【作者】 张立荣；

【导师】 杨文香； 刘大群；

【作者基本信息】 河北农业大学 ， 植物病理学， 2011， 博士

【摘要】 由小麦叶锈菌(Puccinia triticina)引起的小麦叶锈病是一种在世界范围内普遍流行的气传性病害,严重威胁小麦生产。选育并合理利用抗病品种是防治麦类锈病危害最经济、安全、有效的途径之一。因此,利用和发掘抗叶锈资源,延长品种的抗叶锈性寿命,成为小麦叶锈病防治的一项任务。本研究选择当前在我国抗性较好的小麦抗叶锈病近等基因系TcLr24为研究对象,应用同源序列法和cDNA-AFLP技术对其进行抗病相关基因的研究,利用荧光实时定量PCR进行表达分析,同时利用BSMV VIGS技术对基因进行了初步的功能验证。主要结果如下:1根据已克隆植物抗病基因NBS保守结构域设计引物,从携带小麦抗叶锈病基因Lr24的回交6代近等基因系材料TcLr24中获得了1个通读的小麦抗病基因同源片段RGA1。该片段含有NB-ARC保守结构域,具有抗病基因NBS特征结构域(P环、激酶2、激酶3a等)。2以获得的RGA1片段为靶序列,利用RACE技术获得了2822bp的TcLr24 cDNA全长RGA1。RGA1包含2397bp的开放阅读框,编码799个蛋白质氨基酸序列,RGA1编码产物具有NBS、LRR等抗病基因保守结构域,与多个已知植物抗病基因的功能相应区域一致。Southern杂交结果表明,其在TcLr24基因组中为单拷贝。实时荧光定量PCR对RGA1基因的表达情况研究表明,该基因为组成型表达。BSMV VIGS功能验证没有发生表型改变。3本研究利用cDNA-AFLP技术筛选了84对引物能够在小麦近等基因系TcLr24和感病对照Thatcher之间揭示出多态性,根据基因表达与否以及表达量,获得了9种不同差异表达的多态性条带类型,分别代表了不同状态下测试材料的表达状况。4成功克隆和测序了121个差异表达片段(Transcript-derived fragments,TDFs),测序结果在NCBI上应用BLASTx比对进行推导的蛋白质序列的相似性分析,结果共有32条为功能已知的同源序列,占总数的27%;比对上功能未知的序列37条占总数的30%;没有比对上的序列45条占总数的37%。已知功能序列的TDFs涉及初级代谢、转运相关、抗病与防御、转录相关、信号传导相关、细胞结构、能量代谢、蛋白储运等。5从差异表达并测序的片段中选取了9个进行荧光实时定量PCR分析,证实了这些基因差异表达是由于叶锈菌诱导表达的结果,发生在小麦与叶锈菌互作的早期。与cDNA-AFLP表达基本一致,说明利用cDNA-AFLP分析基因的表达是可靠的。6利用RACE的方法获得了3个激酶基因,包括TaSTK (1475bp),TaRLPK (2398 bp),TaLRRK (2038 bp),分别编码丝氨酸/苏氨酸蛋白激酶(408aa),类受体激酶(656aa),LRR-受体激酶(595aa)。InterProScan分析表明,它们所编码的蛋白都具有典型的蛋白激酶的核心区域,属于激酶类基因,并且这三个蛋白的磷酸化作用需要依赖ATP。经Psort预测显示,TaSTK编码的蛋白最可能分布在叶绿体基质中,几率为85.4%,而TaRLPK和TaLRRK编码的蛋白分布在质膜中的可能性较大,几率分别为51.4%和46%,表明TaRLPK和TaLRRK编码的蛋白是膜蛋白。氨基酸比对及进化树分析表明,TaSTK,TaRLPK,TaLRRK分别与大麦、水稻、小麦等激酶具有较高的相似性。7利用BSMV VIGS基因沉默技术对3个激酶基因进行了初步的功能分析。结果表明3个基因被沉默后发生了不同程度的表型变化,并且组织学观察的结果与其表型变化一致。其中,TaSTK基因被沉默的小麦叶片接种叶锈菌后表型由“;”转变成“1”,组织学中观察到,接种叶锈菌120hpi,寄主细胞坏死,但菌丝仍然扩展明显。而TaRLPK和TaLRRK基因被沉默的小麦叶片在接种叶锈菌后表型由“;”转变成“2”,表现为中抗反应。120hpi组织学观察发现,叶锈菌侵染后形成少量坏死,但是这些坏死不能完全抑制病原菌的生长,表现出对叶锈菌的中度抗性反应。研究表明这三个激酶基因参与了小麦抗叶锈性。更多还原

