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) an...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.展开更多
目的:外生菌根真菌松茸Tricholoma matsutake(S.Ito et Imai)Sing.又名松口蘑,是一种极其珍稀昂贵的野生食用蘑菇资源。为了保护利用松茸资源的生物多样性,需要研究发展适用松茸的基因组DNA的提取方法。方法:对传统的液氮冷冻研磨的细...目的:外生菌根真菌松茸Tricholoma matsutake(S.Ito et Imai)Sing.又名松口蘑,是一种极其珍稀昂贵的野生食用蘑菇资源。为了保护利用松茸资源的生物多样性,需要研究发展适用松茸的基因组DNA的提取方法。方法:对传统的液氮冷冻研磨的细胞破壁方法加以改进,采用液氮冷冻与室温融化交替处理松茸菌丝体与其它供试真菌细胞,进而萃取、浓缩、沉淀DNA。结果:利用本文研究的方法分离纯化出了满足分子生物学实验要求的高质量细胞总DNA。结论:该法相对简便、安全,可行、可靠,对供试真菌培养物需求量较少,既适用于担子真菌松茸,也适用于供试的其它真菌类群如酵母菌、丝状霉菌,应用前景广阔。展开更多
文摘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.
文摘目的:外生菌根真菌松茸Tricholoma matsutake(S.Ito et Imai)Sing.又名松口蘑,是一种极其珍稀昂贵的野生食用蘑菇资源。为了保护利用松茸资源的生物多样性,需要研究发展适用松茸的基因组DNA的提取方法。方法:对传统的液氮冷冻研磨的细胞破壁方法加以改进,采用液氮冷冻与室温融化交替处理松茸菌丝体与其它供试真菌细胞,进而萃取、浓缩、沉淀DNA。结果:利用本文研究的方法分离纯化出了满足分子生物学实验要求的高质量细胞总DNA。结论:该法相对简便、安全,可行、可靠,对供试真菌培养物需求量较少,既适用于担子真菌松茸,也适用于供试的其它真菌类群如酵母菌、丝状霉菌,应用前景广阔。
