绿色木霉Tv-1511组蛋白乙酰化酶编码基因TvGCN5的功能分析

doi:10.13560/j.cnki.biotech.bull.1985.2021-0998

摘要/Abstract

摘要：

绿色木霉在植物促生抗逆、生物防治、纤维素酶生产和生物质利用等方面有着广泛用途。组蛋白乙酰化酶在基因表达调控中发挥着重要作用,GCN5是最具代表性的组蛋白乙酰化酶之一。本文针对绿色木霉TvGCN5基因的功能进行了分析研究。通过敲除绿色木霉组蛋白乙酰化酶TvGCN5基因建立绿色木霉Tv-1511-△GCN5缺失突变菌株,同时通过构建TvGCN5基因过表达载体获得绿色木霉GCN5基因过表达菌株Tv-1511-GCN5-OE,以研究绿色木霉Tv-1511组蛋白乙酰化酶编码基因TvGCN5在促生抗逆、产酶等方面的功能。结果表明,相比较野生型,TvGCN5过表达的Tv-1511-GCN5-OE菌株,在胁迫应答方面,具有更强的盐胁迫和高温胁迫耐受性;在对植物促生方面,具有更强的IAA生产能力,促生效果更好;在产酶方面,具有更更强的多种纤维素酶的生产和分泌能力。而缺失型菌株Tv-1511-△GCN5,相比较野生型,则在这些方面普遍降低,效果减弱。因此,TvGCN5基因在木霉促生抗逆、次级代谢以及产纤维素酶等方面可能起着重要作用。

关键词: 绿色木霉, 组蛋白乙酰化酶, 基因编辑, 基因功能, 生理特性

Abstract:

Trichoderma viride presents a wide range of applications in plant growth promotion and stress resistance,biological control,cellulase production and biomass utilization. Histone acetylation plays an important role in regulating transcriptional expression of gene. GCN5 is the most representative histone acetylase. First,the function of TvGCN5 gene in Trichoderma viride Tv-1511 was analyzed,then the deletion mutants（Tv-1511-△GCN5）was constructed by deleting TvGCN5 gene,and the overexpression mutants（Tv-1511-GCN5-OE）of GCN5 gene in the Tv-1511 was obtained by constructing TvGCN5 gene overexpression vector,for studying the functions of TvGCN5 in the Tv-1511 in growth-promoting and stress resistances as well as enzyme production. The results showed that the over-expression of TvGCN5 resulted in the longer mycelia diameter and more biomass under salt stress and high temperature stress compared with wild-type Tv-1511. Furthermore,the Tv-1511-△GCN5 strains obtained enhanced growth-promoting effect of plant by increasing the synthetic capacity of IAA. Notably,the higher production of cellulase was observed in the Tv-1511-GCN5-OE strain. The deletion mutant（Tv-1511-△GCN5）demonstrated the decrease of all above aspects,i.e.,the effects was weaker. Thus,TvGCN5 gene plays an important role in plant growth promotion,stress tolerance and cellulase production.

Key words: Trichoderma viride, histone acetylase GCN5, gene editing, gene function, physiological characteristics

张豪, 李哲, 郭凯, 黄艳华, 郝永任. 绿色木霉Tv-1511组蛋白乙酰化酶编码基因TvGCN5的功能分析[J]. 生物技术通报, 2022, 38(5): 136-148.

ZHANG Hao, LI Zhe, GUO Kai, HUANG Yan-hua, HAO Yong-ren. Functional Analysis of TvGCN5 Gene Encoding Histone Acetylase from Trichoderma viride Tv-1511[J]. Biotechnology Bulletin, 2022, 38(5): 136-148.

图/表 10

图1 TvGCN5基因敲除片段及过表达载体的构建 A,B：TvGCN5基因敲除片段的构建;C,D：TvGCN5基因过表达载体的构建

Fig. 1 Construction of knockout fragment and overexpression vector of TvGCN5 gene A,B：Fragment construction of knockout TvGCN5 gene. C,D：Construction of overexpression vector of TvGCN5 gene

图2 荧光定量PCR和Western blot鉴定TvGCN5基因过表达和缺失的木霉工程菌 A：荧光定量PCR鉴定木霉工程菌中TvGCN5基因的转录水平;B,C：Western blot鉴定木霉工程菌中TvGCN5基因的蛋白表达水平

Fig.2 Identification of overexpressing or deleting TvGCN5 gene in Trichoderma engineering strains by fluores-cence quantitative PCR and Western blot A：TvGCN5 gene transcription level in Trichoderma engineered strains by fluorescence quantitative PCR. B,C：Protein expression level of TvGCN5 in Trichoderma engineered strains by Western blot

图3 绿色木霉Tv-1511及其工程菌中的组蛋白乙酰化修饰水平的变化 A：试剂盒定量检测绿色木霉及其工程菌中组蛋白H3的乙酰化水平;B,C：Western blot检测绿色木霉及其工程菌中组蛋白H3的乙酰化水平

