担子菌交配型基因的克隆及功能研究进展

担子菌中已有２０多个交配型基因被克隆,Ａ、Ｂ位点的交配型基因具有明显不同的结构特征。Ａ位点的交配型基因编码两类含相似同源结构域的蛋白质,两种蛋白质形成异源二聚体,调节依赖Ａ因子的发育过程;而Ｂ位点的交配型基因编码信息素及其受体。交配型基因编码的同源结构域蛋白质在植物、动物和菌类中都具有广泛的保守性。     

Relationship between Monokaryotic Growth Rate and Mating Type in the Edible Basidiomycete Pleurotus ostreatus

The edible fungus Pleurotus ostreatus (oyster mushroom) is an industrially produced heterothallic homobasidiomycete whose mating is controlled by a bifactorial tetrapolar genetic system. Two mating loci (matA and matB) control different steps of hyphal fusion, nuclear migration, and nuclear sorting during the onset and progress of the dikaryotic growth. Previous studies have shown that the segregation of the alleles present at the matB locus differs from that expected for a single locus because (i) new nonparental B alleles appeared in the progeny and (ii) there was a distortion in the segregation of the genomic regions close to this mating locus. In this study, we pursued these observations by using a genetic approach based on the identification of molecular markers linked to the matB locus that allowed us to dissect it into two genetically linked subunits (matBα and matBβ) and to correlate the presence of specific matBα and matA alleles with differences in monokaryotic growth rate. The availability of these molecular markers and the mating type dependence of growth rate in monokaryons can be helpful for marker-assisted selection of fast-growing monokaryons to be used in the construction of dikaryons able to colonize the substrate faster than the competitors responsible for reductions in the industrial yield of this fungus.     

Blue Light Overrides Repression of Asexual Sporulation by Mating Type Genes in the Basidiomycete Coprinus cinereus

Monokaryotic mycelia of the homobasidiomycete Coprinus cinereus form asexual spores (oidia) constitutively in abundant numbers. Mycelia with mutations in both mating type loci (Amut Bmut homokaryons) also produce copious oidia but only when exposed to blue light. We used such an Amut Bmut homokaryon to define environmental and inherent factors that influence the light-induced oidiation process. We show that the Amut function causes repression of oidiation in the dark and that light overrides this effect. Similarly, compatible genes from different haplotypes of the A mating type locus repress sporulation in the dark and not in the light. Compatible products of the B mating type locus reduce the outcome of light on A-mediated repression but the mutated B function present in the Amut Bmut homokaryons is not effective. In dikaryons, the coordinated regulation of asexual sporulation by compatible A and B mating type genes results in moderate oidia production in light. Copyright 1998 Academic Press.     

Mating System and Basidiospore Formation in the Lignin-Degrading Basidiomycete Phanerochaete chrysosporium

Prototrophic strains recovered from crosses between auxotrophic strains of the lignin-degrading basidiomycete Phanerochaete chrysosporium were induced to fruit. The progeny of most of these self-crosses were prototrophic, indicating that the nuclei of the original prototroph were wild-type recombinants rather than complementary heterokaryons and that the binucleate basidiospores of this organism are homokaryotic. Various wild-type strains were shown to have multinucleate cells lacking clamp connections and to possess a variable number of sterigmata per basidium. Colonies arising from single conidia of various wild-type strains were all capable of producing fruit bodies and basidiospores. In addition, single basidiospores from three wild-type strains all produced fruit bodies and basidiospores. Nonfruiting as well as fruiting isolates were obtained from single basidiospores of five other wild-type strains. Basidiospores from these fruiting isolates always yielded colonies that fruited, again indicating that the spores are homokaryotic. Nonfruiting isolates from the same strain did not produce basidiospores when allowed to form a heterokaryon, implying that these isolates do not represent mating types. All this evidence indicates that P. chrysosporium has a primary homothallic mating system. In addition to fruiting and nonfruiting phenotypes, basidiospores from strain OGC101, a derivative of ME-446, gave rise to colonies which did not grow on cellulose (Cel⁻). The fruiting, nonfruiting, and Cel⁻ phenotypes differed from each other and from the parental wild-type strain in a variety of characteristics, including growth, conidiation, and evolution of ¹⁴CO₂ from ¹⁴C-side chain-labeled lignin, indicating that strain OCG101 is a heterokaryon.     

