【作者】 刘丹；

【导师】 陈旭辉；

【作者基本信息】 沈阳农业大学 ， 微生物学， 2022， 硕士

【摘要】 兰科植物具有重要的经济、文化、生态和科研价值。兰科植物种子微小,无胚乳,种子自身贮存的营养无法满足萌发的营养需求,往往需要依靠菌根真菌的共生为其提供营养才能完成种子萌发以及原球茎的生长发育,因此建立兰科植物与菌根真菌的共生对兰科植物的正常生长及生存尤为重要。我国具有丰富的兰科野生植物资源,然而目前关于兰科菌根真菌菌种资源、与兰科植物的共生关系以及在兰科生物多样性保护中的意义还知之甚少,需要进行更多的研究。本研究以羊耳蒜成熟种子为试验材料,采用组织培养技术对能够促进其种子萌发的真菌菌株进行筛选,成功建立羊耳蒜种子与胶膜菌属真菌的共生萌发体系;利用转录组高通量测序技术获取羊耳蒜种子与胶膜菌属真菌在共生和非共生条件下不同萌发阶段的转录组信息;运用生物信息学研究方法,对各阶段样本的基因表达情况和基因差异表达进行详细分析,并对潜在的共生基因进行初步筛选。以期通过该论文的研究,明确胶膜菌属真菌对羊耳蒜种子萌发的促进作用,丰富兰科种子的共生萌发真菌菌种资源,从分子水平上初步探索羊耳蒜种子的共生萌发调控机制。筛选得到的共生相关基因可以为后续深入阐明兰科植物与菌根真菌共生关系的本质并合理利用兰科菌根真菌资源提供理论基础。本论文的研究结果如下:(1)从7株胶膜菌属真菌菌株中筛选得到1株(编号:65号)对羊耳蒜种子萌发的促进效果最好的菌株,共生萌发时羊耳蒜种子的萌发率可达85.24%,显著高于其它菌株。利用该菌株成功建立了其与羊耳蒜种子的共生萌发体系。石蜡制片和显微观察发现,能够在共生萌发不同萌发阶段的羊耳蒜种子中检测到菌丝团,验证了共生体系已成功构建。(2)以接种65号胶膜菌属真菌菌株的共生萌发为处理组,相同培养条件下不接种胶膜菌属真菌的非共生萌发为对照组,对羊耳蒜种子萌发阶段1的两组样本进行转录组分析。结果表明,过滤序列均在4500万条以上,mapping率超过95%,数据质量较好可进行后续分析。两组样本的差异基因数量较多,与对照组相比,大部分差异表达基因均以上调表达为主,主要功能注释在生物过程,代谢途径主要为淀粉和蔗糖代谢通路,表明共生真菌对羊耳蒜种子萌发初期的基因调控主要是为了营养供给。筛选到11条凝集素相关基因、12条结瘤素相关基因和8条GRAS家族相关基因,其中TRINITY＿DN43374＿c4＿g3、TRINITY＿DN50768＿c3＿g1以及TRINITY＿DN36162＿c0＿g1基因的上调表达较为显著,推测这三个基因可能参与羊耳蒜种子与胶膜菌属真菌共生萌发体系的建立,因此在后续研究中可以重点关注。(3)由于在不接种胶膜菌属真菌的非共生萌发条件下,羊耳蒜种子一直没有出现任何明显的萌发迹象,因此随后对共生萌发条件下羊耳蒜种子4个不同萌发阶段的样本转录组进行了差异比较分析,并分别得到不同萌发阶段的原球茎转录组数据和共生胶膜菌属真菌转录组数据。其中,原球茎的转录组测序数据质量较好,过滤序列均在4400万条以上,mapping率超过95%,符合试验后续分析的需要;不同阶段原球茎的基因表达差异较大,代谢通路主要富集于苯丙烷生物合成、植物丝裂原活化蛋白激酶信号途径及光合作用途径,差异基因功能主要集中于细胞成分,表明这些阶段的基因调控均与植物自身的形态发育有关;共筛选出20条凝集素相关基因、15条结瘤素相关基因以及12条GRAS家族相关基因,其中TRINITY＿DN42119＿c1＿g4、TRINITY＿DN19608＿c0＿g1、TRINITY＿DN37564＿c3＿g6和TRINITY＿DN30240＿c0＿g1基因的表达量在不同萌发阶段差异显著,且总体上呈现逐渐上升趋势。对共生体中的胶膜菌属真菌的转录组数据分析表明,不同萌发阶段胶膜菌属真菌的基因表达变化差异不大,推测在兰科种子萌发过程中,真菌的作用主要是与兰科种子建立共生关系并启动种子萌发,启动机制一旦完成,在随后的萌发阶段两者之间的相互作用相对稳定,无明显的特异性调控。然而,由于胶膜菌属真菌的转录组测序数据质量不高,这一结论需要更详尽的数据进行验证。更多还原

