桦纤孔菌有性生殖相关特征及交配型位点分析

对桦纤孔菌菌株MDJCBS88的显微形态、菌丝及担孢子核相进行了观察。采用棉籽壳培养基对担孢子萌发形成的菌株进行栽培试验，筛选出不形成子实体或子实体发育不完整的菌株，将这些菌株在平板上进行了亲和试验，分析桦纤孔菌的有性生殖方式；并基于基因组序列进行交配型基因克隆验证，分析桦纤孔菌的交配型位点结构。显微观察发现，桦纤孔菌菌丝没有锁状联合结构，菌丝细胞无核到多核；子实层担孢子可含0-4个不等的细胞核，不同时期弹射的担孢子含有的细胞核数量不同。桦纤孔菌担孢子萌发率极低，能萌发的担孢子多为早期弹射的担孢子；培养基也影响担孢子的萌发率，与PDA培养基和CYM培养基相比，桦木屑培养基最适合桦纤孔菌担孢子萌发，萌发率为4.55%。从担孢子萌发的96个菌株中获得了2个不结实菌株和9个结实不产孢菌株，占11.5%，这些菌株间亲和试验出现不同的表现特征，包括形成产孢子实体，产生菌丝纽结，相互融合和相互拮抗等现象，认为桦纤孔菌的有性生殖以次级同宗结合为主，并受交配型基因控制。交配型位点克隆测序后分析发现，桦纤孔菌交配型A位点共14 034 bp，含有一个MIP基因和两组HD1和HD2基因；交配型B位点包含3个疑似信息素受体基因和1个信息素前体编码基因。

Characteristics related to sexual reproduction and mating type locus of Inonotus obliquus

Microscopic morphology, hyphae and basidiospore nuclear phase of Inonotus obliquus MDJCBS88 were observed. The strains derived from basidiospores were cultivated on medium with cottonseed shell, and the cultures that did not form fruiting bodies or did not produce basidiospores were screened for compatibility test and observation of sexual reproduction mode. Mating type genes were cloned and sequenced, and mating site structure was analyzed. Microscopic observation showed that hyphae of I. obliquus have simple septa without clamp connection, and the mycelial cell contains no nuclei or multinuclei. Basidiospores in hymenium may contain 0-4 nuclei, and the number of nuclei in the basidiospores ejected at different stages was different. The germination rate of basidiospores was very low, and germination mainly occurred in early ejected basidiospores. The culture medium also affected the germination rate of basidiospores. Compared with PDA medium and CYM medium, birch sawdust medium was the most suitable for the germination of I. obliquus basidiospores, and the germination rate was 4.55%. Among the 96 strains derived from basidiospores, 11 sterile strains were obtained, accounting for 11.5% of the total strains. The compatibility test among these sterile strains showed different pheno-characteristics such as kink of mycelia, fusion of mycelia and antagonistic phenomenon. It was concluded that the sexual reproduction of I. obliquus was affected by mating type loci, and the sexual reproduction mode was mainly secondarily homothallism. Cloning and sequencing mating type loci found that the mating type A locus of I. obliquus was composed of 14 034 bp, containing a MIP gene and two groups of HD1 and HD2 genes. Mating type B locus contained three suspected pheromone receptor genes and one pheromone precursor coding gene.