Novel Haloperoxidase from the Agaric Basidiomycete Agrocybe aegerita Oxidizes Aryl Alcohols and Aldehydes

Agrocybe aegerita, a bark mulch- and wood-colonizing basidiomycete, was found to produce a peroxidase (AaP) that oxidizes aryl alcohols, such as veratryl and benzyl alcohols, into the corresponding aldehydes and then into benzoic acids. The enzyme also catalyzed the oxidation of typical peroxidase substrates, such as 2,6-dimethoxyphenol (DMP) or 2,2′-azinobis-(3-ethylbenzothiazoline-6-sulfonate) (ABTS). A. aegerita peroxidase production depended on the concentration of organic nitrogen in the medium, and highest enzyme levels were detected in the presence of soybean meal. Two fractions of the enzyme, AaP I and AaP II, which had identical molecular masses (46 kDa) and isoelectric points of 4.6 to 5.4 and 4.9 to 5.6, respectively (corresponding to six different isoforms), were identified after several steps of purification, including anion- and cation-exchange chromatography. The optimum pH for the oxidation of aryl alcohols was found to be around 7, and the enzyme required relatively high concentrations of H₂O₂ (2 mM) for optimum activity. The apparent K_m values for ABTS, DMP, benzyl alcohol, veratryl alcohol, and H₂O₂ were 37, 298, 1,001, 2,367 and 1,313 μM, respectively. The N-terminal amino acid sequences of the main AaP II spots blotted after two-dimensional gel electrophoresis were almost identical and exhibited almost no homology to the sequences of other peroxidases from basidiomycetes, but they shared the first three amino acids, as well as two additional amino acids, with the heme chloroperoxidase (CPO) from the ascomycete Caldariomyces fumago. This finding is consistent with the fact that AaP halogenates monochlorodimedone, the specific substrate of CPO. The existence of haloperoxidases in basidiomycetous fungi may be of general significance for the natural formation of chlorinated organic compounds in forest soils.     

Mating tests in Agrocybe cylindracea sensu lato. Recognition of Agrocybe wrightii as a novel species

Mating compatibility tests among seven collections of A. cylindracea (Basidiomycetes, Agaricales) from distant geographic origins were carried out with the aim of determining if A. cylindracea includes one or more taxons from a biological perspective. Fruit body production was used to obtain spore prints, monosporic cultures and to evaluate the fructification potential of compatible crossings. Protoplast isolation, purification and regeneration were used to attempt obtaining monokaryotic isolates from 2-spored specimens. Mating tests evidenced inter-fertility of Asian, American and European strains and the inter-sterility of the Argentinean strain WT-54. These results, in addition to mitochondrial small subunit variable domains phylogeny and morphological data, lead us to consider this strain as a novel species, A. wrightii. The European, Asian and the rest of the American strains belong to A. cylindracea. The concept of A. cylindracea sensu lato includes at least two different species: A. cylindracea and A. wrightii.     

Wide Distribution of Mitochondrial Genome Rearrangements in Wild Strains of the Cultivated Basidiomycete Agrocybe aegerita

We used restriction fragment length polymorphisms to examine mitochondrial genome rearrangements in 36 wild strains of the cultivated basidiomycete Agrocybe aegerita, collected from widely distributed locations in Europe. We identified two polymorphic regions within the mitochondrial DNA which varied independently: one carrying the Cox II coding sequence and the other carrying the Cox I, ATP6, and ATP8 coding sequences. Two types of mutations were responsible for the restriction fragment length polymorphisms that we observed and, accordingly, were involved in the A. aegerita mitochondrial genome evolution: (i) point mutations, which resulted in strain-specific mitochondrial markers, and (ii) length mutations due to genome rearrangements, such as deletions, insertions, or duplications. Within each polymorphic region, the length differences defined only two mitochondrial types, suggesting that these length mutations were not randomly generated but resulted from a precise rearrangement mechanism. For each of the two polymorphic regions, the two molecular types were distributed among the 36 strains without obvious correlation with their geographic origin. On the basis of these two polymorphisms, it is possible to define four mitochondrial haplotypes. The four mitochondrial haplotypes could be the result of intermolecular recombination between allelic forms present in the population long enough to reach linkage equilibrium. All of the 36 dikaryotic strains contained only a single mitochondrial type, confirming the previously described mitochondrial sorting out after cytoplasmic mixing in basidiomycetes.     

