油酸诱导对灵芝三萜生物合成途径中关键酶编码基因的影响
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摘要
通过实时荧光定量PCR技术考察油酸诱导对灵芝三萜生物合成途径中关键酶基因转录水平的影响。主要选择SQS、CYP51和14α-LDM3个关键酶基因做试验。在不同浓度油酸、不同诱导时间和最佳诱导条件下,3个基因响应油酸诱导的变化情况各不相同。结果表明,油酸可使SQS和CYP51两个灵芝三萜生物合成途径中的关键酶基因上调表达,从而使灵芝菌丝体胞内三萜产量大幅度提高。油酸对14α-LDM酶基因主要为抑制作用,从而抑制麦角甾醇这一代谢途径,间接提高灵芝三萜产量。
To characterize the effect of Oleic acid on Ganoderma lucidum biosynthesis, the quantitative real-time PCR was used to measure transcription levels of several genes in the synthesis pathway. Three genes were choosed,which is SQS, CYP51 and 14α-LDM. Under different concentrations of oleic acid, different time of induction and the optimal inducing condition, the transcription levels of three genes were different. The results showed that oleic acid could increase the transcription levels of SQS and CYP51 genes which are the key enzyme genes in the synthesis pathway, so that the production of triterpenoids increased. Oleic acid can inhibit the transcription levels of 14α-LDM, so the metabolic pathway of ergosterol will be inhibited, indirectly increase the production of triterpenoids.
To characterize the effect of Oleic acid on Ganoderma lucidum biosynthesis, the quantitative real-time PCR was used to measure transcription levels of several genes in the synthesis pathway. Three genes were choosed,which is SQS, CYP51 and 14α-LDM. Under different concentrations of oleic acid, different time of induction and the optimal inducing condition, the transcription levels of three genes were different. The results showed that oleic acid could increase the transcription levels of SQS and CYP51 genes which are the key enzyme genes in the synthesis pathway, so that the production of triterpenoids increased. Oleic acid can inhibit the transcription levels of 14α-LDM, so the metabolic pathway of ergosterol will be inhibited, indirectly increase the production of triterpenoids.
引文
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[2] TANG Y J, ZHONG J J. Modeling the kinetics of cell growth and ganoderic acid production in liquid static cultures of the medicinal mushroom Ganoder ma lucidum[J]. Biochemical Engineering Journal,2004, 21(3):259-264.
[3] WANG X M, YANG M, GUAN S H, et al. Quantitative determination of six major triterpenoids in Ganoderma lucidum and related species by high performance liquid chromatography[J]. J Pharm Biomed Anal, 2006, 41(3):838-844.
[4] QIAO Y, ZHANG X M, QIU M H. Two novel lanostane triterpenoids from ganoderma sinense[J].Molecules, 2007, 12(8):2038-2046.
[5] KEYPOUR S, RAFATI H, RIAHI H, et al. Qualitative analysis of ganoderic acids in Ganoderma lucidum from Iran and China by RP-HPLC and electrospray ionisation-mass spectrometry(ESI-MS)[J].Food Chemistry, 2010, 119(4):1704-1708.
[6]方庆华.灵芝真菌发酵生产灵芝酸和灵芝多糖的研究[D].上海:华东理工大学, 2000.
[7]余素萍.灵芝深层发酵生产生物活性物质的研究[D].南京:南京农业大学, 2004.
[8] KIM H M, PAIK S Y, RA K S, et al. Enhanced production of exopolysaccharides by fed-batch culture of Ganoderma resinaceum DG-6556[J]. Journal of Microbiology, 2006, 44(2):233-242.
[9] ZHAO W, XU J W, ZHONG J J. Enhanced production of ganoderic acids in static liquid culture of Ganoderma lucidum under nitrogen-limiting conditions[J]. Bioresour Technol, 2011, 102(17):8185-8190.
[10] KANG D, MUTAKIN M, LEVITA J. Computational study of triterpenoids of Ganoderma lucidum with aspartic protease enzymes for discovering HIV-1 and plasmepsin inhibitors[J]. International Journal of Chemistry, 2015, 7(1):61-62.
[11] TRAN H B, A YAMAMOTO, S MATSUMOTO, et al. Hypotensive effects and angiotensin-converting enzyme inhibitory peptides of reishi(Ganoderma lingzhi)auto-digested extract[J]. Molecules, 2014, 19(9):13473-13485.
[12] FANG X, SHI L, REN A, et al. The cloning,characterization and functional analysis of a gene encoding an acetyl-CoA acetyltransferase involved in triterpene biosynthesis in Ganoderma lucidum[J]. Mycoscience, 2013, 54(2):100-105.
[13]徐晓兰.灵芝三萜和金银花绿原酸生物合成途径关键酶基因的挖掘及分析[D].北京:北京协和医学院,2013.
[14]孙冰沁,秦可欣,何国庆,等.基于油酸诱导的高产三萜灵芝菌丝体发酵条件优化[J].食品工业科技,2016, 37(24):233-237, 244.
[15]赵艳.灵芝关键成分三萜酸的深层发酵技术研究[D].长沙:中南林业科技大学,2011.
[16]高建莉,禹志领,李绍平,等.灵芝三萜类成分研究进展[J].中国食用菌, 2005, 24(4):6-11.
[17]梁翠霞.小分子化合物添加促进灵芝酸的发酵生产及其作用机制探索[D].上海:上海交通大学, 2010.
[18] LIVAK K J, SCHMITTGEN T D. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T))Method[J].Methods, 2001, 25(4):402-408.
[19]魻ZBEK B, GAYIK S. The studies on the oxygen mass transfer coefficient in a bioreactor[J]. Process Biochemistry, 2001, 36(8/9):729-741.
[20]翁雪清,施巧琴,吴松刚.氧载体、表面活性剂及H2O2对L-phe发酵影响的研究[J].氨基酸和生物资源, 2012, 34(2):39-42.