【作者】 黄雪玲；

【导师】 康振生；

【作者基本信息】 西北农林科技大学 ， 植物病理学， 2009， 博士

【摘要】 由Puccinia striiformis f.sp. tritic引起的小麦条锈病是世界范围内小麦(Triticum aestivum)生产上的重要病害。小麦成株抗性与小麦对条锈病的持久抗性有着密切的关系,是持久抗性的重要组成部分。为了对小麦成株抗性机制有更好的了解,我们采用抑制差减杂交的方法,选取条锈菌接种后12h,24h及36h的小麦材料及各时间点对照材料,构建了条锈菌诱导的成株期小麦“兴资9104”的基因表达谱。通过该实验,我们得到1250个阳性克隆,阳性克隆比例为68.5 %。对所有阳性克隆提取质粒DNA,经序列测定、去除载体序列及低质量序列后,共得到1188条高质量的ESTs。ESTs长度范围为100-800bp,多为100-400bp,其中17个序列长度大于600bp。对所有EST用Cap3软件进行聚类拼接,共获得427个Unigenes,包括245个Contigs和182个Singlets。多数congtigs包含2-3个序列,有一个contig包含了33个序列。经Blastx对所得Unigenes与NCBI非冗余蛋白质数据库进行比对分析和功能注释,发现在所有的Unigenes中,比对后没有显著性匹配(no hits)的占37.4%,这一类序列有可能是新的基因,也有可能是因为片段太短而造成没有匹配的序列。267个unigenes (62.6%)与蛋白质数据库中的序列存在着显著到极显著的相似性。在这267个ungenes中,232个表现出与已知功能序列的同源性,35个与未知功能序列具同源性。将已知功能的232个unigenes进行功能分类,其中防御相关的基因为第一大类,占已知功能基因的24%,其中胁迫相关的蛋白、病程相关蛋白及一些蛋白激酶出现的频率较高;占第二类的是与能量相关的基因,占15.7%,其中,参与光合作用及叶绿体结构建成的基因最多,如1,5-二磷酸核酮糖羧化酶/加氧酶(Rubisco),Rubisco活化酶;蛋白质合成及代谢相关的基因分别占13.1%和8.6%,居第三、四位;信号传导相关的基因占8.2%,膜转运类基因占6.4%,蛋白质修饰和储存类基因占3.4%,其它几类所占比例较小,分别是转录相关基因2.6%,编码次生代谢物质的基因2.2%,细胞生长分化相关基因1.9%,转座子0.7%。通过比较我们的实验结果与前人对小麦高温成株抗锈性及小麦条锈小种专化抗性的研究,我们认为小麦成株抗性的机制不同于小种专化抗性,表现在更多的防御途径参与了成株抗性的反应,并且活性氧相关的基因表达在成株抗性品种中产生较少或者是产生较晚。利用Real Time PCR检测技术,我们分析了一些抗病相关基因在接种后不同时间点的转录变化。结果表明,多数基因在成株期和苗期小麦的表达趋势基本一致,但在成株期的表达呈现诱导表达水平上升快,表达量高的特点。同时我们发现,大多数抗病相关基因在成株期未接种对照中的表达量高于苗期未接种对照。这很有可能是成株抗性产生的原因之一。小麦对条锈病的小种专化抗性是小麦抗病性的重要方面,在抗病育种中占有重要的地位。为了解小麦小种专化抗性的分子机制,我们利用寡核苷酸芯片对8个具小种专化抗性的单基因系的基因表达谱进行了分析,以期寻找具有相同遗传背景的单基因系中所共有的抗性机制及各个单基因系所特有的基因表达特征。通过对这八个单基因系的转录分析,我们鉴定了28个转录参与了所有基因型的小种专化抗性,其中19个为非亲和组合较未接种对照所特有的基因,包括假想的抗病蛋白,过氧化物酶、蓝铜结合蛋白、苯丙氨酸解氨酶及细胞色素P450等。另外9个基因为非亲和组合较亲和组合所特有的基因,包括钙调素结合蛋白、蓝铜结合蛋白、过氧化物酶及β-1,3-葡聚糖酶等等。同时我们也鉴定了每个基因型中特有的抗病相关的转录,这表明不同基因型有特定的转录事件产生。结果证实了已知的小种专化抗性中R基因介导的路径,包括氧化爆发,病程相关蛋白的表达及苯丙烷途径的活性。然而,还有几个为未知功能的转录,有待于进一步的研究。更多还原

【Abstract】 Stripe rust, caused by Puccinia striiformis f. sp. Tritici (Pst), is a destructive disease of wheat (Triticum aestivum) worldwide. Adult plant resistance is relative to the durable resistance to stripe rust in wheat and adult plant resistance might be one of the most important component of durable resistance. To gain a better understanding of the mechanism of adult plant resistance, the suppressive subtractive hybridization (SSH) approach was used to identify wheat genes induced by Pst inoculation at adult plant stage. A total of 1,250 positive cDNA clones were obtained and sequenced. After the contig analysis with the Cap3 assembler, 427 unique sequences were obtained and compared to the NCBI no-redundant protein database using the BlastX program. The sequences were putatively categorized as genes belonging to signal transduction, transcription regulation, protein synthesis and storage, membrane transport, and cell growth and division. Based on the putative functions of the induced genes, we propose a special defense-related pathway that is triggered during the expression of adult-plant resistance in XZ after we compared our results with the research on high-temperature adult-plant (HTAP) resistance and race specific resistance in the literature. The time-course expressions using quantitative RT-PCR confirmed the induction of seven selected genes by Pst infection and determined their expression patterns.The result indicated that the expression pattern in adult plant was roughly similar to that in seedlings except the transcription level was higher and faster increased in adult plants.It is noteworthy that the expression level of most defense-related genes was higher in mock inoculated adult plant than that in mock inoculated seedlings. This might be one of the reasons for APR.The race specific resistance of wheat to stripe rust is one of important resistance in wheat breeding. In order to understand the molecular mechanism of race specific resistance, we constructed and used a custom focused oligonucleotide microarray to perform a meta-analysis of the transcriptional response involved in race-specific resistance conferred by Yr1, Yr5, Yr7, Yr8, Yr9, Yr10, Yr15, and Yr17. By profiling the response of the eight resistance genes in a common background genome, we identified 28 transcripts as significantly involved in the race-specific resistance phenotype across all genotypes, in which 19 transcrips, such as putative disease resistance protein, peroxidase, blue copper-binding protein and phenylalanine ammonia-lyase were specific to the uncompatible interaction compared to the uninoculated mock. Nine transcripts were specific to the uncompatible interaction compared to compatible interaction. These tanscripts includes calmodulin-binding protein, blue copper-binding protein, peroxidase and beta-1, 3-glucanase. Unique defense-related transcripts significant in each genotype were also identified, which highlighted some transcriptional events specific to certain genotypes. The results confirm the activity of known R-gene mediated pathways in the race-specific resistance response, including an oxidative burst that likely contributes to a hypersensitive response (HR), as well as pathogenesis-related (PR) protein expression and activity of the phenylpropanoid pathway. However, several identified transcripts remained unknown and may prove interesting candidates for further characterization.更多还原