Patterns of Mating and Mitochondrial DNA Inheritance in the Agaric Basidiomycete Coprinus Cinereus

Patterns of mating and mitochondrial DNA (mtDNA) inheritance were investigated for the Basidiomycete, Coprinus cinereus in order to better understand the relationship of reproductive biology and mtDNA evolution in fungi. Results showed that the unique mating system of basidiomycetes can lead to the formation of mitochondrial mosaics (i.e., colonies composed of sectors differing in mtDNA). Mitochondria do not migrate along with nuclei during mating. Intracellular mixed or recombinant mtDNA molecules were not observed. Interestingly, it was found that mating asymmetry, caused by nonreciprocal nuclear migration, may be an important part of the reproductive biology of C. cinereus.     

Expression of Proteins and Glycoproteins Encoded by the Haploid Nuclei in the Dikaryotic State in the Basidiomycete Agrocybe aegerita

The total proteins and concanavalin A-binding glycoproteins of the cultivated mushroom Agrocybe aegerita were studied in homokaryotic siblings and in dikaryotic strains. The glycoproteins exhibited considerable variability compared with the proteins; the genetic diversity detected in homokaryons in the glycoprotein analysis was 30-fold higher than the genetic diversity revealed by protein analysis, and the glycoprotein patterns could be used to characterize individual genotypes. We found that the expression of glycoproteins in haploid nuclei was significantly asymmetric when the nuclei were paired in dikaryons. The expression levels of the two component nuclei depended on their genotypes, and each haploid nucleus was characterized by its level of expression. Furthermore, some specific glycoproteins that were not detected in all of the homokaryons were newly synthesized in the dikaryotic strains. Among these was a glycoprotein designated gpAa-65, which was identified in all of the dikaryotic strains and appeared to be a good molecular marker of the dikaryotic state.     

pH Regulates White-Opaque Switching and Sexual Mating in Candida albicans

As a successful commensal and pathogen of humans, Candida albicans encounters a wide range of environmental conditions. Among them, ambient pH, which changes frequently and affects many biological processes in this species, is an important factor, and the ability to adapt to pH changes is tightly linked with pathogenesis and morphogenesis. In this study, we report that pH has a profound effect on white-opaque switching and sexual mating in C. albicans. Acidic pH promotes white-to-opaque switching under certain culture conditions but represses sexual mating. The Rim101-mediated pH-sensing pathway is involved in the control of pH-regulated white-opaque switching and the mating response. Phr2 and Rim101 could play a major role in acidic pH-induced opaque cell formation. Despite the fact that the cyclic AMP (cAMP) signaling pathway does not play a major role in pH-regulated white-opaque switching and mating, white and opaque cells of the cyr1/cyr1 mutant, which is defective in the production of cAMP, showed distinct growth defects under acidic and alkaline conditions. We further discovered that acidic pH conditions repressed sexual mating due to the failure of activation of the Ste2-mediated α-pheromone response pathway in opaque a cells. The effects of pH changes on phenotypic switching and sexual mating could involve a balance of host adaptation and sexual reproduction in C. albicans.     

Mating Type Interaction in Closterium peracerosum-strigosum-littorale: Mating Induced Protoplast Release

Mating type interaction between heterothallic strains of Closteriumperacerosum-strigosum- littorale was studied during the conjugationprocess. When vegetative cells of opposite mating types weremixed under the nitrogen depleted mating conditions, the formationof conjugation-papilla and the release of the protoplast withinthe distended papilla from the gametangial cell were shown inboth the paired and unpaired cells. The protoplasts of pairedcells fused to form zygotes, while those of unpaired cells broke.When cells were mixed at a ratio of around 5 to 3 (mating typeplus to mating type minus), sexual activation occurred at thehighest frequency. Mixing at a ratio of 1 to 3, however, inducedthe highest frequency of disrupted cells. Mating type plus cellswere found to be broken specifically when direct contact withmating type minus cells was prevented. This disruption seemedto be mediated by a factor secreted from mating type minus cells. (Received March 20, 1981; Accepted August 18, 1981)     

