Preliminary Studies On Molecular Mechanism Of Substrate-decomposing Ability Degeneration In Agaricus Bisporus

Agaricus bisporus (J.E.Lange) Imbach is a kind of edible mushroom cultivated and consumed worldwide, and has high economic value and important ecological significance. As2796 is the only main variety of A.bisporus used in China during more than 10 years, so the study on the molecular mechanism of the degeneration characteristics of this variety has important theoretical and practical significance. In this subject, the normal strain As2796, as well as two substrate-decomposing ability degeneration mutants derived from which, 2796-3 and 2796-5, were used as two comparison groups. The main research contents and results are showed as below.(1) The 2 degeneration strains were transferred on PDA medium for dozens of generations, and their degeneration characters were comfirmed to be stable. The normal and abnormal strains were mixed and cultured togather, and the results exclued the factor of microorganisms infection, suggesting the degeneration to be caused by gene variation. Different liquid media were designed for strains culturing, and 12 mycelia samples of 3 strains in 4 kinds of liquid media were harvested for further study.(2) The technique of SRAP with 153 pairs of primers was used for the analysis of DNA differences between Agaricus bisporus As2796 and its mutants, 2796-3 and 2796-5. As a result, no difference was found between two kinds of strains. Based on the result, it was guessed that the variation might occur in few genes and was difficult to be found at DNA level.(3) The technique of isozyme PAGE was used for the analysis of the 3 strains cultured in 4 kinds of liquid media. The results showed that the esterase (EST) and polyphenol oxidase (PPO) isozyme phenotypes made obvious differences between the normal and degeneration strains in some kinds of media, and the differences occurred in both of the two degeneration strains, suggesting there to be relationship between the isozyme activity and the substrate-decomposing ability degeneration of A.bisporus.(4) The technique of mRNA differential display reverse transcription-PCR (DDRT-PCR) with 24 pairs of primers was used to analyze mRNA differences between the wild type of A. bisporus strain As2796 and its substrate-decomposing ability degeneration strains 2796-3 and 2796-5 cultured in 4 kinds of different liquid media. As a result, 8 pairs of primers showed repeatable differences, and 9 special fragments were cloned. Sequencing analysis showed that 9 special fragments represented 5 different genes. Some of them have high homologies with an ATP-binding cassette transporter subfamily A protein, carbohydrate esterase family 4 protein, chitin deacetylase, acetyl xylan esterase II and some other hypothetical proteins of several kinds of organisms.(5) The full-length cDNA library of A.bisporus As2796 was constructed. The library had a content of 3.3×106 cfu/mL, an insertion range of about 0.7-4.0Kb, an average cDNA length of 1.5Kb, a recombination efficience of about 82%, and full-length ratio of about 35%. The coding sequences (CDS) of 5 different genes found in DDRT-PCR were got from the library by PCR amplification, which proved the validity of the library for amplifying full-length cDNA or CDS of A.bisporus genes in need.(6) Using the total DNAs of A.bisporus As2796 and 2 degeneration strains as templates, the CDS with introns and about 1200bp upstream regulative sequences of 5 different genes found in DDRT-PCR were amplified, cloned and sequenced. The comparison of the sequences among 3 strains showed that the genes and upstream sequences have no repeatable difference, so the degeneration might be caused by the mutation of regulative protein genes.(7) The conditions for A.bisporus proteome two-dimensional electrophoresis (2-DE or 2D-PAGE) were studied and optimized, and the proteome 2D-PAGE technique of A.bisporus was built up, resulting in clear 2D-PAGE patterns. It was showed that most of the proteins of A.bisporus focus in the range of molecular weight (MW) 20-120KDa and isoelectric point (pI) 5.2-7.8.(8) The technique of 2D-PAGE was used to analyze the protein expression differences between the wild type Agaricus bisporus As2796 and its substrate-decomposing ability degenerated strains 2796-3, 2796-5. As a result, two proteins were found to be up- and down-expressed, respectively, with obvious and repeatable profiles in both degenerated strains when grown in a liquid culture. Based on the results of MALDI-TOF-MS analysis and database searching, these two proteins were identified as an actin and a putative NADH dehydrogenase iron-sulfur protein 3, respectively, and both of which had close relationship with intracellular signal conduction or transcription regulation.In this work we found some gene fragments and proteins (enzymes) related to the substrate- decomposing ability degeneration of A.bisporus for the first time. Based on our study, the molecular mechanism of substrate-decomposing ability degeneration in A.bisporus was preliminarily supposed to be a course of gradual chain regulation, i.e. the degeneration phenotype was not caused by the variation of some certain structural protein genes, but the mutation of regulative protein genes, which changed the gene expression network, resulting the series of differences at mRNA, protein, cell and organism level, and finally brought the degeneration of substrate-decomposing ability. This supposition supported a good basic for the further research on the molecular mechanism of substrate-decomposing ability degeneration of A.bisporus.