【作者】 刘新锐；

【导师】 谢宝贵；

【作者基本信息】 福建农林大学 ， 微生物学， 2020， 博士

【摘要】 金针菇Flammulina filiformis是一种美味的食用菌,其子实体由菌盖和菌柄组成。在我国众多食用菌中,金针菇最早实现工厂化栽培,其栽培技术成熟,在实验室条件下也容易获得子实体,是研究食用菌各种性状的一个较理想材料,此外,菌柄在商品销售的金针菇子实体中占了绝大部分,显示了菌柄的重要性。虽然一些研究人员对金针菇菌柄伸长的特性进行了研究,但一些结果存在矛盾,并且控制其菌柄伸长的关键基因还未知,鉴于此,本研究对金针菇菌柄不同发育阶段的伸长特性进行研究,并通过转录组学揭示菌柄伸长区的分子特征,挖掘控制菌柄伸长相关的基因,并进行功能分析。主要结果如下:(1)菌柄伸长是伞菌菌柄生长发育的一个重要特征。在工厂化栽培条件下,金针菇菌柄的主要伸长区域位于菌柄的顶端,随着子实体的发育,伸长区域在扩大,但不超过2 cm,移除菌柄的部分组织,意外发现菌柄的主要伸长区域发生转移。金针菇菌柄伸长依赖于菌盖的存在,在子实体发育早期,菌盖是菌柄伸长发育所必须的,随着子实体发育的成熟,菌柄伸长对菌盖的依赖性降低,但在孢子弹射期,菌柄的伸长明显加快。由于形成的子实体数量众多,在没有人工干预下,金针菇菌柄生长的同步性差,在子实体发育早期进行套袋处理,可以提高子实体生长的整齐度。(2)转录组学揭示菌柄伸长区的分子特征。对金针菇原基、菌柄中间组织(非伸长区)、菌柄顶端(伸长区)和菌柄生长点等4个样品进行RNA-seq,通过比较转录组,显示菌柄伸长区涉及细胞的繁殖分裂、细胞壁与细胞膜结构组成、以及对环境压力响应的基因富集,此外,还涉及钙离子修饰菌柄伸长区细胞壁相关的钙结合基因。通过4个样品的比较转录组,初步获得在菌柄伸长区特异表达的13个基因,但只有5个基因在公共数据库中有少量信息注释,余下基因的功能处于未知状态。(3)原基和菌柄伸长期是菌柄生长发育的重要阶段。根据4个样品转录组数据,选择1个在原基和菌柄伸长区同时上调表达的未知功能基因Ff7426进行功能分析。通过RT-q PCR检测Ff7426在原基和菌柄不同发育时期的表达量,结果显示Ff7426在原基和菌柄顶端的转录水平高,与RNA-seq分析的结果相一致。Ff7426转化子的性状显示,Ff7426对菌丝的生长速度几乎无影响,干扰Ff7426使金针菇原基形成推迟,并且子实体形成的数量少,此外菌盖开伞的时间延迟,促进了菌柄生长;而Ff7426的过表达,原基形成的数量增多,且子实体成熟时间提早(菌盖开伞),而菌柄长度变短。(4)在4个样品比较转录组分析的基础上,选择1个在菌柄伸长区特异上调表达的未知基因Ff428进行分析。应用RT-q PCR检测Ff428在原基和菌柄不同发育时期的转录水平,结果与RNA-seq分析的相一致。Ff428转化子的性状显示,Ff428不影响菌丝的生长速度,干扰Ff428使金针菇原基形成的时间推迟,子实体发育的数量少,菌柄短,此外,菌柄与菌盖交接处易折断;而Ff428的过表达,促进原基的形成和菌柄长度的增加。(5)Septins蛋白影响细胞的形态。在金针菇基因组中,鉴定到6个septin蛋白编码基因,其中核心基因有5个,随机选择其中的2个核心基因Ff.cdc3和Ff.cdc12进行功能分析。RT-q PCR检测显示,Ff.cdc3和Ff.cdc12在原基阶段有一个明显的上调表达,在菌柄生长发育期,其转录表达主要位于菌柄的顶端。这2个基因的功能相似,对菌丝的生长速度影响小,而干扰这2个基因使金针菇原基形成推迟,同时容易产生畸形原基,并且菌柄较短,但Ff.cdc3和Ff.cdc12的过表达可促进原基形成和菌柄伸长。此外,这2个基因影响细胞对环境胁迫的响应,以及影响一些septin家族之间的转录表达。金针菇菌柄伸长是一个复杂的过程,涉及的因素众多,不仅受到环境的影响,还与自身相关基因的响应有关。菌盖是繁殖后代的场所,本研究讨论了菌盖与菌柄之间的关系,当条件适宜时,金针菇菌柄长度可意外地超过35 cm。在对Ff7426、Ff428、Ff.cdc3和Ff.cdc12的转化子性状分析可得出,菌盖开伞后,菌柄的生长发育将停滞。同时这些基因都影响原基的形成。更多还原

