【摘要】 以蛇足石杉Huperziaserrata内生真菌盘长孢状刺盘孢Cg01菌株为研究对象,利用PEG介导的同源重组转化体系,对Cg01组蛋白甲基化酶基因(histone methyltransferases,HMT)CgClr4和组蛋白去乙酰化酶基因(histonedeacetylase,HDAC)CgClr3、CgSir2进行基因敲除与回补,并通过实时荧光定量PCR(RT-qPCR)检测了回补株中对应基因表达量以及高效液相色谱HPLC检测突变体菌株中石杉碱甲huperzineA(HupA)产量。结果显示3个基因敲除突变体菌株ΔCgClr4、ΔCgClr3、ΔCgSir2的HupA产量分别为255μg/L、270μg/L、244μg/L,与野生型菌株相比分别下降了21.3%、16.6%、24.7%。在基因回补突变体菌株ΔCgClr4/CgClr4、ΔCgClr3/CgClr3、ΔCgSir2/CgSir2中,相应回补基因表达均与野生型无显著性差异,其HupA产量分别为351.9μg/L、334.7μg/L、331μg/L,回补菌株的HupA产量回复到野生型水平。结果表明这3个基因均具有调控内生真菌盘长孢状刺盘孢Cg01合成HupA的作用,为研究蛇足石杉内生真菌中石杉碱甲的合成调控机制提供了理论基础和新的思路。更多还原

【Abstract】 A histone methyltransferases(HMT) gene CgClr4 and two histone deacetylase(HDAC) gene, CgClr3 and CgSir2, were knocked out and then complemented in the endophytic fungus Colletotrichum gloeosporioides strain Cg01 in Huperzia serrata by the PEG-mediated genetic transformation system. The expression of the corresponding genes in the complemented strains were detected by RT-qPCR and huperzine A(HupA) production in the mutant strains were analyzed by high performance liquid chromatography(HPLC).It was found that the HupA production of the 3 gene knockout mutant strains CgClr4, CgClr3 and CgSir2 were 255μg/L, 270μg/L and 244μg/L respectively, decreased by 21.3%, 16.6% and 24.7% as compared with wild type(WT). There is no significant difference between the gene expression of the corresponding gene in the complemented strains CgClr4/CgClr4, CgClr3/CgClr3 and CgSir2/CgSir2 and that in the wild type strain, with the HupA production to be 351.9μg/L, 334.7μg/L and 331μg/L, respectively. The HupA production of complemented strains was restored to that of the wild type strain. The results showed that these three genes had the function of regulating the biosynthesis of HupA, which provided a theoretical basis and a new insight for studying the synthetic mechanism of HupA in endophytic fungi of Huperzia serrata.更多还原