小麦条锈菌吸器转录组分析及PSTha5a23基因的功能研究

发布时间：2018-05-27 15:06

本文选题：小麦条锈病 + 条形柄锈菌　；参考：《西北农林科技大学》2016年博士论文

【摘要】：小麦条锈病是由条形柄锈菌(Puccinia striiformis f.sp.tritici)侵染的一种真菌病害,在全世界小麦种植区域均有不同程度发生。我国小麦条锈病发生面积达大、危害严重,成为影响小麦生产可持续发展的关键因素。然而,由于小麦条锈菌是专性寄生菌,通过吸器吸收营养,长期以来,人们对吸器的转录组与吸器的功能研究很少,限制了对该病害防控技术的深入系统研究。因此,从转录组水平上全面系统了解小麦条锈菌互作的基因表达特征,克隆抗条锈相关基因并利用病毒诱导的基因沉默技术分析其功能,为阐明小麦与条锈菌互作的分子机理奠定基础。因而,本论文拟在前期完成的条锈菌基因组测序基础上,分析条锈菌感染小麦时小麦条锈吸器的转录组,预测候选效应蛋白(effector candidates),研究效应蛋白PSTha5a23的功能。主要研究结果如下:1.本实验通过采用条锈菌CYR32感染的小麦叶片匀浆经用20μm尼龙滤膜过滤后,分离得到的吸器,分离得到吸器的纯度62%,提取得到的吸器RNA浓度为83ng/μL。2.采用Seolxa RNA-seq测序技术对小麦条锈菌吸器转录组测序,得到了13211个基因,其中,新发现特异性吸器转录组(Gene expression)序列1547条。3.条锈菌unigene的GO注释分类结果表明,共有883条unigene被注释到生物学过程(biological process)、细胞组分(cellular component)和分子功能(molecular function)3类中。其中,293条unigene归入生物学过程,85条unigene归入细胞组分,491条unigene归入分子功能。4.将条锈菌unigene序列映射到KEGG数据库的参考代谢通路(pathway)中,共有1489条unigene被注释,涉及20个代谢通路,其中有16条不重复代谢通路。5.Signal P和TMHMM分析,得到参与跨膜运输的新基因51个,利用PredGPI在线分析程序实现蛋白质锚定修饰的预测,得到49个新效应蛋白基因,为研究小麦条锈菌吸器与小麦互作的分子机理提供了新的基因。6.效应蛋白基因经过生物学试验筛选得到PSTha5a23基因抑制小麦的免疫反应。PSTha5a23基因是小麦条锈菌特有的基因,该基因在小麦条锈菌种内比较保守。它的N端有信号肽序列,定位在细胞质。在本氏烟中过表达PSTha5a23基因可抑制Bax、PAMP-INF1、MKK1和NPK1诱导的免疫反应。PSTha5a23基因过表达也能抑制小麦的PTI反应。沉默PSTha5a23基因对小麦条锈菌的致病表型影响不大,但是过表达PSTha5a23基因可增强小麦条锈菌的致病力。这些研究结果表明PSTha5a23是小麦条锈菌分泌的一个重要的抑制小麦免疫反应的抑制子。
[Abstract]:Wheat stripe rust is a fungal disease infected by Puccinia striiformis f. Sp. Triticizer, which occurs in different degrees in wheat planting areas all over the world. Stripe rust of wheat in China is a key factor affecting the sustainable development of wheat production due to its large area and serious damage. However, because wheat stripe rust is a specific parasite and absorbs nutrients through the suction apparatus, for a long time, there has been little research on the transcriptome and the function of the suction organ, which limits the in-depth and systematic study on the disease prevention and control technology. Therefore, the gene expression characteristics of wheat stripe rust interaction were comprehensively and systematically understood at the transcriptional level, and the genes related to stripe rust resistance were cloned and their functions were analyzed by virus-induced gene silencing. It provides a basis for elucidating the molecular mechanism of wheat interaction with stripe rust. Therefore, on the basis of genome sequencing of stripe rust, this paper analyzed the transcriptional set of wheat stripe rust absorber when stripe rust was infected with wheat, predicted the candidate effector and studied the function of the effector PSTha5a23. The main results are as follows: 1. In this experiment, wheat leaf homogenate infected by CYR32 was filtrated with 20 渭 m nylon membrane, and the purity of the absorber was 62%. The concentration of RNA extracted was 83ng/ 渭 L. 2. A total of 13211 genes were obtained by Seolxa RNA-seq sequencing of wheat stripe rust sequencers. Among them, 1547 specific hauster-transcriptional gene expression sequences were found. The results of go annotation classification of unigene showed that a total of 883 unigene were annotated into biological process, cellular component and molecular functional molecular function)3. Among them, 293 unigene were classified into biological process, 85 unigene were classified into cellular component, 491 unigene were classified as molecular function. The unigene sequence of Rust was mapped to the reference metabolic pathway in KEGG database. A total of 1489 unigene were annotated, involving 20 metabolic pathways. Among them, 16 non-repeated metabolic pathways. 5. Signal P and TMHMM analysis revealed 51 new genes involved in transmembrane transport. The prediction of protein anchoring modification was realized by PredGPI online analysis program, and 49 new effector protein genes were obtained, which provided a new gene .6for studying the molecular mechanism of the interaction between wheat stripe rust haustener and wheat. The effector protein gene was screened out by biological experiment. The PSTha5a23 gene inhibited the immune response of wheat. PSTha5a23 gene was unique to wheat stripe rust, which was conserved in wheat stripe rust species. Its N-terminal signal peptide sequence, located in the cytoplasm. Overexpression of PSTha5a23 gene in the tobacco could inhibit the overexpression of Baxo PAMP-INF1 MKK1 and NPK1. PSTha5a23 gene overexpression could also inhibit the PTI response of wheat. Silencing PSTha5a23 gene had little effect on the pathogenic phenotype of wheat stripe rust, but overexpression of PSTha5a23 gene could enhance the pathogenicity of wheat stripe rust. These results suggest that PSTha5a23 is an important inhibitor of wheat immune response secreted by wheat stripe rust.
【学位授予单位】：西北农林科技大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S435.121.42

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