【作者】 李安政；

【导师】 林芳灿；

【作者基本信息】 华中农业大学 ， 微生物学， 2006， 博士

【摘要】 香菇（Lentinula edodes）是一种异宗结合的食用蕈菌，具有由两个不连锁的交配型因子（A和B）控制的四极性交配系统。本研究以13个香菇菌株为材料，采用培养基转换培养（OWE-SOJ技术）和核迁移试验等方法对各菌株孢子单核体交配型因子组合进行了准确鉴定，并对其中可能产生了次级重组体的HL01菌株进行了进一步的分析和验证。同时，采用随机扩增多态性DNA／集团分离析（RAPD／BSA）、代表性差异分析（RDA）和简并引物PCR等分子标记技术，搜寻、鉴定了与香菇交配型因子连锁的分子标记，并对所得标记的序列结构、基本属性等作了初步探讨。所得主要研究结果如下：在对供试13个香菇菌株进行常规交配型分析的基础上，采用OWE-SOJ技术和核迁移试验对各菌株孢子单核体的交配型因子组合进行了鉴定。结果表明，应用OWE-SOJ技术，可以观察到3种形态明显不同的菌落，即由A≠B≠配对形成的具锁状联合的绒毛状菌落、由A≠B=配对形成的无锁状联合的栅栏型菌落及由A=B≠配对形成的无锁状联合的绒毛状菌落。核迁移试验表明，核迁移仅出现于A=B≠反应中而不出现于A≠B=反应中。由于这两种方法均可清楚区分通常无明确形态差异的A=B≠和A≠B=反应，因此，用于香菇孢子单核体交配型因子的准确鉴定是行之有效的。在对上述13个香菇菌株进行交配型分析的过程中发现了一个表现特殊的菌株HL01。在其担孢子随机两两配对的132个组合中，不亲和组合与亲和组合之比为82：50，x²值为11.00，极显著地偏离不亲和组合与亲和组合之比为3：1的理论x²值。用4种标准测交菌株进行交配试验表明，在该菌株的189个供试担孢子中，有161个可分别归于4个标准测交菌株A₁B₁、A₂B₂、A₁B₂和A₂B₁所代表的4种正常交配型，但其余疑似次级重组的28个则不能归于其中任何一类。用两两配对法对这28个担孢子进一步进行交配型分析，鉴定出了A₁B₃、A₁B₄、A₂B₃、A₂B₄、A₃B₂和A₃B₅等6种交配型的单核体，表明HL01的A、B交配型因子内部均发生了重组。A、B因子的重组率分别为8.47％、11.64％；A因子至少含有2个亚单位，B因子则可能由不止2个亚单位构成。随后的结实试验显示，至少含有一个这些次级重组体的可亲和配对均具有结实能力。为鉴定与香菇交配型因子相关的分子标记，我们采用了RAPD／BSA、RDA和简并引物PCR等方法。其中，利用简并引物PCR获得了一条与香菇HL01菌株B₂交配型因子共分离的DNA片段（773bp）。序列分析显示该片段属于信息素受体，表明香菇中B因子的遗传基础与两种模式蕈菌裂褶菌（Schizophyllum commune）和灰盖鬼伞（Coprinus cinereus）是一样的。同时，利用简并PCR法得到香菇中MIP基因片段（419bp）。已证实在其它许多蕈菌中，MIP基因与A交配型因子是紧密连锁（＜1kb）的，因此，本研究利用简并PCR法所获得的香菇的MIP基因片段，为进一步证实香菇中A因子与MIP基因的连锁关系打下了初步的基础。最后，我们对已获得的B因子片段及MIP基因片段用TAIL-PCR和反向PCR方法进行了步移。对B₂因子片段由773bp延长到3119bp，通过BLAST搜索GenBank中的类似序列，可以推定我们已经找到一个近乎完整的香菇B因子信息素受体基因组DNA序列。对MIP基因片段，则由449bp延长到2134bp，根据BLAST分析的结果可以推断在香菇中MIP基因与A交配型因子连锁的可能性是很大的，从而为A因子的克隆、测序等工作的开展奠定了较好的基础。更多还原

