Effects of pH on the Lentinan Content and the Transcriptional Levels of Genes Related to the Synthesis of Key Enzymes in Lentinula edodes

doi:10.13560/j.cnki.biotech.bull.1985.2018-0842

Abstract

Abstract: Lentinula edodes is a fungus as both medicine and food. Its main metabolite,lentinan,has great medicinal value. It is of great significance to study the metabolic pathways of lentinan biosynthesis for improving the content of lentinan. With Xin808 as the material,phenol-sulfuric acid method was used to measure the lentinan content under different pH fermentation conditions,and fluorescence quantitative PCR and three software（GeNorm,NormFinder and BestKeeper）were adapted to screen the stably expressed reference genes. Based on those,the transcriptional expression levels of 3 key enzyme genes in polysaccharide metabolic pathways were analyzed. The results showed that the biomass and intracellular polysaccharide of mycelium increased with the increase of pH,while the extracellular polysaccharide decreased. 18S and rpl4 were determined as the most stable and unstable reference genes,respectively. In the range of pH 5.0-7.0,the relative expression level of UGP was obviously higher than that of PGM and PGI,low-level and stable expression profile of PGM was detected,and the relative expression of PGI was low,but fluctuated with the variation of pH value. In sum,the variation of pH value resulted in the various effects on the biomass and polysaccharide content of L. edodes,and there was no correlation between the transcriptional expression level of 3 key enzyme genes and polysaccharide content in this study.

Key words: lentinan, pH value, reference gene, relative expression level

WANG Jie, ZHANG Yang, QIN Peng, HE Mao-lan, LI Jin, GU Yun-fu, ZENG Xian-fu, XIANG Quan-ju. Effects of pH on the Lentinan Content and the Transcriptional Levels of Genes Related to the Synthesis of Key Enzymes in Lentinula edodes[J]. Biotechnology Bulletin, 2019, 35(2): 39-45.

References

[1] 郭玉明, 钟厚贤. 香茹提取物对白血病细胞HL-60增殖的抑制作用[J]. 农产品加工(学刊), 2006, 4(8):19-21.
[2] 王贺祥. 食用菌学[M]. 北京:中国农业大学出版社, 2007.
[3] 李月梅. 香菇的研究现状及发展前景[J]. 微生物学通报, 2005, 29(7):149-152.
[4] Yu SH, Weaver V, Martin K, et al.The effects of whole mushrooms during inflammation[J]. BMC Immunology, 2009, 10(1):1-13.
[5] Chandra LC, Smith BJ, Clarke SL, et al.Differential effects of shiitake- and white button mushroom-supplemented diets on hepatic steatosis in c57bl/6 mice[J]. Food & Chemical Toxicology, 2011, 49(12):3074-3080.
[6] 韩玉竹, 刘恩岐, 李彦岩, 等. 芽孢杆菌活性多糖的分离纯化及合成基因研究[J]. 食品科学, 2014, 35(11):179-184.
[7] Lai X, Wu J, Chen S, et al.Expression, purification, and characterization of a functionally active mycobacterium tuberculosis udp-glucose pyrophosphorylase[J]. Protein Expression & Purification, 2008, 61(1):50-56.
[8] Looijesteijn PJ, Boels IC, Kleerebezem M, et al.Regulation of Exopolysaccharide Production by Lactococcus lactis subsp. cremoris by the Sugar Source[J]. Applied & Environmental Microbiology, 1999, 65(11):5003-5008.
[9] Peng L, Qiao S, Xu Z, et al.Effects of culture conditions on monosaccharide composition of ganoderma lucidum, exopolysaccharide and on activities of related enzymes[J]. Carbohydrate Polymers, 2015, 133:104-109.
[10] 赵俊杰. 香菇液体发酵及多糖提取工艺研究[D]. 郑州:郑州大学, 2014.
[11] Simonic J, Stajic M, et al.Optimization of submerged cultivation conditions for extra and intracellular polysaccharide production by medicinal ling zhi or reishi mushroom ganoderma lucidum(w. curt. :fr. )p. karst. (aphyllophoromycetideae)[J]. Nternational Journal of Medicinal Mushrooms, 2008, 10(4):351-360.
[12] Fang QH, Zhong JJ.Effect of initial pH on production of ganoderic acid and polysaccharide by submerged fermentation of ganoderma lucidum[J]. Process Biochemistry, 2002, 37(7):769-774.
[13] 黄真池, 欧阳乐军, 等. 桉属植物内参基因的筛选及评估[J]. 西北农林科技大学学报:自然科学版, 2013, 41(10):67-72.
[14] 张宁, 魏周玲, 杜利娟, 等. TMV和PVX胁迫下云烟和番茄实时荧光定量内参基因筛选[J]. 西南大学学报:自然科学版, 2016, 38(7):13-18.
[15] Meng L, Feldman L.A rapid TRIzol-based two-step method for DNA-free RNA extraction from Arabidopsis siliques and dry seeds[J]. Biotechnology Journal, 2010, 5(2):183-186.
[16] Chen A, He S, Li F, et al.Analyses of the sucrose synthase gene family in cotton:structure, phylogeny and expression patterns[J]. BMC Plant Biology, 2012, 12(1):85.
[17] Chen A, Chen X, Wang H, et al.Genome-wide investigation and expression analysis suggest diverse roles and genetic redundancy of pht1 family genes in response to pi deficiency in tomato[J]. BMC Plant Biology, 2014, 14(1):61.
[18] Xiang Q, Li J, Qin P, et al.Identification and evaluation of reference genes for qrt-pcr studies in lentinula edodes[J]. PLoS One, 2018, 13(1):e0190226.
[19] Vandesompele J, Preter KD, et al.Accurate normalization of real-time quantitative rt-pcr data by geometric averaging of multiple internal control genes[J]. Genome Biol, 2002, 3(7):2002.
[20] Pfaffl MW, Tichopad A, Prgomet C, et al.Determination of stable housekeeping genes, differentially regulated target genes and sample integrity:bestkeeper--excel-Based tool using pair-wise correlations[J]. Biotechnology Letters, 2004, 26(6):509-515.
[21] Andersen CL, Jensen JL, Orntoft TF.Nomalization of real-time quantitative reverse transcription PCR data:a model-based variance estimation approach to identify genes suited for nomalization, applidto bladder and colon cancer data sets[J]. CancRes, 2004, 64(15):5245-5250.
[22] 徐思炜, 张君胜, 周雯. 香菇液体菌种培养条件优化[J]. 江苏农业科学, 2014, 42(12):290-292.
[23] Wan HJ, Zhao ZG, Qian CT, et al.Selection of appropriate reference genes for gene expression studies by quantitative real-time polymerase chain reaction in cucumber[J]. Analytical Biochemistry, 2010, 399(2):257-261.