PEG介导的环链虫草菌遗传转化体系建立及hsp30基因抗逆功能分析OA
Establishment of a PEG-mediated genetic transformation system for Cordyceps cateniannulata and analyses of hsp30 gene function in stress tolerance
环链虫草 Cordyceps cateniannulata 是一种对农林害虫具有重要的生防潜力的昆虫病原真菌.虽然完成了该菌转录组测序工作,但当前该菌遗传转化体系尚未建立,严重制约了其功能基因研究的开展.本研究首次成功制备了环链虫草的原生质体,并建立了 PEG 介导的原生质体遗传转化体系,实现了外源 GFP 基因的稳定表达.新克隆了环链虫草hsp30家族的 2 个基因Cchsp30a与Cchsp30b(GenBank 登录号分别为 PX352604 和 PX352605);并利用所建立的遗传转化体系获得Cchsp30a与Cchsp30b增强表达突变株.表型分析表明,突变株在热激(48℃)、氧化胁迫(甲萘醌)、盐胁迫(NaCl)、细胞壁胁迫(刚果红)下的孢子萌发率,以及在紫外辐射下的芽管生长,均显著优于野生型,证实了Cchsp30基因在广谱抗逆性中的正向调控作用.进一步的转录组分析揭示,热激条件下,突变株中大量差异表达基因显著富集于 ABC 转运蛋白等胁迫相关通路.其中,ABCC 转运蛋白亚家族的基因在突变株中差异表达最为突出,暗示Hsp30可能通过调控该亚家族成员的转录,进而影响细胞的物质转运与逆境适应能力.综上所述,本研究不仅建立了环链虫草的遗传操作体系,而且初步明确了Cchsp30的广谱抗逆功能,并发现其可能通过调控ABCC转运蛋白亚家族的转录参与逆境适应过程,这些发现为进一步探索Cchsp30在该昆虫病原真菌抗逆性机制中的作用提供了线索.
Cordyceps cateniannulata is an entomopathogenic fungus with significant biocontrol potential against agricultural and forestry pests.Although the transcriptome sequencing of this fungus has been completed,the lack of establishment of a genetic transformation system has severely hindered functional gene research.In this study,protoplasts of C.cateniannulata were successfully prepared and a PEG-mediated protoplast genetic transformation system was established,achieving stable expression of the exogenous GFP gene.Two novel hsp30 genes,Cchsp30a and Cchsp30b,were cloned from C.cateniannulata(GenBank accession numbers:PX352604 and PX352605,respectively).Using the established transformation system,overexpression mutant strains of Cchsp30a and Cchsp30b were obtained.Phenotypic analysis revealed that,compared to the wild-type strain,the mutant strains exhibited significant enhancement of spore germination rates under heat shock(48 ℃),oxidative stress(menadione),salt stress(NaCl),and cell wall stress(Congo red),and enhanced germ tube elongation under UV radiation.These results confirm the positive regulatory role of Cchsp30 genes in broad-spectrum stress tolerance.Further transcriptome analysis under heat stress showed that a large number of differentially expressed genes(DEGs)in the mutants were significantly enriched in stress-related pathways,particularly the ABC transporter pathway.Notably,genes belonging to the ABCC transporter subfamily were most prominently differentially expressed in the mutants,suggesting that Hsp30 might enhance cellular stress adaptation by modulating the transcription of ABCC subfamily members,thereby influencing substance transport.In summary,this study not only established a genetic manipulation system for C.cateniannulata,but also but also preliminarily characterized the broad-spectrum stress resistance function of Cchsp30 and revealed its potential involvement in stress adaptation through regulating the transcription of ABCC subfamily transporters.These findings provide clues for further exploring the role of Cchsp30 in the stress resistance mechanisms of this entomopathogenic fungus.
王定锋;李良德;李慧玲;周文鲜;张辉
福建省农业科学院茶叶研究所,福建 福州 350013福建省农业科学院茶叶研究所,福建 福州 350013福建省农业科学院茶叶研究所,福建 福州 350013福建省农业科学院茶叶研究所,福建 福州 350013福建省农业科学院茶叶研究所,福建 福州 350013
PEG 介导环链虫草原生质体遗传转化hsp30基因ABC 转运蛋白
PEG-mediatedCordyceps cateniannulataprotoplastgenetic transformationhsp30 geneATP-binding cassette(ABC)transporters
《菌物学报》 2026 (6)
189-209,21
福建省自然科学基金(2022J01477)福建省农业科学院青年英才项目(YC2021008) This work was supported by the Natural Science Foundation of Fujian Province(2022J01477)and the'Fund for Hundred'Talent Program of Young Science Elite of the Fujian Academy of Agricultural Sciences(YC2021008).
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