【作者】 邓麟；

【导师】 康振生；

【作者基本信息】 西北农林科技大学 ， 植物病理学， 2010， 硕士

【摘要】 由小麦条锈菌(Puccinia striiformis f.sp. tritici)引起的小麦条锈病是小麦生产上一种重要的真菌病害,对我国小麦生产造成严重威胁。培育和种植小麦抗病品种是防治条锈病最经济、有效、安全的举措。因此,克隆小麦中参与与条锈菌互作的相关基因,分析其表达特征和功能,以阐明小麦与条锈菌互作的分子机理,可为小麦抗条锈品种选育和遗传改良提供理论基础。本研究结合同源克隆、RACE、电子克隆、数据库挖掘等基因克隆手段,在小麦品种水源11中克隆出13个可能参与了小麦与条锈互作的基因,并对部分基因进行了生物信息学和表达谱分析以及功能的初步验证,研究的主要内容和结果如下:(1)为研究小麦SNARE蛋白在小麦与条锈菌互作中的功能,利用同源克隆和RACE获得小麦TaNpsn11和TaNpsn13基因全长cDNA,通过检索TIGR小麦数据库克隆得到另外9个SNARE基因,分别为3个R-SNARE:Tav-Snare13、TaVamp725和TaSec22;2个Qa-SNARE:TaSyp132和TaSyp22;2个Qb-SNARE:TaNpsn12和TaBet1;1个Qc-SNARE:TaSyp51,以及一个Qb+c-SNARE:TaSnap28。(2)对植物特有的NPSN家族基因进行了表达特征分析,发现TaNpsn11~13各自在小麦的根、茎、叶组织中差异表达,其可能在器官的分化和发育过程中起着一定作用;与条锈菌互作中3个基因在非亲和组合中表达高于亲和组合,表明TaNpsn11~13参与了小麦对条锈菌的抗性;四种外源植物激素处理后,TaNpsn11均上调表达,TaNpsn12只受水杨酸(SA)和脱落酸(ABA)上调表达,而TaNpsn13被茉莉酸甲酯(MeJA)和ABA上调表达,被乙烯ETH下调表达,表明TaNpsn11~13可能是通过不同的信号途径参与小麦对条锈菌的抵抗;非生物胁迫下,TaNpsn11~13均不受到机械伤害胁迫的诱导,而对干旱和低温胁迫上调表达,并且干旱的诱导强度高于低温胁迫,对于高盐胁迫,TaNpsn11表现为抗性,而TaNpsn12、13表现为敏感;最后,成功改造适用于SNARE蛋白亚细胞定位的载体pRTL2-GFP-C1。(3)运用电子克隆技术并通过RT-PCR验证获得了小麦TaHin1的全长cDNA;其开放阅读框642 bp,编码213个氨基酸,理论分子量23.24 KDa,等电点8.67,含信号肽和HIN1结构域,可能为分泌蛋白;表达谱分析表明,TaHin1在小麦根、茎、叶组织中表达差异不显著;与条锈菌互作时,仅在非亲和组合中被诱导表达;外源SA、MeJA诱导TaHin1上调表达,ABA诱导其下调表达,而ETH不诱导其表达,表明其可能通过SA、JA信号途径参与了小麦对条锈菌的防御反应,ABA在防御反应中起负调控作用;高盐、干旱、机械损伤以及低温等非生物胁迫下,TaHin1均上调,暗示其在小麦对非生物胁迫的抗逆应答过程中也发挥着一定作用。(4)通过TIGR小麦数据库检索和RT-PCR验证,获得一条编码小麦肌动蛋白解聚因子(ADF)的全长cDNA,命名为TaAdf;其cDNA全长812 bp,其中ORF 417 bp,编码138个氨基酸,蛋白理论分子量16.10 KDa,等电点5.65;基因组序列含3个外显子,其中第一外显子仅包含起始密码子;利用中国春缺体/四体系将其定位于小麦6D染色体;洋葱表皮亚细胞定位显示其在整个细胞均有分布;原核表达蛋白大小约16 KDa,与理论值相符;表达谱分析发现TaAdf在不同组织差异表达,可能与小麦组织分化和发育相关;在与条锈菌互作中,亲和组合表达高于非亲和组合,暗示其与小麦对条锈菌的感病性相关;外源植物激素处理后,表达量主要被ABA上调,表明其可能通过调节气孔的开闭参与条锈菌对小麦的致病过程;非生物胁迫下,TaAdf对低温和干旱表现出明显抗性,对高盐和机械伤害胁迫无反应;VIGS结果显示TaAdf沉默以后,小麦对条锈菌的抗性加强。更多还原

