【作者】 刘天明；

【导师】 吉万全； 胡银岗；

【作者基本信息】 西北农林科技大学 ， 作物遗传育种， 2006， 硕士

【摘要】 小麦条锈病是影响小麦生产的最重要的病害之一。它可导致大规模的粮食减产,严重威胁我国的粮食安全。利用抗病品种是控制该病的最佳途径,但是目前生产上多数抗条锈病品种抗病基因未知,致使应用抗条锈病品种进行抗源合理布局困难。因此,从分子水平上了解小麦抗条锈病基因的表达,抗病机理及寻找新的抗病基因,对获得稳定、持久的抗病品种有重要的意义。本研究将cDNA-AFLP、抑制消减杂交技术,半定量RT-PCR扩增技术,电子克隆技术及生物信息学技术结合起来,以小麦抗条锈病种质N95175为实验材料,开展了小麦抗条锈病相关基因的表达研究,主要结果如下:(1)利用cDNA-AFLP的方法对抗条锈病相关基因进行差异表达分析,将获得的EST利用NCBI(www.ncbi.nih.gov)的BLAST分析软件分别对GenBank的dbEST数据库和蛋白数据库进行比对分析,获得的9条EST中有1条与病菌侵染胁迫下的EST有很高的同源性(同源性大于86%);6条EST分别与数据库中的锌指蛋白、转录调节因子、两种假定蛋白、催化酶类似的一种转位因子和一种未命名的蛋白等有较高的同源性。锌指蛋白、转录调节因子及转位因子据推测可能与小麦抗条锈病相关,而获得的假定的蛋白及未命名的蛋白是否与抗病相关,还需要进一步研究。(2)利用抑制消减杂交技术,对小麦抗条锈病相关基因差异表达进行研究。将测序后的EST利用BLASTN序列比对工具对GenBank的dbEST数据库进行同源检索分析,大部分与来源于高粱、水稻、小麦等作物在生物与非生物胁迫(包括病菌侵染、盐处理、激素处理、氧胁迫、干旱胁迫及元素缺乏等)下的EST有较高的同源性。EST序列利用ESTFastAnnotator进行相关蛋白质及基因功能的在线注释和分析,将未获得信息的序列再利用BLASTX对GenBank的非冗余蛋白质数据库进行同源性检索,并分析抗条锈病相关基因的功能。所得到功能已知的EST中部分与抗病相关基因片段(蛋白激酶、锌指蛋白、细胞色素P450、热激蛋白70、咖啡酸-O-甲基转移酶等)有较高的同源性。(3)利用半定量RT-PCR方法研究了抗条锈病相关基因片段锌指蛋白和热激蛋白70在小麦抗条锈病反应过程中基因表达,结果表明两个基因在条锈菌侵染后不同时间段内基因表达量是变化的。利用电子克隆的方法获得了热激蛋白70基因的cDNA全长序列。更多还原

【Abstract】 Stripe rust in wheat is one of the most important diseases. It could lead to grain output reduced in large-scale and cause a serious threat to Chinese food security. Using stripe rust-resistant varieties is the best way to control the disease. Currently, most of the stripe rust-resistant genes of disease-resistant varieties are unknown, So that it is difficult to achieve rational distribution of the rust-resistant varieties. Therefore, understanding disease-resistant gene expression, disease-resistant mechanisms and the search for new disease-resistant genes in molecular level are very important to access the development of stable and lasting disease-resistant varieties. cDNA-AFLP, Subtractive Stress Hybridization, Semi-QRT-PCR, In silico cloning and bio-informatics technologies were combined to research germplasm N95175 with stripe rust resistance and analysis of the Stripe rust resistance related gene expression. The main results are as follows :(1) cDNA-AFLP technology was adopted in analysis of stripe rust-resistance related genes expression in N95175. Using BLASTN software and BLASTX software separately to analyze EST sequences in the GenBank dbEST database and protein sequence databases in NCBI. Based on the sequences homologous comparison and function querying, the results showed: one of nine ESTs was high homologous (>86%) with pathogen infection EST; three ESTs were high homologous with zinc finger protein, transcriptional regulator and transposable element which were presumed to related to stripe rust resistance; three ESTs were high homologous with hypothetical protein and unnamed protein which functions were still need further study.(2) Suppression subtractive hybridization was adopted to analyze stripe rust-resistance related genes differential expression in N95175.EST sequences were analyzed by BLASTN software. The results indicated that the ESTs were high homologous with biotic and abiotic intimidate (contains pathogen-infection, salt-treated, hormone-treated, oxidatively-stressed, drought-stressed and trace elements更多还原