【Abstract】 Flammulaina filiformis is a kind of delicious edible mushroom,with the fruiting body composed of pelius and stipe.F.filiformis was the earliest among the many edible mushrooms that realized industrial production.Its cultivation techniques are mature,even in the lab,and its fruiting body can be easily produced,so it is a proper material for studying the traits of edible mushroom.Besides,the stipe is a dominant part of the commercially sold fruiting body,indicating its importance.Although,some researchers had studied some characteristics of the elongating stipe,some discrepancies still existed in their results,and key genes that control stipe elongation are still unknown.Based on this background,elongating characteristics of F.filiformis stipe at different development stages were studied,and transcriptomics were used to reveal the molecular characteristics in the elongating region,followed by identification of genes involved in stipe elongation control,and the functions of these genes were verified.The main results are as follows:(1)Stipe elongation is an important characteristic of stipe development in Agarics.Under factory cultivation conditions,the dominant elongating region of F.filiformis stipe laid at the top of stipe.Along with the development of fruiting body,the elongating region expanded,but was limited in 2 cm.When a part of tissue was removed from the stipe,the dominant elongating region moved to other region,to our surprise.Stipe elongation depended on the presence of pelieus.In the early stage of fruiting body development,pelieus was necessary for stipe elongation.Along with the fruiting body being mature,stipe elongation became less dependent on pelieus.However,during spore dispersion period,the elongation of the stipe was significantly accelerated.Due to the large number of fruiting bodies formed,the F.filiformis stipe had poor growth synchronization without control.But a bagging to fruiting bodies at the early development could enhance the uniformity of stipe growth.(2)Transcriptomics was used to reveal molecular characteristics of stipe elongation.Samples of primordia,tissues in the middle stipe(non-elongating region),apical stipe(elongating region)and growth point were collected for RNA-seq.Comparative transcriptomics revealed gene enrichment in cell reproduction and division,cell wall and cell membrane structure,facing environmental stress during stipe elongation.Besides,calcium-binding genes were enriched in cell wall modification of stipe elongation region by calcium ion.Preliminary analysis revealed that 13genes were specifically expressed in the elongation region of stipe,by comparing four samples,but only five of them had been annotated with a little bit information in the public databases,while the functions of other genes were still unknown.(3)Primordium and stipe elongation stages are important stages for stipe growth.Base on the transcript datas of four samples,a functional unknown gene Ff7426 was selected for functional analysis,which was up-regulated in expression at primordium and stipe elongation region.The results shown by RT-q PCR of Ff7426 in primordium and different stipe development were in accordance with RNA-seq.The phenotype of Ff7426 transformants showed the gene had no effect on growth rate of the mycelium.Interfere RNA of Ff7426 delayed primordium formation,resulted in less fruiting bodies,and inhibited the expansion of pelieus which resulting in increased stipe length.Moreover,over-expression of Ff7426 promoted primordium formation and fruiting body maturation(pelieus expansion),thus,resulting in shortened stipes.(4)According to the results of comparative transcriptomics among four samples,an unknown gene Ff428 was selected for functional analysis,which was specifically up-regulated expression in elongating region.The results shown by RT-q PCR of Ff428 in primordium and different stipe development were in accordance with RNA-seq.The characteristics of Ff428 transformants showed that the gene had no effect on growth rate of the mycelium.Interfere RNA of Ff428 delayed formation of primordium,resulted in less fruiting bodies,shortened stipes.Moreover,the connection between stipe and pelieus was fragile.And the over-expression of Ff428 promoted the primordium formation and stipe elongation.(5)Septins affected cellular morphology.In the genome of F.filiformis,six septin-encoding genes were identified,of which five were core genes.Ff.cdc3 and Ff.cdc12 were randomly chosen for functional verification.RT-q PCR results showed that Ff.cdc3 and Ff.cdc12 were significantly up-regulated expression in primordium stage.During stipe development,their transcription was mainly in the apical region of stipe.Both genes had similar functions.They showed minor effects on mycelial growth rate.The RNAi mutants showed delayed primordium formation,produced abnormal primordium and shortened stipes.Moreover,over-expression of both the genes promoted primordium formation and stipe elongation.On the other hand,both the genes showed response to environmental stress and affected the transcription of some other members in the septin family.Stipe elongation is a complex process,related with many factors,including environmental factors and its own gene expression.Pelieus is the place to reproduce its offspring.The relationship between pelieus and stipe was discussed here.When conditions are appropriate,the length of the stipe can be as astonishing as over 35 cm.Analyses of Ff7426,Ff428,Ff.cdc3 and Ff.cdc12 suggested the stipe would be arrested development after pelieus mature,while these genes affected primordium formation simultaneously.更多还原