Application Of PCR Technique In Study On The Mating Type Factor Of Auricularia Auricula

Auricularia auricula is a popular edible mushroom fungus with long cultivated history, and has important medical and healthy functions.The hybrid strain H2J3, obtained by mating of two protoplasted monokaryons H₂ and J3 of Auricularia auricula, was cultivated. Fifty-nine isolates were collected by discharging spores from the fruit-body of H2J3.Based on the mating type phenotype, the F₁ isolates were divided into two groups, and two isogenic pools, A₁ and A₂, were constructed by mixing equally the DNA of ten strains of each group respectively. One hundred and seventy-six special single primers were designed according to CAAX motif of pheromone precursor and conserved amino acid sequences of homeodomain encoded by A mating type genes in advanced fungi. PCR results showed that primer XY₁₆₁ could produce a special 1462bp DNA marker only in A₁ isogenic pool. Then the special DNA band was cloned and sequenced. The marker was probably located in downstream of pheromone gene by sequence analysis. BLAST results indicated that the DNA sequence similar to marker XY₁₆₁1462 was not found, but its translated protein sequence was similar to short chain dehydrogenase. The mating type locus of Pleurotus ostreatus and Cryptococcus neoformans also contain dehydrogenase genes. Above results suggested that DNA sequences within mating type locus are probably various among different fungi, but the protein sequences encoded by them share similar structure and function.Only employing combination of degenerate primer br1-F and br1-R, designed on STE3 pheromone receptor of Schizophyllum commune, can we obtain a special 811bp DNA band among parental strain H₂ and twenty-four F₁ monokaryons, including nine monokaryons with H₂ mating type and fifteen ones with J₃ mating type, accounting for 15.3% and 25.4% of all the testing monokaryons respectively. After cloning and sequencing to the special DNA band from strain 45 and 59, the BLAST program was used to search their similar sequences. Although any similar DNA sequences were not hit, theirtranslated protein sequences had similar ones-----fungal STE3 pheromone receptor. Thenumber of similar sequences was ninety-two, and their e value ranged from 2e-35 to 8.1. The pheromone receptor of Auricularia auricula was similar to counterparts of tetrapolar basidiomycetes on the basis of BLAST results. Sequence analysis displayed that the DNA sequence probably contain four introns. The SOSUI program predicted that putative protein is a membrance protein including five transmembrance helices. The partial sequence of pheromone receptor gene displayed high recombination among F₁ monokaryons, the possible reason is that this gene is independent of mating type locus,and is unlinked to mating type locus. Bipolar mushroom fungus, Pholiota nameko, also shares the character.Six couples of degenerate primers were designed in terms of conserved amino acid sequence of mitochondrial intermediate peptidase encoded by MIP gene that was tightly linked to mating type locus in some homobasidiomycete. Special DNA bands amplified by MIP primers could be got in some homobasidiomycete, but that was not the case in heterobasidiomycete. We could not obtain any DNA band in Auricularia auricula too, when employing MIP primers. This result consists with the fact that Auricularia auricula is a typical heterobasidiomycete.