【作者】 张翔；

【导师】 谢宝贵；

【作者基本信息】 福建农林大学 ， 微生物学， 2014， 硕士

【摘要】 草菇是在亚洲广泛栽培的一种商业菇,然而草菇的发展受到诸多因素的制约,其中包括一直以来草菇的遗传类型存在争议,这在一定程度上限制了草菇育种。在这样的背景条件下,我们以5个野生草菇菌种和2个栽培草菇菌种的单孢菌株做为最初的试验材料进行草菇交配型基因的研究,主要目的在于为阐明草菇的遗传类型提供数据支持。首先根据实验室已经测序的草菇单孢菌株PYd21的A因子基因及其侧翼保守基因mip的序列设计引物,将试验菌株的mip基因和A因子的基因进行共分离,然后进行酶切,在每种草菇菌种中各找到2个酶切具有多态性的单孢菌株,共14个单孢菌株做为进一步试验的材料。将已经选出来的14个单孢菌株进行长片段PCR,Sanger法完成测序,最后,共得到了 8种A因子基因的序列,其中包括2个基因组已测序的PYd15和PYd21单孢菌株。将这8个序列进行ORF以及一些功能的预测,分析比较预测结果,发现这8个A因子基因序列非常不保守,不能根据A因子的内部序列设计引物来扩增草菇未知的A因子的基因。通过同源搜索,在PYd21中找到4个信息素受体基因,没有发现信息素基因。并且发现PYd21和PYdI5这2个单孢菌株的4个信息素受体基因的序列比对后,其对应的基因基本一样。然后,我们根据4个信息素受体基因的序列设计引物,分别对选出来的6株单孢菌株进行PCR,测序,初步对比后发现它们并不具有多态性,只有个别的SNP位点。所以,B因子在草菇的交配过程中,可能没有相应的调控功能。将第一步筛选出来的14个单孢菌株进行种内配对,然后将A因子基因序列不同的8个单孢菌株进行两两配对,配对完成后,将挑选出来的杂交种进行分子验证。结果:A因子基因不同,信息素受体基因相同的菌株可以进行配对。更多还原

【Abstract】 The Straw mushroom Volvariella volvacea is an important commercial mushroom and widely cultivated in Asia.However,the development of V.vovacea is limited by factors such as breeding of V.volvacea is limited due to an unclear sexual pattern.We decided to study monospore isolates from 5 wild strains and 2 cultivated strains of V.Volvacea to clarify the mating type system of V.volvacea.Primers used for cosegregation of the mip gene and A mating loci were based on the A mating locus sequence and the flanking,conserved mip gene of V.volvacea monospore strain PYd21.Amplicons were digested and two monospore isolates which displayed polymorphism were selected from each strain,resulting in a total of 14 monospore strains for further testing.The A mating loci of the 14 selected strains were amplified using long distance PCR,and sequenced with Sanger.Eight types of A mating loci sequences were obtained,including the sequence of PYdls and PYd21.Then comparatively analysis and predict the structure of eight obtained sequences.The results showed the sequences of the eight A mating loci were not conservative,so the primers that were designed according to known straw mushroom A mating loci can not be used to amplify the gene unknown A mating loci.By homology search,we found the B mating locus encodes 4 pheromone receptors with no pheromone gene in PYd21.Then the primers that were designed according to the sequence of 4 pheromone receptor gene of PYd21,were used to amplify B mating locus of 6 monospore strains with different A mating locus(except PYd15 and PYd21),sequenced,comparison analysis,the results showed that 4 pheromone receptor gene do not have polymorphism in different monospore strains,only individual SNP sites.So B mating locus in the mating process of straw mushroom,may not have the function.The previously selected 14 monospore isolates were crossed,and the 8 strains with different A mating type genes were reciprocally crossed,following the crossing,the hybrid strains were screened with the molecular method.The results showed the stains with different A mating locus,identical pheromone receptor gene can be crossed.更多还原