【作者】 杨艳会；

【导师】 陈新建； 张重义；

【作者基本信息】 河南农业大学 ， 作物遗传育种， 2011， 博士

【摘要】 地黄(Rehmannia glutinosa Libosch.)属玄参科多年生草本植物,是我国传统的大宗中药材。地黄栽培历史悠久,产量与品质俱佳,畅销国内外,系著名“四大怀药”之一。然而地黄种植之后需8-10年方可再种,地黄连作障碍问题表现非常突出,严重制约着地黄的生产,连作障碍已成为地黄生产中亟待解决的问题。目前,有关地黄连作障碍的报道主要集中在对该现象的解释、根系分泌物的自毒作用及其生理活性等方面,对其形成的分子机理尚不清楚。本研究以“温85-5"地黄品种为供试材料,连作(连续种植2年,SP)为处理、正茬(种植1年,FP)为对照,在块根膨大前期,利用新一代Solexa/Illumina测序技术,分别构建根、叶及整体植株三套(包括连作和正茬)转录组文库；结合数字化基因表达谱(Digital Gene Expression Profiling, DGE)和生物信息学分析,分别建立连作地黄根和叶数字化基因表达差异谱,筛选响应连作障碍的关键基因；构建正茬和连作地黄sRNA文库,鉴定了新型miRNAs,建立了连作地黄miRNA差异表达谱,确定响应地黄连作障碍的miRNAs及其靶基因；利用qRT-PCR(quantitative real-time PCR)分析了关键基因和特异miRNAs的表达模式；初步揭示了地黄响应连作障碍的分子机理。主要研究结果如下：1.通过对地黄根和叶转录组文库测序,每个样品均测到超过4000万条raw reads序列。利用生物信息学软件分析和拼接后分别获得99,708条(根,R)、94,544条(叶,L)、87,665条(根和叶拼接)地黄转录组序列：同时,利用Blast和ESTscan软件分析得到了50,653条CDS序列。87,665条地黄转录组序列Nr, Swissprot数据库同源性比较表明,有超过4万条(40%)基因序列与其他生物的已知基因具有一定程度的同源性：COG分类显示,48,227条(55.01%)序列具有功能注释；GO分类显示,54,321条(61.97%)序列具有分子功能：KEGG分析显示,21,138条(24.11%)序列参与了119个谢路径。在地黄基因组未知的情况下,本研究建立的地黄转录组,为揭示地黄响应连作障碍的分子机理奠定了技术和信息基础。2.构建了连作(R2)和正茬(R1)地黄根部表达序列标签库。R2和R1均获得超过500万条高质量clean tags,筛选了2,817个响应连作的差异表达基因。通过Nr、GO和KEGG基因功能分析,候选了114个响应连作障碍的关键基因,利用qRT-PCR方法分析了16个关键基因的表达模式。结果表明：钙信号途径和MAPK信号途径参与了地黄化感物质的信号感知、传导和放大(产生乙烯),同时地黄体内生长素和玉米素合成及运输、DNA复制、RNA合成、核苷酸合成、蛋白质合成和运输、呼吸作用、各种营养代谢等生命过程的核心途径均受到连作的严重伤害。通过综合分析,初步构勒了地黄根部响应连作“毒害”的分子机理。3.构建了连作(L2)和正茬(L1)地黄叶片表达序列标签库。L2和L1均获得超过500-万高质量Clean tags,构建了L2与L1的数字化基因表达差异谱,筛选了1,954个表达差异的基因。通过Nr, GO和KEGG基因功能分析候选了115个响应地黄连作障碍的关键基因。利用qRT-PCR法分析了10个关键基因的表达模式。结果表明：连作叶片生命活动的核心代谢途径,如：DNA复制、RNA合成、蛋白质合成、光合作用、呼吸作用等受到了影响和扰动,抑制或关闭了一系列相关基因的正常表达,进而导致“毒害”事件的发生。4.构建了连作(SP,second year planting)和正茬(FP, first year planting)地黄小RNAs库。从这两个库中鉴定了448个保守miRNAs;和22个新型miRNAs家族,建立了SP和FP的miRNAs差异表达谱,获得了312个差异表达的miRNAs(保守miRNAs和10个新型miRNAs)。 Nr、GO和KEGG功能分析显示,连作地黄体内表达差异miRNAs及其靶基因参与了植物转录调节、生长发育、信号传导、逆境响应等生物学过程。利用qRT-PCR法分析了16个表达差异的miRNAs表达模式。结果表明：连作引起体内miRNAs表达水平改变,同时出现了特异响应的miRNAs。在根和叶差异表达序列标签库中追踪这些差异表达miRNAs的靶基因,我们发现,在所有能够追踪到的靶基因表达水平与其相应miRNAs的表达情况有着惊人的一致,即：miRNAs表达水平高,其靶基因表达就低,反之亦然。许多靶基因处于控制核心代谢途径的位置。在地黄遗传信息严重匮乏的情况下,本研究利用新一代测序技术(Solexa/Illumina sequencing),首次构建了地黄及其根、叶转录组文库,建立了连作地黄功能基因数字化差异谱及其miRNAs差异谱,筛选了响应地黄连作障碍的关健基因及其miRNAs。研究结果从不同层次、不同角度、相互印证,构成了一个完整的证据链条：连作由体内钙和MAPK信号途径感知、传导,启动了特异miRNAs的表达,改写了基因正常表达程序,阻碍了核心代谢途径,使植株全方位受害,根部受害更甚。本研究初步构勒了连作地黄“毒害”的作用机理,为深入研究地黄连作障碍成因、解读连作障碍的分子机理,开发消减连作危害的有效技术奠定了理论基础。同时,本研究对深入理解地黄生长发育的分子机理与调控机制提供了技术与信息平台,同时也为研究其它植物连作障碍的分子机制提供有益的借鉴。更多还原

【Abstract】 Rehmannia glutinosa (Rehmannia glutinosa Libosch.) is a large quantity of traditional Chinese medicine and perennial herbaceous plant, classified under the family Scrophulariaceae. The species has been extensively cultivated for a long history. Because of its higher production and superior medical value, R. glutinosa sells well both in the domestic market and abroad and is one of famous Four Huai Medicines. However, the land must be given up for the other purpose for a period of8-10years after one season of R. glutinosa cultivation because the problem of continuous cropping obstacles is rarely serious, which has become an urgent problem to be resolved in agriculture production. Researches on continuous cropping of R. glutinosa are mainly focused on identifying the causative factors for this yield decline, the physiological activity and autotoxicity of the root exudates. At present, the molecular basis of