不同温度培养对香菇漆酶活性及转录表达的影响
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摘要
以香菇新808为材料,研究不同温度培养下漆酶活性及其基因表达调控特征.通过平板培养法测定4种温度(20℃、25℃、28℃和30℃)对香菇菌丝圈及氧化圈的影响,通过液体发酵测定漆酶活性,并采用qPCR技术检测漆酶同工酶基因的相对表达量.结果表明:不同温度条件下,漆酶的活性及转录表达存在明显差异.25℃培养条件下香菇菌丝圈及氧化圈最大,漆酶活性最强(114.11 U/mL);而30℃时的菌丝圈和氧化圈最小,漆酶活性低(47.52 U/mL).不同温度培养下漆酶同工酶基因出现差异性表达,以25℃为对照组,20℃培养条件下9个漆酶基因(Llac1-3、Llac5和Llac7-11)表达量上调,28℃处理下除Llac5和Llac7基因以外,其余8个漆酶基因表达量发生上调,而30℃处理组6个漆酶基因(Llac1-2、Llac4、Llac7-8和Llac10)表达量下调.表明20℃处理下有助于漆酶基因的表达,而30℃处理下则抑制了其同工酶基因的表达.
The laccase activity and its gene expression regulation characteristics under different temperature cultures(20℃, 25℃, 28℃and 30℃) were studied with the Lentinus edodes Xin808 to analyze the effect of its diameter of the mycelium ring and oxidation ring. The laccase activity was measured by liquid fermentation, and the relative expression level of lac was detected by qPCR technique. The results showed that, the growth rate of Lentinus edode mycelium, formation of oxidation ring, and laccase activity were all higher under 25℃ cultivation temperature. While 30℃ inhibited the growth of mycelium and the formation of oxidation ring and resulted into the lowest laccase activity. Moreover, the Lac genes exhibited differential expression profiles cultured in different temperature. Compared to the 25℃(control), the relative expression level of 9 laccase genes(i.e. Llac1-3, Llac5 and Llac7-11) were up-regulated cultured at 20℃. Besides, the expression level of genes such as Llac5, Llac7, and the rest 8 laccase genes were up-regulated at 28℃. While 6 genes(including Llac1-2, Llac4, Llac7-8 and Llac10) were down-regulated under 30℃ treatment. It can promote Llac genes expression cultured at 20℃, while 30℃ treatment inhibit the expression of the isozyme genes.
The laccase activity and its gene expression regulation characteristics under different temperature cultures(20℃, 25℃, 28℃and 30℃) were studied with the Lentinus edodes Xin808 to analyze the effect of its diameter of the mycelium ring and oxidation ring. The laccase activity was measured by liquid fermentation, and the relative expression level of lac was detected by qPCR technique. The results showed that, the growth rate of Lentinus edode mycelium, formation of oxidation ring, and laccase activity were all higher under 25℃ cultivation temperature. While 30℃ inhibited the growth of mycelium and the formation of oxidation ring and resulted into the lowest laccase activity. Moreover, the Lac genes exhibited differential expression profiles cultured in different temperature. Compared to the 25℃(control), the relative expression level of 9 laccase genes(i.e. Llac1-3, Llac5 and Llac7-11) were up-regulated cultured at 20℃. Besides, the expression level of genes such as Llac5, Llac7, and the rest 8 laccase genes were up-regulated at 28℃. While 6 genes(including Llac1-2, Llac4, Llac7-8 and Llac10) were down-regulated under 30℃ treatment. It can promote Llac genes expression cultured at 20℃, while 30℃ treatment inhibit the expression of the isozyme genes.
引文
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[18] 秦澎, 辜运富, 曾先富, 等. 香菇漆酶高产菌株筛选及漆酶基因的表达研究 [J]. 菌物学报, 2017, 36: 1243
[19] 黄书文, 刘爱和, 吴建雄. 不同培养温度与时间对香菇生殖生长的影响 [J]. 中国食用菌, 2014, 33: 65.
[20] 刘尚旭, 董佳里, 张义正. 糙皮侧耳菌木质素降解酶的比较研究 [J]. 四川大学学报: 自然科学版, 2000, 37: 594.
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[22] 万云洋, 杜予民, 杨建红,等. 漆树漆酶两种同工酶分离纯化与特性研究 [J]. 武汉大学学报:理学版, 2003, 49: 201.
[23] 沈柯宇, 张西蓓, 秦澎, 等. 4种重金属对灵芝漆酶活性及转录表达的影响 [J]. 应用与环境生物学报, 2017, 23: 448.