【Abstract】 Yellow rust,caused by Puccinia striiformis f.sp. tritici, is one of the most detrimental diseases of wheat, imposing great threat to wheat production in China. It has been proven that breeding and growing resistance cultivars is the most economic and environmental friendly way to control the disease. Therefore, cloning genes related to the interactions between wheat and stripe rust fungus, along with their expression pattern and functional analyses, could help to illuminate the mechanism of host-pathogen interactions and provide solid foundation for wheat resistance breeding and modification. In this study, 13 genes which might be related to the host-pathogen interactions in wheat cultivar Suwon 11 were cloned by combining homologous cloning, RACE, in-silicon cloning and database exploration. Some of them were studied by bioinformatics analysis, expression pattern and functional analysis. The main contents and conclusions are as follows:1. To study the roles of SNARE proteins in wheat-stripe rust interactions, several wehat SNARE genes were cloned and analysed through bioinformatics. Firstly, TaNpsn11 and TaNpsn13 were obtained by combining homologuous cloning and RACE. Meanwhile, TIGR wheat annotation database provided a source for SNARE gene cloning; after RT-PCR verification, 9 wheat SNARE genes were characterized, including three R-SNAREs: Tav-Snare13、TaVamp725 and TaSec22;two Qa-SNAREs:TaSyp132 and TaSyp22;two Qb-SNAREs:TaNpsn12 and TaBet1;one Qc-SNARE:TaSyp51 and one Qb+c-SNARE:TaSnap28.2. Expression pattern of plant specific SNARE family--NPSN family was studied by qRT-PCR. Differentitial expression of TaNpsn11~13 in wheat tissues revealed their diverse roles in differentiation and development of wheat organs. When challenged by stripe rust pathogen, TaNpsns were significantly up-regulated in incompatible interactions, indicating that they might play important roles in wheat’s resistance to the pathogen. Among the four exogenous phytohormones, TaNpsn11 could be up-regulated by all phytohormones; TaNpsn12 was just up-regulated by salicylic acid (SA) and abscisic acid (ABA); TaNpsn13 was up-regulated by ABA and methy-jasmonate (MeJA) while down-regulated by ethylene (ETH). These results showed that TaNpsn11~13 might function in wheat resistance to stripe rust disease through different signaling pathways. TaNpsns could not be induced by wounding stress while significantly up-regualted by drought and low temperature. In response to high salinity,TaNpsn11 showed resistance while others showed sensitivity. At last, pRTL2-GFP-C1, a vector suitable for subcellular localisation of plant SNAREs, was successfully modified.3. A novel gene which might encode HIN1 in Suwon 11 was cloned by in-silico cloning. The gene, designated as TaHin1, contained a 642 bp open reading frame (ORF) in length, encoding 213 amino acid residues with a molecular weight 23.24 KDa and PI value 8.67. TaHIN1 might be a secreted protein, containing a signal peptide and a HIN1 domain. In the expression pattern analysis, TaHin1 expressed in wheat tissues without difference. Challenged by stripe rust fungus, TaHin1 was induced only in incompatible interaction, up-regualted by SA and MeJA while down-regulated by ABA. ETH could not induce the expression. TaHin1 was obviously up-regulated by various abiotic stresses. The results indicated that TaHin1 might facilitate wheat defense through SA and JA pathways, while ABA played a negative role. And it also played roles in wheat’s response to abiotic stresses.3. A novel gene which might encode a wheat actin-depolymerizing factor (ADF)was obtained from TIGR wheat annotation database, designated as TaAdf. The full-length cDNA was 812 bp, contained a 642 bp ORF, encoding 138 amino acid residues with a molecular weight 16.10 KDa and PI value 5.65. Its genomic DNA contained three exons in which the first exon just contained the initiation codon ATG. It was localised on chromosome 6D by Chinese Spring Nullisomic-Tetrasomics. Subcellular localisation showed its distribution in the whole cell. A 16 KDa band was observed in protokaryotic expression, in accordance with predicated weight. In the analysis of expression patterns, TaAdf expressed differentially in wheat tissues, revealing its role in differentiation and development of wheat organs. Challenged by stripe rust pathogen, TaAdf expressed higher in compatible interaction than in incompatible one. Meanwhile it was mainly up-regualted by ABA, revealing that TaAdf might mediate the wheat susceptibility to stripe rust disease by controlling the stoma through ABA signaling pathway. Among abiotic stresses, drought and low temperature signaicantly up-regulated its expression, while high salinity and wounding had no effect. After the silience of TaAdf in wheat through VIGS, enhanced resistance to stripe rust fungus was observed.更多还原