Cys_(2)His_(2)型转录因子GL26016对灵芝菌丝生长和灵芝酸合成的影响

A Cys2 His2 Zinc Finger Transcription Factor GL26016 Modulates Cell Growth and Ganoderic Acid Biosynthesis in Ganoderma lingzhi

克隆灵芝(Ganoderma lingzhi)基因gl26016,全长为1 169 bp,开放阅读框为1 107 bp,序列分析表明该基因编码Cys_(2)His_(2)(C_(2)H_(2))型转录因子。采用CRISPR/Cas9方法缺失该基因483 bp序列,通过PCR扩增及测序表明成功获得突变菌株△GL26016。对△GL26016与野生型菌株(WT)的菌丝生长和灵芝酸含量进行检测,结果表明:两者相比,菌丝形态和菌丝体干重差异不显著,△GL26016中灵芝酸含量较高。△GL26016中灵芝酸GA-Mk、GA-S、GA-Me在第6天的含量分别为(5.09±0.36)、(5.36±0.11)、(3.45±0.26)μg·mg^(-1),分别是WT菌株积累量的1.51、1.47、1.93倍。研究结果表明GL26016参与灵芝酸生物合成调控。

The complete gene sequence of the gl26016 gene of Ganoderma lingzhi was cloned,with a full length of 1169 bp and an open reading frame of 1107 bp.Protein sequence analysis showed that gl26016 encodes a Cys2His2(C2H2)type transcription factor.To investigate the function of gl26016,a 483 bp fragment was deleted in gl26016 by the CRISPR/Cas9 technology,and the gl26016 disrupted mutant strain was verified by PCR and sequencing.The results showed that the gl26016-disrupted strain(△GL26016)was successfully obtained.Both the wild-type strain(WT)and the△GL26016 strain showed similar dry mycelium weight and mycelium morphology.However,the accumulation of ganoderic acids in△GL26016 was higher than that in WT.On the 6th day of liquid static culture,the contents of GA-Mk,GA-S and GA-Me in△GL26016 were(5.09±0.36),(5.36±0.11),and(3.45±0.26)μg·mg^(-1),respectively,which were 1.51-folds,1.47-folds and 1.93-folds of those in WT,respectively.These results suggested that GL26016 plays an important role in GA biosynthesis in G.lingzhi.