Fig.3 Changes in the levels of histone acetylation in T. viride Tv-1511 and its engineered strains A：Quantitative detection of histone H3 acetylation level in T. viride Tv-1511 and its engineered strains by assay kit. B,C：Detection of histone H3 acetylation level in T. viride Tv-1511and its engineered strains by Western blot

图4 绿色木霉Tv-1511原始菌株及其工程菌耐盐能力的分析 A,B：木霉培养皿耐盐（300 mmol/L NaCl）实验及菌落直径定量统计;C：盐胁迫（300 mmol/L NaCl）下木霉液体培养菌丝体干重

Fig.4 Analysis of the salt tolerances of T. viride Tv-1511 original strain and its engineered strains A,B：Petri dish test of the salt tolerance of Trichoderma and quantitative statistics of colony diameter. C：Dry weight of Trichoderma in liquid culture mycelium under salt stress

图5 绿色木霉Tv-1511原始菌株及其工程菌耐高温能力的分析 A,B：木霉培养皿耐热（35℃）实验及菌落直径定量统计;C：高温胁迫（35℃）下木霉液体培养菌丝体干重

Fig.5 Analysis of the high temperature tolerance of T. vir-ide Tv-1511 original strain and its engineered strains A,B：Petri dish test of the heat tolerance of Trichoderma and quantitative statistics of colony diameter. C：Dry weight of Trichoderma liquid culture mycelium under high temperature stress

图6 绿色木霉Tv-1511原始菌株及其工程菌对拟南芥促生能力的分析 A：绿色木霉及其工程菌对拟南芥生长的促进作用;B：绿色木霉及其工程菌处理后拟南芥植株鲜重的定量统计;C：绿色木霉及其工程菌处理后拟南芥植株叶片面积的定量统计

Fig.6 Analysis of the growth-promoting effects of T. viride Tv-1511 original strain and its engineering strains on Arabidopsis thaliana A：Promoting effects of T. viride and its engineering strains on the growth of A. thaliana. B：Quantitative statistics of fresh weight of Arabidopsis treated with T. viride and its engineered strains. C：Quantitative statistics of leaf areas of Arabidopsis treated with T. viride and its engineering strains

图7 绿色木霉Tv-1511原始菌株及其工程菌对椒样薄荷促生能力的分析 A：绿色木霉及其工程菌对椒样薄荷生长的促进作用;B：绿色木霉及其工程菌处理后椒样薄荷植株鲜重的定量统计;C：绿色木霉及其工程菌处理后椒样薄荷植株叶片面积的定量统计

Fig.7 Analysis of the growth-promoting effects of T. viride Tv-1511 original strain and its engineering strains on peppermint（Mentha piperita） A：Promoting effects of Trichoderma viride Tv-1511 and its engineering strains on the growth of peppermint. B：Quantitative statistics of fresh weight of peppermint treated with Trichoderma strains. C：Quantitative statistics of leaf area of peppermint treated with Trichoderma strains

图8 绿色木霉Tv-1511原始菌株及其工程菌对黄瓜促生能力的分析 A：绿色木霉及其工程菌对黄瓜生长的促进作用;B：绿色木霉及其工程菌处理后黄瓜植株鲜重的定量统计;C：绿色木霉及其工程菌处理后黄瓜植株叶片面积的定量统计

Fig.8 Analysis of the promoting effects of T. viride Tv-1511 original strain and its engineering strains on cucum-ber（Cucumis sativus L.） A：Promoting effects of Trichoderma viride Tv-1511 and its engineering strains on the growth of cucumber. B：Quantitative statistics of fresh weight of cucumber treated with Trichoderma strains. C：Quantitative statistics of leaf areas of cucumber treated with Trichoderma strains

图9 绿色木霉Tv-1511原始菌株及其工程菌发酵液中IAA含量的检测 A：IAA标准品的色谱图;B：绿色木霉及其工程菌发酵液中IAA含量的定量统计

Fig.9 Detection of IAA contents in the fermentation broths of T. viride Tv-1511 original strain and its engineer-ing strains A：Chromatogram of IAA standard. B：Quantitative statistics of IAA content in the fermentation broth of T. viride Tv-1511 and its engineering strains

图10 绿色木霉Tv-1511原始菌株及其工程菌产纤维素酶活性的分析 A：绿色木霉及其工程菌发酵液中滤纸酶活性的检测;B：绿色木霉及其工程菌发酵液中羟甲基纤维素酶活性的检测;C：绿色木霉及其工程菌发酵液中外切葡聚糖酶活性的检测;D：绿色木霉及其工程菌发酵液中β-葡萄糖苷酶活性的检测

Fig.10 Analysis of cellulase activities of T. viride Tv-1511 original strain and its engineering strains A：Detection of filter paper enzyme activity in the fermentation broth of T. viride Tv-1511 and its engineering strains. B：Detection of hydroxymethyl cellulase activity in the fermentation broth of T. viride Tv-1511 and its engineering strains. C：Detection of exoglucanase activity in the fermentation broth of T. viride Tv-1511 and its engineering strains. D：Detection of β-glucosidase activity in the fermentation broth of T. viride Tv-1511 and its engineering strains

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