【作者】 葛瑛；

【导师】 朱延明；

【作者基本信息】 东北农业大学 ， 植物学， 2010， 博士

【摘要】 松嫩平原西部地区是世界三大碱土集中分布区之一,面积高达373万hm2。在这样的土壤环境下,植物遭受着钠离子毒害、碳酸氢根离子毒害和pH升高引起的土壤物理性质变化,严重影响农业生产。迫切需要通过基因工程技术改良作物的耐碱能力。挖掘耐碱基因,揭示其分子机理,是耐碱分子育种的重要前提。野大豆可以耐受9.02的pH环境,是进行植物耐碱功能基因组学的理想材料。本研究以采自吉林省白城市盐碱地的耐碱野大豆为试材,结合基因芯片技术构建碱胁迫基因转录谱,采用生物信息学手段,结合野大豆基因组数据,分析碱胁迫应答基因的共表达特征、染色体聚簇和上游调控区特征,揭示碱胁迫应答分子机制,筛选耐碱候选基因。通过本研究,将加深对植物耐碱分子机理的认识,为作物耐碱分子育种提供基因资源和奠定理论基础。本研究的主要研究结果如下:1.野大豆碱胁迫基因转录谱的建立用50mmol/L NaHCO3模拟碱胁迫,处理野大豆G07256,提取RNA,与Affymetrix公司的基因芯片进行杂交,获得了杂交质量高、重复性好的野大豆碱胁迫基因转录谱。该转录谱包括胁迫前和胁迫后0.5h、1h、3h、6h、12h、24h共7个时间点,根部23741个和叶部22200个转录本。采用实时荧光定量PCR技术对芯片杂交结果进行了验证。随机选择的24个基因的变化趋势与芯片结果一致,表明芯片杂交结果能够真实地反映基因的转录水平变化。2.野大豆碱胁迫应答基因的获得采用RankProd统计方法分析每两个时间点间基因表达量的差异显著性(p < 0.01, pfp < 0.15),筛选出在某两个时间点间发生了显著变化的基因,即碱胁迫应答基因,根部2493个,叶部2310个。通过功能富集分析发现,根中参与代谢过程基因显著为碱胁迫应答基因,叶中参与代谢过程和膜转运相关的基因显著为碱胁迫应答基因。在6h出现碱胁迫基因应答高峰。在根中,参与细胞结构建成、疾病与防御、次生代谢和转录相关途径的基因显著早期应答,在转录相关基因中富集了WRKY转录因子。在叶中,参与细胞结构建成、蛋白质合成、能量和次生代谢相关基因显著早期应答。利用碱胁迫前6h转录谱构建基因调控网络,鉴定出28个碱胁迫应答关键基因。3.野大豆碱胁迫下共表达基因及特征为揭示碱胁迫应答基因的共表达特征,采用适合短时间序列表达数据的聚类方法STEM进行了聚类分析。发现根中有11类基因,叶中有10类基因具有显著的共表达特征(p < 0.001)。共表达基因通常包含类似功能的基因,上游调控区具有共同的调控元件。共表达特性与其在基因组上的分布不存在相关性。发现共表达基因启动子中有21个motif对基因的共表达起重要作用,其中有12个是已知的顺式作用元件。4.碱胁迫应答miRNA筛选及调控作用分析通过将miRNA前体序列与芯片上探针组和大豆基因序列进行BLASTn比对,发现有38个转录本能够代表miRNA前体。获得了根和叶中各11条碱胁迫应答miRNA,其中7条尚未有胁迫应答的报道,为新发现的碱胁迫应答miRNA。预测miRNA靶基因,发现碱胁迫应答的miRNA主要通过调控转录因子和激酶基因影响植物的碱胁迫应答。5.野大豆耐碱候选基因筛选通过碱胁迫应答基因与分子标记的关联分析,发现了1个功能未知基因和1个具有亮氨酸拉链和丝苏氨酸磷酸转移酶活性的基因位于耐盐碱分子标记附近。根据基因的碱胁迫应答情况及功能注释,从碱胁迫应答基因中筛选出2个耐盐碱分子标记附近基因,435个有明确结构域且尚未在生物学实验中确定基因功能的基因,和9个野大豆物种特异性的功能未知基因,作为下一步耐盐碱基因工程研究的候选基因资源。更多还原

