灵芝线粒体及其对菌丝生长、主要活性成分影响的分析

线粒体是为细胞提供能量（ATP）的细胞器,携带着自己的DNA——mtDNA,已有多种灵芝属菌株的线粒体基因组相继报道,但对于种内的线粒体基因组的分析较少,对核相同、线粒体不同的菌株间差异的研究也鲜有报道。本研究对两株灵芝线粒体基因组进行组装注释,根据差异片段构建分子标记进行种间分类。结果显示：两株灵芝线粒体基因组大小分别为49 233bp、61 563bp的闭环结构,含有15个常见蛋白编码基因,rRNA大小亚基基因及26个携带氨基酸的tRNA基因,其差异区段主要为内含子序列、大亚基及基因间区。根据cob、cox2基因序列能够进行灵芝种间区分,用于灵芝种间鉴定。本研究还根据灵芝119、灵芝无孢的单核菌株构建同核异质体（TY-119、TY-W）,分析线粒体对菌落形态、菌丝生长速度及多糖、三萜成分的影响,结果显示：同核异质体TY-W与TY-119菌落形态上有一定差异,同核异质体TY-W菌丝生长速度为4.77mm/d,是TY-119菌丝生长速度4.50mm/d的1.06倍,同核异质体TY-119菌丝、子实体阶段多糖含量分别为4.45mg/g、12.14mg/g是TY-W菌丝体多糖含量（3.23mg/g）的1.38倍、子实体多糖含量（10.24mg/g）的1.19倍;同核异质体TY-W菌丝、子实体阶段的三萜含量分别为6.82mg/g、11.45mg/g是同核异质体TY-119菌丝体三萜含量（9.26mg/g）的0.74倍,子实体三萜含量（9.10mg/g）的1.26倍。利用高效液相色谱分析同核异质体中灵芝酸含量显示同核异质体TY-W灵芝酸A、灵芝酸E、灵芝酸F含量分别为3.77μg/mL、14.29μg/mL、12.91μg/mL;是TY-119灵芝酸A含量（2.59μg/mL）的1.46倍、灵芝酸E含量（13.65μg/mL）的1.17倍、灵芝酸F含量（12.72μg/mL）的1.06倍。对同核异质体菌丝、子实体阶段多糖、三萜合成通路关键基因（pgm、ugp、gls、hmgs、hmgr、mvd、fps、sqs）表达量进行检测,显示两菌株间多数基因具有显著性差异。结果表明线粒体的不同会影响灵芝菌落形态、菌丝生长速度及多糖、三萜的含量,有助于我们进一步研究线粒体基因组。

Analyses of mitochondrial genome and its influence on mycelial growth and active component of Ganoderma lingzhi

Mitochondrion is an important organelle carrying its own DNA, being essential for conversion of ATP into energy in eukaryotic cells. Many mitochondrial genomes of the genus Ganoderma have been reported so far. However, the intraspecific differences in nuclear and mitochondrial genomes of Ganoderma species are rarely reported. In this study, mitochondrial genomes of two Ganoderma lingzhi strains were assembled and annotated, and molecular markers were developed based on differential mitogenome fragments for intraspecific classification. The results showed that the mitogenome of G. lingzhi is a closed ring structure ranged from 49 233 bp to 61 563 bp in size, containing 15 common protein coding genes, small and large subunit genes of rRNA and 26 amino acids carrying tRNA genes. The main differential fragments located in introns, large subunit and intergenic regions. The phylogenetic tree was constructed based on different fragment sequences, and the results indicated that the sequences of cob gene and cox2 gene could be used in interspecies identification of Ganoderma. Monokaryotic commercial strain 119(Fujian, China) and sporeless strains W(Fujian, China) of G. lingzhi were used in construction of the isonuclear alloplasmic TY-W and TY-119 and the effects of mitochondria on the morphology, mycelial growth rate and triterpenoid composition of their fruiting bodies were evaluated. It was found that some differences appeared in the colony morphology of isonuclear alloplasmic TY-W and TY-119. The mycelial growth rate of TY-W was 4.77 mm/d, 1.06 times faster than the 4.5 mm/d growth rate of homonuclear heterogeneous TY-119. The content of polysaccharide in mycelial stage and fruiting stage of TY-W was 4.45 mg/g and 12.14 mg/g respectively, being 1.38 times and 1.19 times the content in mycelial stage(3.23 mg/g) and fruiting stage(10.24 mg/g) of TY-119. Triterpenoids in mycelial stage and fruiting stage of TY-W was 6.82 mg/g and 11.45 mg/g, respectively, being 0.74 times and 1.26 times the content in mycelial stage(9.26 mg/g) and fruiting stage(9.10 mg/g) of TY-119. Based on high performance liquid chromatography(HPLC) analysis, the content of ganoderic acids A(3.77μg/mL), E(14.92μg/mL) and F(12.91μg/mL) of TY-W in fruiting stage is respectively 1.46 times, 1.17 times and 1.01 times that of TY-119. The key genes’(pgm, ugp, gls, hmgs, hmgr, mvd, ls, fps and sqs) expression levels of polysaccharide and triterpenoid synthesis pathways in isonuclear alloplasmic fruiting bodies showed significantly different between TY-W and TY-119. In conclusion, the differences of mitochondria influenced the colony morphology, mycelial growth rate and the polysaccharide and triterpenoid content as well as key genes expression in G. lingzhi strains. Further studies are needed to improve our understanding of fungal mitochondrial genome.