【Abstract】 The edible fungus Lentinula edodes is a heterothallic homobasidiomycete whose matingis controlled by a bifactorial incompatibility mating system determined by two unlinkedfactors （the A and B mating type factors）. In this study, 13 L. edodes strains were used andmating types of monokaryons derived from each of these strains were exactly identifiedby OWE-SOJ （Oak Wood Extract Agar-Squeezed Orange Juice Agar） technique and bynuclear migration testing. Further analysis and confirmation were carried out upon HL01strain whose progeny may occur recombinants. In addition, taking advantage ofRAPD/BSA, representational difference analysis（RDA） and degenerate PCR, wescreened and identified DNA markers linked to mating type factors of L. edodes, andmade preliminary investigation of the sequences structure and basic properties of themarkers obtained. The main results are as follows.Based on the regular mating types analysis of the 13 L. edodes strains, OWE-SOJtechnique and nuclear migration testing were used for identification of mating typefactors of monkaryons derived from each of these strains. The result showed that, usingOWE-SOJ technique, three kinds of typical colony could be observed, e.g. fluffy colonywith clamp connection formed by A≠B≠pairs, barrage colony without clamp connectionformed by A≠B=pairs, and fluffy colony without clamp connection formed by A=B≠pairs. Furthermore, nuclear migration test demonstrated that nuclear migration occurredonly in A=B≠but not in A≠B=. Therefore both of the two methods can discriminatedefinitely mating reactions between A=B≠and A≠B=, and it is effective to use them toexactly identify mating type factors of L. edodes.In the process of mating type analysis of the 13 L. edodes strains, one strain HL01appeared exceptive phenotype: 132 random pairs in monokaryons derived from onesporocarp of HL01 were made and the proportion of incompatibility to compatibility was82:50, of which the chi-square value was 11.00, extremely distorted the theoreticalchi-square value that consists with 3:1. Using the four standard tester strains, weidentified the mating type of 189 spore monokaryons derived from HL01 and found that161 out of them could be classified into four normal mating types （A₁B₁, A₂B₂, A₁B₂ andA₂B₁） excepting the other 28 monokaryons that probably occurred secondaryrecombination. By crossing in all pairwise combinations, the 28 monokaryons werefurther analysed and found six mating types（A₁B₃, A₁B₄, A₂B₃, A₂B₄, A₃B₂ and A₃B₅）,indicating that secondary recombination occurred in both A factor and B factor. And Afactor is composed of at least two subloci with 8.47% recombination frequency; B factor is composed of more than two subloci with 11.64% recombination frequency. Thesubsequent fruiting test revealed that all the compatible pairs contained at least one of therecombinants had the ability to produce fruiting body.To screen and identify the molecular markers related to mating type factors of L.edodes, several DNA marker techniques, such as RAPD-BSA, RDA and degenerate PCRwere employed. Among these methods, by means of degenerate PCR we obtained one773bp DNA fragment cosegregating with B₂ mating-type factor in L. edodes strain HL01.Sequencing analysis revealed it belongs to pheromone receptor, suggesting the geneticbasis for B factor in L. edodes is the same as in the two model mushroom species,Schizophyllum commune and Coprinus cinereus, structure and function of whose Bincompatibility factors have been studied in detail. Also, by degenerate PCR we identifiedpartial DNA fragment （419bp） of MIP gene coding mitochondrial intermediate peptidasethat has been shown tightly linked to A mating type loci （＜1kb） in most other mushroomspecies, including two model organisms. Thus the MIP fragment obtained from HL01establishes preliminary basis for further verification of the linked relationship between Amating loci and MIP gene in L. edodes.Finally, we made chromosome walking using TAIL-PCR and inverse PCR based onpartial DNA fragments of B factor and MIP gene obtained above. It walking from 773bpto 3119bp for B2 factor, 449bp to 2134bp for MIP gene. BLAST searches demonstratedthat nearly entirely sequences of B factor of L. edodes are obtained and that the MIP geneare most likely linked to A mating loci, which provides appropriate basis for cloning andsequencing of A factor in L. edodes更多还原