the species’ sensitivity to its own exudate remains unclear. In our study, R. glutinosa cultivar "Wen85-5" was planted as an experimental material, at the same time, whole plants, roots and leaves from normal growth (the first year planting, FP) as control samples and continuous cropping (second year planting, SP) as treatments. The samples were collected at the earlier tuberous root expansion stage of R. glutinosa. Exploring Solexa/Illumina sequencing technology, we established the transcriptomes of plants (including roots and leaves), roots and leaves from continuous cropping R. glutinosa. With Digital Gene Expression Profiling (DGE) analysis and bioinformatics technology, we analyzed root and leaf different expression gene profiles from SP and FP, collected candidates for continuous cropping-related genes. We compared differential miRNAs expression in SP and FP of R. glutinosa plants, and identified specific miRNAs and its targets which are related to R. glutinosa continuous cropping obstacles. Expression levels of some candidate key genes and miRNAs were massured by qRT-PCR. This preliminarily unraveled the molecular mechanism underline R. glutinosa continuous cropping obstacles. The main results are as follows:At first, using Solexa/Illumina sequencing technology, more than40million raw reads were generated from both roots and leaves of mixed FP and SP R. glutinosa. Based on the bioinformatics analysis with assembler microsofts,99,708(Root, R),94,544(Leaf, L) and87,665(including roots and leaves) transcriptomic sequences were obtained. Furthermore,50,653CDS (coding sequences) of R. glutinosa were found by Blast and ESTscan analysis. Nr and Swissprot annotation of87,665transcriptome sequences showed that about that of40thousand (40%) had homologous with other species.48,227(55.01%) and54,321(61.97%) sequences were classified by COG and GO functional annotation, respectively. Based on KEGG analysis,21,138sequences were involved in119metabolic pathways. With unknown R. glutinosa genome, our study provide with useful technology and information platform to further reveal molecular mechanism of R. glutinosa continuous cropping obstacles. Secondly, more than5million clean tags were obtained from continuous cropping (R2) and normal growth (R1) of R. glutinosa roots by constructing tag libraries. DGE analysis revealed that the transcript profile of continuous cropping R. glutinosa was considerably changed, as indicated by the different significant2,817(1,676up-and1,141down-regulation) expression in R2. Nr, GO and KEGG analysis showed that114of these different expression genes played key roles in continuous cropping obstacle-responsive processes. Based on qRT-PCR, expression patterns of16candidate genes were analyzed. The results suggested that Ca2+and MAPK signal pathways were involved in sense, transduction and magnification of R. glutinosa allelochemicals, disturbing synthesis and transport of auxin and zeatin, DNA replication, RNA synthesis, nucleotide synthesis, protein synthesis and transport, respiration, various nutrition metabolisms and other key pathways. Thus this bascially outlined the molecular mechanism of continuous cropping harm in R. glutinosa.Thirdly, we constructed tag libraries from continuous cropping (the second year crop, L2) and normal growth (the first year crop, L1) of R. glutinosa leaves, respectively. More than5million clean tags were both obtained in two samples. DGE analysis screened1,954different significant genes. Nr, GO and KEGG analysis showed that115different expression candidate genes could be relevant to continuous cropping obstacles in R. glutinosa. Expression profiles of10key genes were analyzed by qRT-PCR. The results suggested that important metabolism pathways, which included DNA replication, RNA synthesis, protein synthesis, photosynthesis, respiration and so on, were interfered in continuous cropping R. glutinosa as repressing and closing a series of key gene expression. This gave rise to harm events of continuous cropping R. glutinosa.Fourthly, the study sequenced R. glutinosa sRNAomes from SP and FP of R. glutinosa plants, respectively. Based on R. glutinosa transciptome data,22novel and448conserved miRNA families were identified. We contrasted the miRNA different profiles of SP and FP libraries and screened312differentially expressed miRNAs (including10novel and302conserved miRNAs) which were predicted to target1,379genes. Nr, GO and KEGG analysis showed the potential targets of these differentially expressed miRNAs involved in the main transcription regulation, plant development, signal transduction, stress response and other biological processes.16different expression miRNA patterns were analyzed by qRT-PCR method. The results elucidated that, miRNA expression levels were changed in continuous cropping R. glutinosa, appearing in specific responsive miRNAs. Comparison with tag libraries of root (R2and R1) and leaf (L2and LI), we found that expression levels of targets were accordance with that of the corresponding different expression miRNAs, i.e. these miRNAs expression levels were higher while that of its targets were lower, and vice verse. Many targets regulated essential metabolism pathways.To sum up, with the shortage of R. glutinosa genetic backgrounds, we firstly constructed transcriptome libraries of plants, roots and leaves of continuous cropping R. glutinosa by next generation sequencing technology (Solexa/Illumina sequencing). At the same time, analysis of different expression genes and-miRNAs profiles obtained candidates of R. glutinosa continuous cropping obstacle-responded genes and specific miRNAs. The data from different research layers and angles, an entirely proof chain come to light that continuous cropping were sensed and conduced by Ca2+and MAPK signal pathways, in turn, specific miRNAs synthesis were directed, gene normal expression programs changed, key metabolism pathways repressed, the symptom (especially the roots) appeared. The study basically described regulated mechanism of continuous cropping obstacles in R. glutinosa. The present results lay the foundations for forming causes R. glutinosa continuous cropping obstacles. It is helpful for disclosing its molecular mechanism and exploiting effective technologies of subtracting its harm. Furthermore, our studies are of theoretical and practical significance for further explaining R. glutinosa growth and development mechanism. These also provide valuable references for researches on continuous cropping obstacles for other plants and for the sustainable development of Chinese medicine resource.更多还原