组蛋白乙酰化对灵芝生长、灵芝多糖和灵芝酸生物合成的影响

doi:10.3864/j.issn.0578-1752.2020.03.014

摘要/Abstract

摘要：

【目的】组蛋白乙酰化修饰对真菌生长发育和次级代谢合成具有重要的调控作用。研究组蛋白乙酰化对静置培养的灵芝生长发育和主要代谢物合成的影响,为表观遗传手段提高灵芝多糖和灵芝酸生物合成提供理论依据。【方法】采用液体振荡-静置两阶段法培养灵芝。在静置培养阶段添加不同浓度（0、0.6、60、120和180 μmol·L ^-1）辛二酰苯胺异羟肟酸（SAHA）,采用常规方法测定或观察灵芝生物量、糖消耗、上层菌丝膜形成、菌丝体形态、灵芝孢子生成以及灵芝酸和灵芝多糖生物合成,利用蛋白免疫印迹技术测定灵芝组蛋白乙酰化水平;利用qRT-PCR技术对灵芝多糖（ugp、gls和pgm）、灵芝酸（hmgr、sqs、se和ls）生物合成关键基因以及灵芝全局调控因子相关基因（vet和LaeA）进行表达分析。【结果】SAHA处理使灵芝组蛋白H4乙酰化水平提高,最高为对照组的1.6倍;抑制了灵芝菌丝体生长和色素产生,改变了菌丝体的形态。灵芝孢子的形成也受到抑制,且SAHA浓度越大,抑制程度越明显。SAHA处理显著增加了灵芝多糖的产量,最高增加50%;灵芝酸生物合成受到抑制,与对照相比降低13%—27%;qRT-PCR分析结果表明SAHA处理下灵芝多糖与灵芝酸合成关键酶基因表达均有不同程度上调,其中灵芝多糖合成关键酶基因提升1.5—3.5倍,灵芝酸合成关键酶基因提升1.8—12.1倍;灵芝全局性调控因子vet和LaeA的表达被抑制,仅为对照组的11.30%—62.4%。【结论】组蛋白乙酰化可通过灵芝全局调控因子调控灵芝生长发育进而影响灵芝酸生物合成,同时组蛋白乙酰化对灵芝多糖生物合也有影响。

关键词: 灵芝, 组蛋白乙酰化, 灵芝酸, 灵芝多糖, SAHA

Abstract:

【Objective】Histone acetylation modification plays an important role in the growth, development and metabolic synthesis of fungi. Few epigenetic studies of higher medicinal fungi were reported at present. In this study, the effects of histone acetylation on the growth and development of Ganoderma lucidum (G. lucidum) and the synthesis of its main metabolites were studied by adding chemical epigenetic inhibitor octanedianiline hydroxamic acid (SAHA), which provided a theoretical basis for improving the biosynthesis of G. lucidum polysaccharides and Ganoderma acid (GA) by epigenetic means.【Method】A two-stage cultivation, liquid fermentation combined with static cultivation, was applied to culture G. lucidum. The cell was treated with different concentration of SAHA (0, 0.6, 60, 120, and 180 μmol?L ^-1) during the liquid static cultivation of G. lucidum. Biomass, sugar consumption, mycelial mat formation, mycelial morphology, sporulation and biosynthesis of GA and G.lucidum polysaccharides were measured or observed by conventional methods. Histone acetylation levels of G. luidum were examined by Western blot, the relative expression levels of polysaccharides biosynthesis genes (e.g. ugp, gls, and pgm), GA biosynthesis genes (e.g. hmg, sqs, se, and ls) and global regulator vet, LaeA gene were detected by qRT-PCR. 【Result】The acetylation level of histone H₄ in G. lucidum treated with SAHA increased to 1.6 times as much as that under control group. SAHA inhibited the growth of G. lucidum mycelia and the production of pigments, and changed the morphology of mycelia. The formation of spores was also inhibited, and the higher the concentration of SAHA, the more obvious the inhibition degree. SAHA treatment significantly increased the yield of G. lucidum polysaccharides, up to 50%, and the biosynthesis of GA was inhibited, which decreased by 13%-27% compared with the control. The results of qRT-PCR analysis showed that the gene expression of the key enzymes in G.lucidum polysaccharides and GA synthesis were up-regulated in different degrees under SAHA treatment. The gene expression of the key enzymes in polysaccharides synthesis were increased by 1.5-3.5 times and that of the key enzymes in GA synthesis by 1.8-12.1 times. The expression of vet and LaeA genes, the global regulators, were inhibited, which was 11.3%-62.4% of the control group.【Conclusion】Histone acetylation could regulate the growth and development of G. lucidum through global regulatory factors, thus affecting the biosynthesis of GA, while histone acetylation also had an effect on G. lucidum polysaccharides biosynthesis.

Key words: Ganoderme lucidum, histone acetylation, ganoderic acid, Ganoderma lucidum polysaccharide, SAHA

张宗源,蒋咏梅,章文贤. 组蛋白乙酰化对灵芝生长、灵芝多糖和灵芝酸生物合成的影响[J]. 中国农业科学, 2020, 53(3): 632-641.

ZHANG ZongYuan,JIANG YongMei,ZHANG WenXian. Effects of Histone Acetylation on Ganoderma lucidum Growth, Polysaccharide and Ganoderic Acid Biosynthesis[J]. Scientia Agricultura Sinica, 2020, 53(3): 632-641.

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基因 Gene	引物序列（5′-3′） Primer sequence (5′-3′)		参考文献 Reference
hmgr	F-TCGCAGTGGCACAGGAGC	R-CCCGGTGTTGGTGTTAGAAG	[36]
sqs	F-TGACGCTTCCTGACGAGA	R-GTGGCAGTAGAGGTTGTA	[36]
ls	F-CTTCCGCAAGCACTACCCG	R-AGCAGATGCCCCACGAGCC	[36]
se	F-ACTTCTGCGGGATCATATTGG	R-TGTAGGATTTGCTCCTTCAGGT	[34]
LaeA	F-CTCCGCCGATTCTACTGG	R-ACGGTCTAGCCGCTCAAA	[34]
vet	F-GGACACGACCCTTGGAACA	R-CGGAAACGCGAACATAGCC	[34]
pgm	F-GGGCCTGAGGAAGAGGGTGA	R-CGGTTTCG GGGGAGAAGTAG	[29]
ugp	F-TGGTCTCGGAACTTCTATGGG	R-CAGTGCTTCTTCTCGTCCTCA	[29]
gls	F-TCGTTTGGG TTGGGTCTGT	R-GAAGCCCTTGTCGCTCTGC	[29]
18S- rRNA	F-TATCGAGTTCTGACTGGGTTGT	R-ATCCGTTGCTGAAAGTTGTAT	[36]