기

Journal of plant biotechnology = 식물생명공학회지, v.49 no.4, 2022년, pp.307 - 315

초록
AI-Helper

개똥쑥은 말라리아 등 다양한 질병의 치료물질인 artemisinin 제공하나, 식물체 내 농도가 낮고, 생산이 불안정하여 국제적 수요에 대응하지 못하고 있다. 재배환경을 인공적으로 제어하는 식물공장 시스템은 계절이나 장소에 제한 없이 약용식물의 공장식 생산체계가 가능하다(Kim 2010). 본 연구에서는 식물공장에서 개똥쑥의 artemisinin의 대량생산이 가능한 최적의 조건을 찾기 위하여 파종부터 수확까지 적색광(R)과 청색광(B)을 혼합한 3종류의 LED (R : B = 6 : 4, 7 : 3, 8 : 2)에서 생장 및 물질생산에 적합한 광조건을 탐색하였다. 개똥쑥의 수확 전, 1,395 ㎼/cm²의 UV-B, 4℃의 저온, 그리고 건조 처리로 식물에 hormesis를 유도하여 artemisinin의 생산 증가를 확인하였다. Artemisinin 생합성에 관여하는 효소들 중에서 ADS, CYP, ALDH1의 발현량을 qPCR로 측정하였고, artemisinin 정량을 통해 전사체와 대사물질의 연관성을 확인하고, artemisinin 생산에 적합한 재배 광조건과 hormesis 처리 조건을 탐색하였다. 3종의 LED 비율 중 8 : 2에서 높은 생체중 및 건물중을 생산했으며, hormesis를 유도하기 위한 3종의 물리 처리에서 이를 통해 7 : 3 식물을 수확전 6시간 건조처리했을 때 artemisinin 함량이 약 2배 증가하였다.

Abstract ▼ AI-Helper

Artemisinin is a secondary metabolite of Artemisia annua that shows potent anti-malarial, anti-bacterial, antiviral, and anti-tumor effects. The supply of artemisinin depends on its content in Artemisia annua, in which various environmental factors can affect the plant's biosynthetic yield. In this study, the effects of different light-emitting diode (LED)-irradiation conditions were tested to optimize the germination and growth of Artemisia annua for the enhanced production of artemisinin. Specifically, the ratio between the red and blue lights in the irradiating LED was varied for investigation as follows: [Red : Blue] = [6 : 4], [7 : 3], and [8 : 2]. Furthermore, additional stress factors like UV-B-irradiation (1,395 ㎼/cm2), low temperature (4℃), and dehydration were also explored to induce hormetic expressions of ADS, CYP, and ALDH1, which are essential genes for the biosynthesis of artemisinin. Quantitative polymerase chain reaction (qPCR) was used to analyze the expression levels of the respective genes and their correlation with the specified conditions. [8 : 2] LED-irradiation was the most optimal among the tested conditions for the cultivation of Artemisia annua in terms of both fresh and dry weights post-harvest. For the production of artemisinin, however, [7 : 3] LED-irradiation with dehydration for six hours pre-harvest was the most optimal condition by inducing around twofold enhancement in the biosynthetic yield of artemisinin. As expected, a correlation was observed between the expression levels of the genes and the contents of artemisinin accumulated.

주제어

표/그림 (11)

그림 Fig. 1 Growth box equipped with three different LEDs for optimal cultivation of Artemisia annua. (A-C) [Red : Blue] = (A) [6 : 4] at 36 ~ 46 μm/M²/s, (B) [7 : 3] at 2 ~ 47 μm/M²/s, and (C) [8 : 2] at 23 ~ 47 μm/M²/s
그림 Fig. 2 (A) UV-B-irradiation, (B) low incubation temperature, and (C) dehydration were applied to Artemisia annua to induce hormetic expressions of favorable genes. The UV-B lamp (1,395 μW/cm²) was p laced 50 cm above t he p lant s ubjects. T he p lant g rowth chamber with fluorescent light (4711 LUX) was set at 4°C to provide a low incubation temperature. For dehydration, the plant subjects were placed in water for six hours and dried on Whatman paper. All treatments were performed for 0, 3, 6, 9, and 12 hours
표 Table 1 Primer sequences for ITS, matK, and rbcL (Moon et al. 2013)
그림 Fig. 3 Biomass production of Artemisia annua cultivated under different LED-irradiation conditions. [Red : Blue] = [6 : 4], [7 : 3], and [8 : 2]; FW = fresh weight; DW = dry weight
표 Table 2 Primer sequences used for the qPCR analysis of ADS (amorpha-4,11-diene synthase), CYP (cytochrome P450 mono- oxygenase), and ALDH1 (alcohol dehydrogenase I) genes, which are essential for the biosynthesis of artemisinin
그림 Fig. 4 Gradient PCR was used to amplify the DNA fragments of ITS, matK, and rbcL for barcode analysis. M: 1kb DNA ladder; annealing temperatures of (a) 75°C, (b) 73.5°C, (d) 67.2°C, (f) 58.9°C, and (h) 55°C; (A) Artemisia annua KS; (B) Artemisia annua Danong
그림 Fig. 5 Phylogenic tree analysis for (a) matK and (b) rbcL genes cloned from (A) Artemisia annua Danong and (B) Artemisia annua KS using the MEGA6.06 software
표 Table 3 The target genes were cloned and sequenced to confirm the suitability of the primer designs for ADS (amorpha-4,11-diene hase), CYP (cytochrome P450 mono-oxygenase), and ALDH1 (alcohol dehydrogenase I)
그림 Fig. 6 DNA products were amplified by gradient PCR for ADS, CYP, and ALDH1 genes. Annealing temperatures for the gradient PCR were as follows: A) 75°C, B) 73.9°C, C) 72.1°C, D) 69.2°C, E) 65.5°C, F) 63°C, G) 61.1°C, and H) 60°C. The analyzed fragment sizes of the target genes were as expected: 94 bp for ADS, 157 bp for ALDH1, and 107 bp for CYP. The fragment sizes were calculated based on the 1kb DNA ladder marker
그림 Fig. 7 qPCR analysis of the target gene expressions upon stresses of (A) ultraviolet light-irradiation (UV), (B) low incubation temperature (LT), and (C) dehydration (DH). The effects of no treatment control (NC; 0-h), 3-h, 6-h, 9-h, and 12-h treatments were investigated under the specified conditions
그림 Fig. 8 Relative contents of artemisinin altered in response to (A) UV-B / UVB, (B) low temperature / LT, (C) dehydration / DH, NC / no treatment control; 3, 4, 9, 12 (hours) for the treatments.

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