【作者】 龙翔宇；

【导师】 郑有良； 魏育明； Thérèse Ouellet；

【作者基本信息】 四川农业大学 ， 生物化学与分子生物学， 2011， 博士

【摘要】 基因表达分析在小麦的众多研究领域中变得日趋重要,在转录水平进行基因表达定量时,都需要应用内参基因对目标基因表达量进行校正和标准化,因此选择合适的内参基因显得十分重要,然而,目前还没有一种理想的小麦内参基因可以完全适用于不同类型的小麦组织。小麦赤霉病是一种世界性病害,是影响小麦高产、稳产和品质的重要因素之一。赤霉病的抗性受数量性状遗传控制,抗性分子标记QTL的研究越来越多,一大批数量性状QTL被定位,并且QTL的位点几乎存在于整个基因组,但是发掘及报道小麦赤霉病抗性相关基因的研究却很少。基因芯片技术是后基因组时代功能分析的重要技术之一,可以快速而高效地获得大量基因在mRNA水平的表达信息,从而能够在整个基因组范围对基因的表达进行分析,该技术已广泛的应用于小麦的基因表达研究。本文利用基因芯片技术发掘小麦内参基因及抗赤霉病相关基因。一、分析小麦基因表达谱芯片,筛选表达较为稳定的小麦内参基因,结果如下：1.收集及分析小麦基因表达谱芯片共333张,包括了盐胁迫、干旱胁迫、铝胁迫、氮胁迫、高温胁迫、锈病菌诱导、白粉病菌诱导、赤霉病菌诱导及不同发育时期的9个实验组,在倍数阈值为2的情况下筛选到基因表达相对稳定的探针117个。通过生物信息学分析,最终确定50个表达相对稳定的候选内参基因。2.应用实时荧光定量PCR技术,检测50个候选内参基因在不同时期根、茎、叶、种子及盐、旱、冻、脱落酸、铝、氮、病等不同胁迫下的表达情况。经geNorm、Normfinder及MINTAB分析处理,表明32个内参基因在各个组织及不同胁迫环境下表达量均相对稳定,其中7个内参基因表现出极高的稳定性,可作为表达水平的分子标记,用于校正目标基因表达量。3.选择7个新鉴定的内参基因与13个传统的内参基因进行表达稳定性分析,发现新鉴定的内参基因较传统的内参基因稳定。而在传统的内参基因中,EF1A表现出很更高的稳定性,传统的内参基因Gapdh和alpha-tubulin出现了很不稳定的表达。二、分析小穗病率、赤霉菌的含量及产生毒素(DON)含量来鉴定不同QTL材料的赤霉病抗性,采用基因芯片技术分析含有不同QTL材料基因表达谱,筛选与抗赤霉病QTL相关的基因,结果如下：1.含有抗赤霉病3BS、DL、5A-QTL材料的抗病性均高于不含有抗赤霉病QTL的材料,抗性亲本HC374的抗性最强,不含有抗赤霉病QTL的材料与感病亲本BW301的抗性基本相似,出现了极差的抗性。2.筛选到与抗赤霉病2DL-QTL相关的基因1个,该基因只在含有抗赤霉病2DL-QTL的材料中表达,并且确定该基因的来源于Wuhan,但是基因功能注释是未知的。3.筛选到与抗赤霉病3BS-QTL相关的基因4个,该基因只在含有抗赤霉病3BS-QTL的材料中低表达或不表达,4个基因的功能注释是未知的。4.筛选到与抗赤霉病5A-QTL相关的基因2个,其中一个基因只在含有抗赤霉病5A-QTL的材料中表达,并且确定基因来源于Nyubay,该基因编码了一个脂质转运蛋白,是一种重要的防御蛋白,在抵抗和适应外界胁迫中有着重要的作用。另一个基因在含有抗赤霉病5A-QTL的材料中诱导表达,其他材料中表达量不变,该基因编码了一个环氧化物水解酶,是生物体系中细胞保护、外源化合物代谢和信号调节的重要酶类。更多还原

【Abstract】 With the development of genomics and bioinformatics, more and more researchers focus on the analysis and applications of gene expression and regulation. Microarray and qRT-PCR are increasingly becoming of the good choice for high-throughput gene expression analysis. To get reliable results from both methods of analysis, it is vital to get accurate normalization of gene expression against one or more control genes. Thus, it is required that more ideal internal control genes are identified for normalization using various methods. Fusarium head blight (FHB) is a devastating fungal disease of common wheat (Triticum aestivum L.) and durum wheat (T. turgidum L. var. durum) worldwide. And FHB is one of the main factors that could reduce wheat yields and change milling, baking and pasta-making properties. The genetic control of FHB resistance has been analyzed through lots of QTL mapping studies. While FHB resistance genes were rarely identified and reported in wheat during recently years. Microarray provides abundant information of gene expression in whole genome, and the technology has been widely used in Wheat, In this study, novel internal control genes and FHB resistance genes were identified and evaluated in wheat using microarray.Novel internal control genes were selected and evaluated using microarray and Q-PCR.1. In this study, fifty candidate genes for internal control were obtained from the analysis of 333 Affymetrix GeneChip Wheat Genome arrays from nine independent experiments (salt toleration, drought toleration, aluminium toleration, nitrogene toleration, high temperature toleration, rust toleration, powdery mildew toleration and different development stages). The two-fold ratio method was used to select potential genes/fragments with minimal changes between samples.2. Expression levels of the 50 selected genes were then evaluated by quantitative real-time PCR with cDNA samples from different tissues, stages of development and environmental conditions. Seven novel internal control genes of 32 which were relatively stable were selected for revising the expression of target genes after comparing their respective expression profiles using NormFinder, geNorm, Pearson correlation coefficients and the 2-fold-change method.3. The expression stabilities of the novel internal control genes from this study were compared with 13 traditional ones for their expression stability. It was observed that 7 of the novel internal control genes were better than the traditional ones in expression stability under all the tested cDNA samples. Among the traditional internal control genes, the elongation factor 1-alpha exhibited strong expression stability, whereas the alpha-tubulin and GAPDH genes had very poor expression stability in the range of wheat samples tested.FHB resistance was evaluated in some wheat lines (Null,2DL,3BS,5A-QTL), and some QTL related resistance genes were selected and analyzed.using Microarray and Q-PCR.1. The disease resistance analysis showed that QTL lines (2DL,3BS,5A-QTL) had higher resistance to FHB than the Null-QTL ones (Null). Parental material (HC374) had the most obvious resistance, while other parental material (BW301) and.3251 (Null) presented worse resistance to FHB.2. One gene related with 2DL-QTL was identified in 2DL-QTL lines. The gene only expresses in lines which included 2DL-QTL. The gene is from Wuhan according to the expression pattern which is same as that in Wuhan. But the gene annotation is unknown till now.3. Four genes related with 3BS-QTL were identified in 3BS-QTL line. Those genes are lower expression or no expression in lines which included 3BS-QTL, and the genes annotation are also unknown up to now.4. Two genes related with 5A-QTL were identified in 5A-QTL line. One gene only expresses in lines which included 5A-QTL, and it is from Nyubay according to the expression pattern which is same as that in Nyubay. This gene encodes a lipid transfer protein (LTP) which plays an important role in resistance to pathogen and adaptation to environmental stresses. Another gene was only induced expression in lines which included 5A-QTL, and encodes an epoxide hydrolase which presents in all living organisms and plays important roles in proceeding metabolism, cellular protective mechanisms and signaling pathways.更多还原