【作者】 陈立佼；

【导师】 黄兴奇； 赵永昌；

【作者基本信息】 云南大学 ， 植物学， 2015， 博士

【摘要】 羊肚菌因具有独特鲜美的口感、丰富的营养及极高的药用价值,是世界级的珍贵药食用真菌,同时也是我国重要的贸易野生菌,其理论研究和应用研究一直是食用菌学的热点。但因其子囊孢子核相多样,培养的微观宏观特性复杂多变,对其基础理论和应用研究均存在诸多困难。虽然在羊肚菌属的系统分类进化、生理生化、菌丝菌核培养及子实体栽培等研究方面已取得一定进展,然而所获成果尚不能解决由于其遗传多态性带来的研究者间观点不一致的问题。从孢子发育入手对羊肚菌有性生殖进行研究,有助于探索羊肚菌遗传多样性的成因,是羊肚菌各方面研究的重要突破口。从分子水平上探索羊肚菌的交配机制,可为深入认识羊肚菌有性生殖过程,特别是人工条件下子实体的发育研究奠定坚实基础,有效推动羊肚菌商业化、产业化栽培研究的发展。本研究论文以半人工栽培较为成功的梯棱羊肚菌(Morchella importuna)为材料研究羊肚菌子囊发育与子囊孢子的形成过程,结合转录组和基因组测序对Elata Clade 组群(M elata、M.importuna和M.septimelata)、Esculenta Clade 组群(M crassipes)和Rufobrunnea Clade 组群(M.rufobrunnea)5 个物种的代表菌株,进行信息素受体基因和交配型基因的克隆测序、结构分析和表达分析,并基于信息素受体基因和交配型基因的分布情况对M.elata、M.importuna 和M.crassipes 三个种子囊孢子群体菌株进行了分类研究。本文通过M.importuna子囊发育过程的Heidenhain’s苏木精染色和DAPI染色观察,首次总结出羊肚菌子囊发育及子囊孢子形成的模式。由于在减数分裂及有丝分裂中,纺锤丝的牵引方向与子囊壁分别是“垂直--垂直--无序”的关系,形成的子囊孢子为非顺序四分体;细胞膜对细胞核的随机包被和子囊孢子内的有丝分裂核剧增作用,使得羊肚菌形成的子囊孢子包括不同类型的多核同核体和多核异核体。M crassipes单孢菌株NH057基因组测序表明,M.crassipes的基因组大小为49.4 Mb,具有12340个基因,总长度为19,005,309bp,其中包含了 10个与有性生殖相关的基因,即2个信息素受体基因、1个M信息素基因、3个交配型基因(MAT1-1-1、MAT1-2-1 和MA T1-2-2)、2 个交配型转化蛋白基因(swi5和 swi10)和2个交配细胞膜融合蛋白基因(FIG1和PRM1)。其中MAT1-2-2是本研究新发现的交配型基因;通过对MAT1-1-1、MAT1-2-1和MAT1-2-2基因位点侧翼序列的基因预测分析及序列比对,发现其不符合同位异源基因的反向相似特征。根据NH057基因组数据及M rufobrunnea转录组数据,利用保守区PCR扩增和 DNA Walking 技术成功克隆得到 YAASM2688、YAASM2689、YAASMCH、YAASMBM和YAASMVR的信息素受体编码基因MPRa和MPRb,及YAASM2689、YAASM2688 和 YAASMCH 的交配型基因 MAT1-1-1 和 MAT1-2-2。对基因及其编码的氨基酸序列进行保守性分析,并对信息素受体氨基酸序列进行了二级结构的预测和比较。结果表明,这两类基因及编码产物都具有物种特异性;同时,对35株羊肚菌进行了基于信息素受体基因的分子发育分析,与基于ITS rDNA、EF1-α和LSU rDNA序列多基因系谱一致性系统发育分析结果比较,表明信息素受体基因的物种特异性可作为系统发育学分析的参考序列。利用MPRa、MPRb、MAT1-1-1、MAT1-2-1和MAT1-2-2(仅M2689)的分布情况,分别将子实体MCH、M2689和M2688的111、130和91个单孢菌株分为12、7和4大类,同核体和异核体孢子的数目及类型呈现多样。不同类型的单孢菌株的亲和性培养结果表明,羊肚菌为异宗配合菌,交配型基因决定其亲和性。利用实时定量PCR对M2689的5个异核体单孢菌株进行了信息素受体基因和交配型基因在菌丝满板期、菌核形成中期和菌核成熟期的表达研究,结果表明各目的基因在同一时期及不同时期存在明显的表达差异,在以上三个时期中,MAT1-2-2、MAT1-1-1和MPRa基因的相对表达量都明显高于MAT1-2-1和MPRb基因,大多菌株中MPRa、MPRb、MAT1-1-1和MAT1-2-2基因在各个时期的表达规律为第Ⅱ时期>第Ⅰ时期>第Ⅲ时期;MA T1-2-1的表达规律均为第Ⅰ时期>第Ⅱ时期>第Ⅲ时期。在梯棱羊肚菌子实体五个发育阶段中,MPRa、MPRb、MAT1-1-1和MAT1-2-2基因表达量并不随发育的进行呈线性变化,没有明显的规律;在同一子实体中,交配型基因的表达量高于信息素受体基因表达量,且MPRb不是子实体发育中较活跃的基因;在大多数子实体中,MPRa、MAT1-1-1和MAT1-2-2基因在菌盖中的表达量高于菌柄,而MPRb在菌柄中的表达量高于菌盖。更多还原