[24] Ohga S, Royse D J. Transcriptional regulation of laccase and cellulase genes during growth and fruiting of Lentinula edodes on supplemented sawdust. [J]. Fems Microbiol Letters, 2001, 201:111.
[2] 谭丽泉, 阮小文, 余梅, 等. 白腐真菌产木质素过氧化物酶影响因子的研究[J]. 工业安全与环保, 2014, 40: 58.
[3] Calò F, Parise M. Waste management and problems of groundwater pollution in karst environments in the context of a post-conflict scenario: The case of Mostar (Bosnia Herzegovina)[J]. Habitat Inter, 2009, 33: 63.
[4] Janusz G, Kucharzyk K H, Pawlik A, et al. Fungal laccase, manganese peroxidase and lignin peroxidase: Gene expression and regulation [J]. Enzym Microbial Technol, 2013, 52: 1.
[5] Martínez A T, Speranza M, Ruiz-Dueňas F J, et al. Biodegradation of lignocellulosics: microbial, chemical, and enzymatic aspects of the fungal attack of lignin [J]. Inter Microbiol, 2005, 8: 195.
[6] 刘忠川, 王刚刚. 真菌漆酶结构与功能研究进展 [J]. 生物物理学报, 2013, 29: 629.
[7] 茅文俊, 李燕, 周陈力,等. 草菇漆酶基因在子实体形成过程中的表达分析 [J]. 食用菌学报, 2016, 23 :1.
[8] 唐菊, 段传人, 黄友莹, 等. 白腐菌木质素降解酶及其在木质素降解过程中的相互作用 [J]. 生物技术通报, 2011 (10): 32.
[9] 孙淑静, 郭艳艳, 吴晓华,等. 不同食用菌品种产漆酶规律的研究 [J]. 中国食用菌, 2014, 33: 38.
[10] Chen L, Gong Y, Cai Y, et al. Genome sequence of the edible cultivated mushroom Lentinula edodes (Shiitake) reveals insights into lignocellulose degradation [J]. Plos One, 2016, 11: 1.
[11] 范芳芳. 白腐真菌漆酶基因表达调控及其功能研究 [D]. 武汉: 华中科技大学, 2013.
[12] Cavallazzi, Kasuya J R P, Soares C M, et al. Screening of inducers for laccase production by Lentinula edodes in liquid medium [J]. Brazil J Microbiol, 2005, 36: 383.
[13] 郭勇, 叶小金, 甘炳成, 等. 不同温度和光照培养条件下3种食用菌菌丝的茵落及菌丝形态的研究 [J]. 西南农业学报, 2011, 24: 2301.
[14] 魏巍, 余梦瑶, 许晓燕,等. 出芝温度对灵芝基质物质含量和酶活的影响 [J]. 中国生育健康杂志, 2016, 33: 49.
[15] 肖楚. 黑木耳漆酶高产菌株筛选及发酵条件、酶学性质的研究 [D]. 哈尔滨: 东北农业大学, 2012.
[16] Ren A, Li M J, Shi L, et al. Profiling and quantifying differential gene transcription provide insights into ganoderic acid biosynthesis in ganoderma lucidum in response to methyl jasmonate [J]. Plos One, 2013, 8: e650271.
[17] 陈霞连, 杨华侨, 黎佳欣, 等. 美容杜鹃RcHsfB3基因的克隆及表达分析 [J]. 四川大学学报:自然科学版, 2017,54: 405.
[18] 秦澎, 辜运富, 曾先富, 等. 香菇漆酶高产菌株筛选及漆酶基因的表达研究 [J]. 菌物学报, 2017, 36: 1243
[19] 黄书文, 刘爱和, 吴建雄. 不同培养温度与时间对香菇生殖生长的影响 [J]. 中国食用菌, 2014, 33: 65.
[20] 刘尚旭, 董佳里, 张义正. 糙皮侧耳菌木质素降解酶的比较研究 [J]. 四川大学学报: 自然科学版, 2000, 37: 594.
[21] 程科. 白腐菌漆酶分离纯化、酶学性质及其对阿特拉津最适降解条件的研究 [D]. 长春:吉林农业大学, 2012.
[22] 万云洋, 杜予民, 杨建红,等. 漆树漆酶两种同工酶分离纯化与特性研究 [J]. 武汉大学学报:理学版, 2003, 49: 201.
[23] 沈柯宇, 张西蓓, 秦澎, 等. 4种重金属对灵芝漆酶活性及转录表达的影响 [J]. 应用与环境生物学报, 2017, 23: 448.
[24] Ohga S, Royse D J. Transcriptional regulation of laccase and cellulase genes during growth and fruiting of Lentinula edodes on supplemented sawdust. [J]. Fems Microbiol Letters, 2001, 201:111.