球毛壳菌CGMCC 6882中Y1Ste20基因敲除重组质粒构建及对菌丝形态的影响
Construction of the Y1Ste20 gene knockout recombinant plasmid in Chaetomium globosum CGMCC 6882 and analysis of its effects on mycelial morphology
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摘要: 【目的】探究基因敲除解脂耶氏酵母Ste20激酶同源基因(Y1Ste20基因)对球毛壳菌CGMCC 6882菌落直径及菌丝形态的调控作用。【方法】采用CRISPR-Cas9基因编辑技术,构建靶向Y1Ste20基因的敲除重组质粒;通过PEG介导的原生质体转化法将该基因敲除重组质粒转入球毛壳菌CGMCC 6882原生质体中,获得Y1Ste20基因敲除菌株,并对其菌落生长特征及菌丝形态进行分析。【结果】利用CRISPR-Cas9基因编辑技术构建Y1Ste20基因敲除重组质粒(pFC332-5S rRNA-Ste20 gRNA),并成功转入球毛壳菌CGMCC 6882原生质体中,获得Y1Ste20基因敲除菌株球毛壳菌XY1104,其在固体培养基上的菌落直径((201.89±9.35)mm)小于球毛壳菌CGMCC 6882((434.35±10.03)mm),且在液体培养基中的菌丝呈细小短杆状,未发生缠绕,而球毛壳菌CGMCC 6882无独立菌丝,易形成菌球。【结论】Y1Ste20基因敲除导致球毛壳菌CGMCC 6882的菌落直径减小,菌丝呈细小短杆状并趋于密集生长,缠绕现象减少,表明Y1Ste20基因能参与调控球毛壳菌CGMCC 6882菌丝生长及形态建成。
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关键词:
- 球毛壳菌CGMCC 6882 /
- Y1Ste20基因 /
- 基因敲除重组质粒 /
- 菌丝形态
Abstract: 【Objective】This study aimed to investigate the regulatory role of the Ste20 kinase homolog gene from Yarrowia lipolytica(Y1Ste20) in Chaetomium globosum CGMCC 6882 after targeted gene knockout, with particular focus on its effects on colony diameter and hyphal morphology.【Methods】A Y1Ste20 gene knockout recombinant plasmid was constructed using CRISPR-Cas9 gene editing technology. The recombinant plasmid was introduced into C.globosum CGMCC 6882 protoplasts via PEG-mediated protoplast transformation to obtain the Y1Ste20 gene knockout strain C.globosum XY1104, and its colony growth characteristics and hyphal morphology were analyzed.【Results】The Y1Ste20 gene knockout recombinant plasmid (pFC332-5S rRNA-Ste20 gRNA) was constructed using CRISPR-Cas9 gene editing technology and successfully transferred into C.globosum CGMCC 6882 protoplasts, yielding the Y1Ste20 gene knockout strain C.globosum XY1104. On solid medium, the colony diameter of strain XY1104 was (201.89±9.35) mm, which was smaller than that of C.globosum CGMCC 6882 ((434.35±10.03) mm). In liquid culture, strain XY1104 exhibited fine, short, rod-like hyphae without entanglement, whereas C.globosum CGMCC 6882 lacked discrete hyphae and readily formed mycelial pellets.【Conclusion】Disruption of the Y1Ste20 gene led to a reduced colony diameter and hyphae that were fine, short, and rod-like, grew densely, and showed reduced entanglement in C.globosum CGMCC 6882. These findings indicate that Y1Ste20 is involved in regulating hyphal growth and morphogenesis in C.globosum CGMCC 6882. -
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