【Abstract】 Wheat leaf rust caused by Puccinia triticina is one of the most devastating diseases worldwide, which is resposible for major damage in wheat and results in both yield losses and downgrading in quality. The use of resistant cultivars is the most economical and environmentally sound method to reduce the damage. So, discovery and utilization of favorable genes in wild source and prolonging resistant cultiver the life of becomes an important task to reduce the damage caused by wheat leaf rust. The study was carried out to investigate resistance related genes from TcLr24 by homology-based method and complementary DNA amplified fragments length polymorphism (cDNA-AFLP) analysis. The expression patterns of the test fragments were conducted through real- time PCR. At the same time the genes were selected for further functional analysis using BSMV VIGS technique. The main results were discrived as follows:1. One through reading fragment was obtained and sequenced from cDNA of TcLr24 according to the conserved sequence of resistance gene analogs (RGA). The deduced amino acids of protein RGA1 contained a NB-ARC conserved domain which were identical to the NBS conserved domains (P-Loop, kinase-2, kinase-3a) of many plant resistance genes.2. The full length sequence of disease resistance homology gene in the cDNA from TcLr24 was 2822bp named as RGA1 which was obtained by the rapid amplification cDNA ends (RACE) based on RGA1 fragment. The predicated RGA1 protein encode 799 amino acids. BLASTp analysis showed that the deduced amino acids of protein contained NBS conserved domain and many leucine-rich repeats (LRR) domains, which were identical to the conserved domains of many plant resistance genes. These sequences appeared to be induced by Puccinia triticina and were genes in the wheat leaf tissue . There is one copy in DNA of TcLr24 by Southern hybridazation. Expression patterns revealed by real-time PCR analyses indicated that RGA1 gene was constitutive genes in the wheat leaf tissue in the incompatible interaction between host and pathogen. Silencing RGA1 using BSMV VIGS reduced the transcript level of gene through real time PCR analysis, of which the gene showed no changed phenotype.3. Eighty-four pairs of primers could amplify differential expression bands among TcLr24 and Thatcher by cDNA-AFLP analysis. Polymorphic bands were grouped into nine different types, and four types of them were supposed to correspond to disease- resistant gene.4. One hundreds and twenty-one differential expression bands were cloned and sequenced. Blastx analysis showed that the thirty-two transcript derived fragments (TDFs) accounted for 27% of all the TDFs showed high homology with the function known gene. Thirty-seven TDFs accounted for 30% were homology with unknown function genes. Fouty-five sequences accounted for 37% were no match with any sequence in the database. All TDFs were classified, energy and metabolism, transporters, disease/defense, transcription, energy gene, cell structure, signal transduction, protein storage were supposed to correspond to the expression of the disease-resistant genes.5. Based on their functional analysis, nine genes were selected for further real time PCR analysis. The results indicated that expression patterns revealed by real time PCR analysis were basically in accord with the cDNA-AFLP analysis. These results demonstrated that the TDFs were most likely pathogen-induced genes.6. Three kinase-encoding genes were obtained using the rapid amplification cDNA ends (RACE) method, including TaSTK (1475bp), TaRLPK (2398 bp), TaLRRK (2038 bp), which respectively encoded Ser/Thr protein kinase, Receptor protein kinase, LRR-receptor protein kinase. InterProScan analysis revealed that three genes were deduced to proteins with protein kinase activity, thus characterizing as kinase-encoding genes. ATP-dependent phosphorylation feature were observed for TaSTK,TaRLPK,TaLRRK. Psort analysis indicated TaSTK had 85.4% probability of target in chloroplast stroma. TaRLPK,TaLRRK had 51.4%, 46% probability of target in membrane. The analysis of multiple alignment and phylogenetic tree demonstrated that TaSTK, TaRLPK, TaLRRK were highly similar to barley, rice, wheat kinase.7. Three kinase genes were carried out for further functional analysis using BSMV VIGS technique. Positive silencing for each gene could be detected using real time PCR analysis, of which wheat plants of three silenced genes each showed changed phenotype. Further histological observation indicated the same results. Plants of silenced gene TaSTK showed larger area of hypersensitive cell death with slight amount sporulation showing infection type of“1”. Wheat plants with silenced TaRLPK, TaLRRK gene, each showed sporulation around with relatively small area of hypersensitive cell death, indicating resistant infection type of“2”. The results implied three kinase genes might play an important role in the defense response.更多还原