杜鹃花类菌根形成及效应表达过程中的差异基因分析

发布时间：2018-08-21 12:02

【摘要】：杜鹃花科(Ericaceae)植物具极高的园艺价值,包括“十大名花”杜鹃花及蓝莓等重要经济作物,是适应贫瘠土壤的典型类群,研究认为这种适应性与该科植物根系形成的一种特殊类型的内生菌根：杜鹃花类菌根(也称欧石楠类菌根,ERM)密切相关。为探讨ERM共生体形成及效应的分子机理,本研究从野生云锦杜鹃(Rhododendron fortunei Lindl.)根系中分离纯化了一种ERM真菌,建立了该真菌与云锦杜鹃根系的共生体系,采用转录组测序技术分析了接种苗根系在转录水平上的变化,获得了大量与菌根形成及氮吸收代谢相关的差异表达基因。主要结果如下：(1)ERM真菌的鉴定真菌分离自浙江华顶山森林公园的野生云锦杜鹃根系,通过菌落、菌丝和孢子形态的观察,以及ITS RNA序列的系统发育树分析,确认该真菌为大孢树粉孢原变种Oidodendron maius var. mains,编号为Om19。(2)ERM共生体系的建立通过真菌回接实验,建立ERM共生体系。供试植物为云锦杜鹃种子播种于1/2 ER培养基生长两个月的实生苗,供试真菌为Om 19；无菌条件下将实生苗移栽至基质中,接种Om19菌块,基质为草炭与沙子按2：1体积比混合,并添加MMN营养液(氮源替换为硝态氮)；接种两个月后采用锥虫蓝染色法观察根系解剖结构,发现根系表皮和皮层细胞内形成菌丝和菌丝节,确认Om19与根系形成ERM共生体。接种苗株高、根长和根数显著提高,表明ERM促进宿主植物对硝态氮的吸收。(3)ERM侵染进程的观察及菌根侵染率的测定通过徒手压片、扫描电镜和半薄树脂切片观察接种Om19后每周根系的解剖形态,追踪侵染过程根系表面和细胞结构的变化,并计算菌根侵染率。徒手压片发现接种第二、三周的根系表面有菌丝及菌丝侵入点,第四周胞内有菌丝和菌丝节,第六周部分根段的细胞完全被侵染；扫描电镜观察发现接种苗根系表面包裹一层稠密的菌丝；半薄树脂切片发现根系结构简单,没有根毛结构,由1层表皮细胞和1-2层皮层细胞及狭小的中柱组成,90%以上含有菌丝的表皮细胞壁加厚不明显；菌根侵染率在第二周逐渐上升,第六周显著提高,达50%以上。(4)接种不同时期ERM促进云锦杜鹃生长效应的观察接种苗和未接种苗的鲜重从第一周到第十周都有不同程度的增长。第三周开始,接种苗的鲜重显著高于未接种苗,从103.5mg增长到第十周的328.0mg,第六周接种苗的鲜重比未接种苗高63%,干重增加47%,植株总氮量提高36.6%。(5)转录组测序分析共生条件下云锦杜鹃根系基因表达由于接种第六周菌根侵染率、植株生物量和总氮含量均显著提高,本研究构建了该时期接种苗和未接种苗根系的转录组文库,获得70720条All-Unigene,分别在NR, NT, Swiss-Port, KEGG, COG和GO等六个数据库中注释到45058,36399,28048,4986,15106和35283条基因。通过FPKM法计算Unigene表达量,获得了在接种苗根系中显著差异表达的基因(DEGs)16892条,其中14364条显著上调表达,4613条注释到KEGG数据库的267条代谢途径中。(6)ERM形成及氮吸收代谢相关的途径DEGs注释结果中与菌根形成相关的代谢途径有：内吞作用411条,醚酯类代谢365条,植物病原菌互作237条,植物激素信号转导223条,类结瘤因子信号途径19条,钙信号途径21条。氮吸收代谢相关的代谢途径有：代谢途径注释到1398条DEGs,氮代谢途径32条,精氨酸与脯氨酸代谢途径34条,矿物质吸收代谢途径23条。(7)与ERM形成及氮吸收代谢相关的基因DEGs中与丛枝菌根(AM)形成相关的同源基因有：ENOD 13条,SYMRK/NORK2条,CCaMK/DMI311条,NUP 16条,表明ERM的形成可能具有与AM类似的信号转导途径。DESs中与氮吸收和代谢相关的基因有：硝酸转运蛋白基因(NRT)8条、氨转运蛋白(AMT)11条,编码谷氨酰胺合成酶俗氨酸合成酶(GS/GOGAT)代谢途径中的关键基因有：硝酸还原酶(NR)4条、亚硝酸还原酶(NiR)4条,谷氨酸合成酶(GOGAT) 11条和谷氨酰胺合成酶(GS)3条。AMT基因的高表达表明ERM植物根系吸收硝态氮的途径可能与AM相似,而NRT基因的上调表达表明ERM可能提高了根系对硝态氮的直接吸收能力。通过qRT-PCR技术分析了4条(SYMRK、NORK、CCaMK和DMI3)与ERM形成和7条(AMT3、NRT1-1、NRT1-2、GOGAT-1、GOGAT-2、GS-1和GS-2)与氮吸收代谢相关基因在接种不同时期的表达模式。结果显示接种后这些基因表达量逐渐上升,在第六周表达量最高(除DMI3外),第八周下降,该现象与菌根侵染过程和效应表达过程追踪的实验结果一致。
[Abstract]:Ericaceae plants have very high horticultural value, including the important cash crops such as "ten famous flowers" Rhododendron and blueberry. They are typical groups adapted to poor soil. It is considered that this adaptability is closely related to a special type of endophytic mycorrhizal formed by the roots of Ericaceae plants: Rhododendron mycorrhizal (also known as Photinia mycorrhizal, ERM). In order to explore the molecular mechanism of ERM symbiosis formation and its effect, an ERM fungus was isolated and purified from the roots of Rhododendron fortunei Lindl. The symbiotic system of the fungus and Rhododendron fortunei Lindl. was established. The changes of the root system at transcriptional level were analyzed by transcriptome sequencing. A large number of differentially expressed genes related to mycorrhizal formation and nitrogen uptake and metabolism were obtained. The main results were as follows: (1) ERM fungi were isolated from the roots of Rhododendron yunnanensis in Huadingshan Forest Park, Zhejiang Province. The fungi were identified as macrospores by morphological observation of colonies, hyphae and spores, and phylogenetic tree analysis of ITS RNA sequences. Establishment of symbiotic system of ERM by fungal inoculation experiment. The seedlings of Rhododendron yunnanensis were sown on 1/2 ER medium for two months, and the fungi were Om19. The seedlings were transplanted to the substrate under aseptic conditions and inoculated with Om19. The root anatomical structure was observed by trypanosome blue staining two months after inoculation. It was found that mycelia and hyphae nodes were formed in the