应用简并引物扩增黑木耳信息素受体基因片段

Application of degenerate primers to amplify fragment of pheromone receptor gene in Auricularia auricula

Degenerate primers, br1-F and br1-R, designed based on the conserved amino acid sequence of STE3 pheromone receptor in Schizophyllum commune, were used to amplify genomic DNA of monkaryotic parental strains(H2, J3) and fifty-nine monokaryons of their F1 progenies in Auricularia auricula. A fragment of the PCR product 811bp in length were amplified from the parental strain H2, nine monokaryons of the H2 mating –type and fifteen ones of the J3 mating –type of F1 progenies. After cloning , sequencing the fragment, and analyzing the sequence by BLAST searching, no homologous sequences were found. However, the putative protein sequences translated from the DNA sequence had homeodomain protein called fungal STE3 pheromone receptor, and seventy-six homologous sequences were hit, with their e values ranged from 2e-35 to 6.6. It is predicted by the use of SOSUI program that the putative protein is a membrance protein including five transmembrance domains. The recombination ratio was 62.7% between the fragment of STE3 pheromone receptor gene and the mating type locus among F1 progenies of Auricularia auricula. This result indicated that the pheromone receptor gene was not linked to the mating type factor, as reported in Pholiota nameko, a bipolar edible mushroom. This study would lay a foundation for cloning the complete pheromone receptor gene and testing its function in the future.

Degenerate primers, brl-F and brl-R, designed based on the conserved amino acid sequence of STE3 pheromone receptor in Schizophyllum commune, were used to amplify genomic DNA of monkaryotic parental strains（H2, J3） and fifty-nine monokaryons of their F1 progenies in Auricularia auricula.A fragment of the PCR product 811bp in length were amplified from the parental strain H2, nine monokaryons of the H2 mating -type and fifteen ones of the J3 mating -type of F1 progenies. After cloning,sequencing the fragment, and analyzing the sequence by BLAST searching, no homologous sequences were found. However, the putative protein sequences translated from the DNA sequence had homeodomain protein called fungal STE3 pheromone receptor, and seventy-six homologous sequences were hit, with their e values ranged from 2e-35 to 6.6. It is predicted by the use of SOSUI program that the putative protein is a membrance protein including five transmembrance domains. The recombination ratio was 62.7% between the fragment of STE3 pheromone receptor gene and the mating type locus among F1 progenies of Auricularia auricula. This result indicated that the pheromone receptor gene was not linked to the mating type factor, as reported in Pholiota nameko, a bipolar edible mushroom. This study would lay a foundation for cloning the complete pheromone receptor gene and testing its function in the future.