Mating Type Inheritance in Glaucoma*

SYNOPSIS. Mating was observed in collections of Glaucoma from 2 localities in Illinois. The collections could be divided into 2 syngens on the basis of mating type reactions. Corticotype analysis showed that syngen 1 was intermediate in meridian number between Glaucoma chattoni and Glaucoma scintillans, while syngen 2 had the same range as Glaucoma scintillans (Lee, unpublished). The major features of mating type inheritance in syngen 1 are that exconjugant clones usually have identical mating types and that mating types I and II appear in an approximately 1:1 ratio in successive generations. This is the result expected in genic mating type inheritance if one of the original parents was a homozygote and the other a heterozygote at the mating type locus. No significant immaturity period was found in these strains. Only 2 viable pairs were obtained from syngen 2 crosses, but results from these suggest that mating type inheritance may be epigenetic and a longer immaturity period may characterize this syngen.     

Cloning and Analysis of Partial B Mating Gene Pheromone Receptor in Agrocybe salicacola (Strophariaceae)

A 4231bp DNA fragment of B mating type pheromone receptor from strain YAASM0711 of Agrocybe salicacola was obtained by using degenerate PCR and DNA walking techniques. The result of alignment and structure prediction of DNA sequences showed that one 1194bp nucleotides gene ASRcb1 encoding B mating pheromone receptor was found, including four introns(72bp, 49bp, 48bp and 41bp) and five extrons (217bp, 113bp, 67bp, 138bp and 449bp). The spliced open reading frame (ORF) contains 984bp nucleotides encoding 327 amino acid residues, and includes seven transmembrane protein regions as in its similar sequences of Coprinus cinerea and Laccaria bicolor pheromone receptors. The genetic evolution of pheromone receptor showed ASRcb1 was clustered with more receptors, which suggested multiple ways of evolution occurred in fungi.     

杨柳田头菇交配型因子与菌丝生长速度关系

以杨柳田头菇（Agrocybe salicacola）菌株711子实体为材料,经担孢子弹射,用稀释分离法获得224个单孢菌株,其中单核菌株210个,交配试验及单孢出菇证明其性遗传模式为四极异宗结合,4种交配型的比例为AxBx∶AxBy∶AyBx∶AyBy为47∶59∶53∶51。研究结果还表明,杨柳田头菇4种交配型的数量、比例与单孢萌发速度、生长速度相关,生长速度较慢的菌株基本上属于一种交配型,只有少数生长慢的菌株属于另外两种交配型。值得注意的是,尽管快（F）-慢（S）配对的异核体与快（F）-快（F）配对的异核体在YPD平板上生长速度无明显差别,但在聚丙烯栽培袋上F-S异核体的生长速度明显快于F-F异核体,这说明交配因子A和B与生长速度基因可能连锁,且存在重组现象,F-S交配的异核体在生长上有优势,可能是人工选择后交配因子亚基减少导致偏分离发生的原因。     

Species-Specific and Mating Type-Specific DNA Regions Adjacent to Mating Type Idiomorphs in the Genus Neurospora

Mating type idiomorphs control mating and subsequent sexual development in Neurospora crassa and were previously shown to be well conserved in other Neurospora species. The centromere-proximal flanks of the A and a idiomorphs, but not the distal flanks from representative heterothallic, pseudohomothallic, and homothallic Neurospora species contain apparent species-specific and/or mating type-specific sequences adjacent to the well-conserved idiomorphs. The variable flank is bordered by regions that are highly homologous in all species. The sequence of ~1 kb immediately flanking the conserved idiomorphs of each species was determined. Sequence identity between species ranged from 20% (essentially unrelated) to >90%. By contrast, the mt-A1 gene shows 88-98% identity. Sequence and hybridization data also show that the centromere-proximal flanks are very different between the two mating types for N. intermedia, N. discreta, and N. tetrasperma, but not for N. sitophila and N. crassa. The data suggest a close evolutionary relationship between several of the species; this is supported by phylogenetic analysis of their respective mt-A1 genes. The origin of the variable regions adjacent to the evolutionarily conserved mating type idiomorphs is unknown.