epidermis and cortex cells of root system, and the ERM symbiosis was formed between Om19 and root system. (3) Observation of the infection process of ERM and determination of mycorrhizal infection rate were carried out by hand pressing, scanning electron microscopy and semi-thin resin section to observe the anatomical morphology of the root every week after inoculation of Om19, to track the changes of root surface and cell structure, and to calculate the mycorrhizal infection rate. Scanning electron microscopy showed that the root surface of inoculated seedlings was surrounded by a dense layer of hyphae; semi-thin resin section showed that the root structure was simple without root hairknot. The mycorrhizal infection rate increased gradually in the second week and increased significantly in the sixth week, reaching more than 50%. (4) The fresh weight of inoculated seedlings and uninoculated seedlings was observed. From the first week to the tenth week, the fresh weight of inoculated seedlings was significantly higher than that of non-inoculated seedlings, from 103.5mg to 328.0mg at the tenth week. The fresh weight of inoculated seedlings in the sixth week was 63% higher than that of non-inoculated seedlings, the dry weight increased by 47%, and the total nitrogen content increased by 36.6%. (5) Transcription sequence analysis of Rhododendron yunnanensis roots under symbiotic conditions. Because of the mycorrhizal infection rate at the sixth week of inoculation, plant biomass and total nitrogen content were significantly increased. A transcriptome Library of 70 720 All-Unigenes was constructed from the roots of inoculated and non-inoculated seedlings. These genes were annotated in NR, NT, Swiss-Port, KEGG, COG and GO databases to 45058, 36399, 28048, 4986, 15106, respectively. Unigene expression was calculated by FPKM method, and 16892 differentially expressed genes (DEGs) were obtained, of which 14364 were significantly up-regulated, and 4613 were annotated into 267 metabolic pathways in KEGG database. (6) DEGs related to ERM formation and nitrogen uptake and metabolism were related to mycorrhizal formation. There are 411 endocytosis pathways, 365 ether ester metabolism pathways, 237 plant pathogenic bacteria interactions, 223 plant hormone signaling pathways, 19 nodulation factor signaling pathways and 21 calcium signaling pathways. The metabolic pathways related to nitrogen uptake and metabolism include: the annotation of metabolic pathways to 1398 DEGs, 32 nitrogen metabolic pathways, and the metabolism of arginine and proline. There are 34 pathways and 23 pathways of mineral absorption and metabolism. The related genes are: nitrate transporter gene (NRT) 8, ammonia transporter protein (AMT) 11, encoding glutamine synthase vulgar acid synthase (GS / GOGAT) metabolic pathway of the key genes are: nitrate reductase (NR) 4, nitrite reductase (NiR) 4, glutamate synthase (GOGAT) 11 and glutamine synthase (GS) 3. High expression of ERM indicated that the pathway of nitrate uptake by roots of ERM plants might be similar to that of AM, and the up-regulation of NRT gene suggested that ERM might enhance the direct uptake of nitrate by roots. The formation of ERM and seven of ERM (AMT3, NRT1-1, NRT1-2, GOGAT-1, GS-1 and GS-2) were analyzed by qRT-PCR. The results showed that the expression of these genes increased gradually after inoculation, reached the highest level at the sixth week (except DMI3) and decreased at the eighth week, which was consistent with the experimental results of mycorrhizal infection and effector expression.
【学位授予单位】：福建农林大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S685.21

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