【Abstract】 Morels(Morchella spp.)are sought after for their unique delicious taste,rich nutrition and high medicinal value.They are precious edible and medicinal fungi,and are also important trade wild mushroom in China.It is difficult to study morels’s genetic characteristics because of multinuclear in ascospores and the diversity of macroscopic and microscopic characterization of culture.Until now,many research findings of taxonomy and molecular biology,physiology and biochemistry about mycelium culture,sclerotia formation and fruiting body cultivation of morels had been obtained.However,researchers have many inconsistent views on them because of genetic variance.Research on sexual reproduction which may reveal the causes of morels’ genetic diversity could become an important breakthrough in morel study.Exploring the mating type of morels can provide solid theoretical basis for the further understanding of its sexual reproduction,especially development of fruiting body on artificial conditions,and then promote the development of morels commercialization,industrialization cultivation research effectively.M.importuna which grown in artificial condition was used for studying the ascus development and ascospore formation.Pheromone receptor genes and the mating type genes have isolated from representative strains of Elata Clade group(M.Elata,M.importuna and M.septimelata),Esculenta Clade group(M.crassipes)and Rufobrunnea Clade group(M.rufobrunnea)respectively.The sequence,structure,as well as expressing analysis have been carried out.The ascospore isolations from M.elata,M.importuna and M.crassipes strains also have been classified based on the distribution of the pheromone receptor genes and mating type genes.The ascus development of M.importuna was observed by Heidenhain’s hematoxylin and DAPI staining.We initially summarized the M.importuna ascus development and ascospore formation patterns.We found out that the spindle orients was "vertical--vertical--disorder" to ascus wall respectively in two meiotic division and mitosis processes.The nuclei were packaged randomly in the ascospore formation.M.importuna could produce diverse homokaryon and heterokaryon ascospores in non-sequential tetrad order.In addition,the nuclei in young spores was fewer than that of mature spores,so the multi-nucleic ascospores were the results of the mitosis after their formation.The 49.4Mb genome sequencing data were obtained from M.crassipes single spore strain NH057,and 12,340 genes with total length of 19,005,309 bp were predicated.There were ten genes were related to sexual reproduction,namely two pheromone receptor genes,M pheromones genes,mating type gene MAT1-1-1,MAT1-2-1 and MAT1-2-2,mating type conversion gene swi5 and swi10,and mating membrane fusion protein gene FIG1 and PRM1.The MAT1-2-2 was the newly discovered mating type genes.One of the most meaningful finding revealed by the sequence prediction analysis and sequence alignment on MAT1-1-1,MAT1-2-1 and MAT1-2-2 gene flanking sequences,was that mating type genes were not in conformity with their reverse sequences.On the basis of NH057 genomic analysis and M.rufobrunnea transcriptomic analysis,the pheromone receptors encoding genes--MPRa and MPRb in YAASM2688,YAASM2689,YAASMCH,YAASMBM and YAASMVR,and mating type genes--MAT1-1-1 and MAT1-2-2 in YAASM2688,YAASM2689 and YAASMCH were cloned and sequenced successfully by using amplification conservative section with degenerate primers and DNA Walking PCR amplification.The analysis on the conservation of all genes and their encoded amino acid sequences were processed,and the secondary structure of pheromone receptors were predicated and compared.The results showed that two types of genes and their coding products were specificity in relation to species.The comparing analysis showed that phylogeny based the sequences of MPRa and MPRb from 35 Morchella strains was mainly consistent with that of ITS rDNA,EF1-α and LSU rDNA.So,pheromone receptor genes could also be used for phylogenetic analysis.The 111,130,and 91 single spore isolations from fruiting body MCH,M2689 and M2688 were classified into 12,7,and 4 major types based on the MPRa,MPRb,MAT1-1-1,MAT1-2-1 and MAT1-2-2(M2689 only)gene respectively,They were classified into different kinds of homokaryon and heterokaryon.Compatibility test on different types of single spore isolations show that morels was heterothallic fungi,and compatibility is mainly determined by mating type genes.The mating type genes and the pheromone receptor genes expression analysis in five single spore heterokaryon strains from M2689 have been done by real-time quantitative PCR.In hypha--covered--plate stage,sclerotiaum--formation--vigorous stage and sclerotiaum--mature stage,the expression of MAT1-1-1 MAT1-2-2 and MPRa gene were significantly higher than that of the MAT1-2-1 and MPRb genes.The expression quantity of MPRa,MPRb,MAT1-1-1 and MAT1-2-2 gene in most strains in sclerotium--formation--vigorous stage were higher than in hypha--covered-plate stage,followed in sclerotiaum-mature stage.However,the expression quantity of MAT1-2-1 gene in all strains in hypha--covered--plate stage was higher than in sclerotium--formation--vigorous stage,followed in sclerotiaum--mature stage.In addition,There was no the gene expression rules in morels fruit body represents the five development stages for MPRa,MPRb,MAT1-1-1 and MA T1-2-2 gene,In the same fruit body,the expression quantity of mating type gene were higher than that of pheromone receptor gene expression,but the MPRb was not the main active gene for fruiting body development;In most of the fruiting body,the relative expression quantity of MPRa,MAT1-1-1 and MAT1-2-2 gene in ascocarp were higher than that in stipe,and that of MPRb expression in the stipe is higher than in ascocarp.更多还原