【Abstract】 Orchids have important economic,cultural,ecological and scientific value.The seeds of orchids are tiny and have no endosperm,and the nutrients stored in the seeds themselves cannot meet the nutritional needs of germination.It is often necessary to rely on the symbiosis of mycorrhizal fungi to provide nutrients for them to complete the seeds germination and the growth and development of protocorms.The symbiosis of mycorrhizal fungi is particularly important for the normal growth and survival of orchids.Our country has rich resources in orchid wild plant.However,little is known about orchid mycorrhizal fungi resources,the symbiotic relationship with orchids,and the significance in orchid biodiversity conservation.More research is needed.In this study,the mature seeds of L.japonica were used as experimental materials,and the fungal strains that could promote the germination of the seeds were screened by tissue culture technology.Using high-throughput transcriptome sequencing technology to obtain the transcriptome information of the seeds of L.japonica and the fungi of Tulasnella at different germination stages under symbiotic and non-symbiotic conditions.Using bioinformatics research methods,the gene expression and gene differential expression of samples at each stage were analyzed in detail,and potential symbiotic genes were initially screened.It is hoped that through the research of this paper,the promoting effect of the fungi of Tulasnella on the germination of L.japonica seeds will be clarified,the symbiotic germination fungal resources of orchid seeds will be enriched,and the regulation mechanism of symbiotic germination of L.japonica seeds will be preliminarily explored at the molecular level.The screened symbiotic-related genes can provide a theoretical basis for the subsequent in-depth elucidation of the nature of the symbiotic relationship between orchids and mycorrhizal fungi and the rational use of orchid mycorrhizal fungi resources.The results of this paper are as follows:(1)One strain(No.65)was selected from the seven Tulasnella fungal strains with the best effect on promoting the germination of L.japonica seeds.The germination rate of L.japonica seeds during symbiotic germination was 85.24%,which was significantly higher than that of other strains.Using this strain,a symbiotic germination system with L.japonica seeds was successfully established.Paraffin section and microscopic observation showed that mycelium could be detected in L.japonica seeds in different germination stages of symbiotic germination,which verified that the symbiotic system was successfully constructed.(2)Taking the symbiotic germination inoculated with No.65 Tulasnella fungus strain as the treatment group and the non-symbiotic germination without Tulasnella fungus as the control group under the same culture conditions,the transcriptome analysis of the two groups of samples from L.japonica seed germination stage 1 was performed.The results show that the number of filtered sequences is more than 45 million,and the mapping rate exceeds 95%.The data quality is good for subsequent analysis.Compared with the control group,most of the differentially expressed genes were mainly up-regulated.The main functions were annotated in biological processes,and the metabolic pathways were mainly starch and sucrose metabolism pathways.Gene regulation in the early stage of L.japonica seed germination is mainly for nutrient supply.11 lectin-related genes,12 nodulin-related genes and 8 GRAS family-related genes were screened,among which TRINITY＿DN43374＿c4＿g3,TRINITY＿DN50768＿c3＿g1 and TRINITY＿DN36162＿c0＿g1 genes were significantly up-regulated.It is speculated that these three genes may be involved in the establishment of the symbiotic germination system of L.japonica seeds and Tulasnella fungi,so they can be focused on in follow-up studies.(3)Since L.japonica seeds did not show any obvious signs of germination under non-symbiotic germination conditions without Tulasnella fungus inoculation,we then conducted a differential comparative analysis of the transcriptomes of samples of L.japonica seeds at four different germination stages under symbiotic germination conditions.The protocorm transcriptome data and the symbiotic Tulasnella fungus transcriptome data at different germination stages were obtained.Among them,the quality of the transcriptome sequencing data of protocorm is good,with more than 44 million filtered sequences,and the mapping rate exceeds 95%,which meets the needs of the subsequent analysis of the experiment;the gene expression of protocorm at different stages varies greatly.Metabolic pathways are mainly enriched in phenylpropane biosynthesis,plant mitogen-activated protein kinase signaling pathway and photosynthesis pathway,and differential gene functions are mainly concentrated in cellular components,indicating that gene regulation at these stages is related to plant morphological development.A total of 20 lectin-related genes,15nodulin-related genes and 12 GRAS family-related genes were screened out.Among them,the expression levels of TRINITY＿DN42119＿c1＿g4,TRINITY＿DN19608＿c0＿g1,TRINITY＿DN37564＿c3＿g6 and TRINITY＿DN30240＿c0＿g1 genes were significantly different at different germination stages,and showed a gradual upward trend in general.Transcriptome data analysis of Tulasnella fungi in symbionts showed that the gene expression changes of Tulasnella fungi in different germination stages were not very different.It is speculated that in the process of orchid seed germination,the role of fungi is to establish a symbiotic relationship with orchid seeds and start the process.Seed germination,once the initiation mechanism is completed,the interaction between the two is relatively stable in the subsequent germination stage,and there is no obvious specific regulation.However,due to the low quality of transcriptome sequencing data for Tulasnella fungi,this conclusion may need to be validated with more exhaustive data.更多还原