【Abstract】 The western Songnen Plain of China, which has 3.73million ha of sodic land, is one of the three major contiguous sodic soil regions in the world. Both sodium and bicarbonate ion pose a toxicity hazard. High pH leads a change of soil physical properties. Through genetic engineering technology of alkali - resistant capability of modified crop is urgently needed. Mining the alkaline resistant genes and understanding its molecular basis is the premise to breed crop plants with enhanced tolerance to high saline-alkaline. The wild soybean (Glycine soja) line used in this study is a ideal material to integrate the functional genomics of alkaline tolerance, which can germinate and set seed in the sodic soil at pH9.02.By Affymetrix? Soybean GeneChip?, we investigated transcriptional profiling in Glycine soja grew in saline and alkaline soil in Baicheng city, Jilin Province, stress response genes were screened. Integrating genome data, genes regulated by alkaline stress were identified and clustered so as to show the co-expression characteristics of stress response genes. Gene function category, location in chromosome and upstream motif were analyzed to investigate alkaline stress response mechanism of plants. This will provide the theoratical basis to elucidate the molecular mechanisms of plant stress response and provide functional genes for plant saline and alkaline tolerant molecular breeding. The main results were summarized as following.1. Alkaline stress-responsive genes expression profile in wild soybean50mmol/L NaHCO3 was used to simulate the alkaline stress. Wild soybean G07256 was stressed and RNA was isolated. The transcriptome profiling was established in wild soybean using Affymetrix soybean gene chip. This profiling includes 7 time points (0, 0.5h, 1h, 3h, 6h, 12h, 24h) and two organs (root and leaf). There were 23741 transcripts in roots and 22200 transcripts in leaves.The Genechip results were tested using real-time PCR. 14 candidate genes’transcription changes accorded with the genechip results. This results showed that the genechip results are reliable.2. Screening of alkaline stress response genesRankProd statistical method was used to identify the significance of differences between the gene expression levels of two time points (p < 0.01, pfp < 0.15). The gene whose expression level changed significantly between two time points was regarded as alkaline stress response gene. 2493 and 2310 stress response transcripts were found in root and leaf separately. Function enrichment test suggested that the differentially expressed genes were related to metabolism processes in roots as well as metabolism and membrane translocation in leaves. 28 key genes were identified in the regulatory network.The number of alkaline response genes shown a peak at 6h. Genes involved in cell structure, desease and defense, secondary metabolism and transcription (WRKY family was enriched in transcript factor) were early responded in root. Genes involved in cell structure, proterin synthesis, energy, and secondary metabolism were early responded in leaf. 28 key genes were identified in gene regulation network.3. Characteristic of wild soybean co-expressed genes regulated by alkaline stressClustering analysis of short time series gene expression data was conducted to reveal the co-expression characteristics of alkaline stress response genes. The co-expression characteristics (p < 0.001) of 11 classes of genes and 10 classes of genes were found in roots and in leaves seperately. Co-expressed genes showed co-functions and common motifs. Co-expression characteristics and its distribution in chromosome have no correlation. 21 motifs were found in up-stream of co-expression genes. 12 motifs were known cis- elements.4. Identification of alkaline stress response miRNA38 probe groups that can represent miRNA precursor were found by BLASTn miRNA precursor, probe groups and soybean gene sequences. 11 miRNAs expressed differentially in roots and leaves, seperately. The report about 7 miRNAs was not found, these miRNAs was regarded as new stress response miRNAs. By predicting miRNA target genes, transcriptional factors and kinase were found being regulated by miRNA.5. Screening of alkaline tolerant candidate genesThrough correlation analysis of alkaline stress response gene and molecular marker, a unknown gene and a gene with serine-threonine protein kinase activity. Two salt tolerant marker gene, 435 unkown genes, 9 soybean or wild soybean specific genes were screened to be candidate genes for the further crops’stress tolerant research according to expression pattern, function notation, domain of stress response genes.更多还原