【作者】 王晓杰；

【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】 由条形柄锈菌（Puccinia striiformis f. sp. tritici）引致的小麦条锈病是一种气传性真菌病害,在全世界小麦种植区域均有不同程度发生。在我国,小麦条锈病发生、危害十分严重,已成为影响小麦生产可持续发展的限制因素。国内外的研究、实践证明,选育和种植抗病品种是防治小麦条锈病最经济、有效、安全的措施。因而,小麦抗条锈病机制以及小麦与条锈菌互作机理等方向的研究一直是植物抗病性及其利用领域中的研究热点。然而,由于条锈菌是专性寄生菌,且尚未发现其有性时代,加之小麦基因组庞大复杂,限制了对该病害的深入系统研究。因此,从转录组水平上全面系统了解小麦与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,并为小麦抗条锈性的合理利用和小麦抗条锈性的遗传改良及条锈病的持久控制提供理论依据和技术支撑。本文以小麦品种水源11分别与条锈菌条中CY31和CY23构成的亲和与非亲和组合为材料,采用cDNA-AFLP技术对其进行表达谱分析,同时分离差异表达的TDF（transcripts-derived fragment）;利用qRT-PCR技术验证cDNA-AFLP表达谱以及分析重要基因的表达特征;通过PCR筛选文库和RACE方法,克隆蛋白激酶、病程相关蛋白及与HR（hypersensitive reaction）相关的基因,并通过生物信息学分析基因的基本特性;运用BSMV-VIGS（virus induced gene silencing）技术对候选基因进行了初步的功能分析。主要研究内容及结果如下:1.分别在小麦接种条锈菌后6 h、12 h、18 h、24 h、36 h、48 h、72 h、96 h、120 h、144 h及168 h取样,抽提总RNA,反转录合成cDNA,然后利用cDNA-AFLP进行差异表达谱分析;利用64对引物检测到亲和与非亲和组合中表达的TDFs分别为54,912和52,992个,其中的33对引物具有较好的多态性。在亲和组合中,2,306个TDFs的表达谱发生了改变（占表达谱的4.2%）,呈上调表达的有1,340个TDFs,下调表达的有966个TDFs。在非亲和组合中约2,437个TDFs表达受到抑制或促进（占表达谱的4.6%）,其中1,787个TDFs呈上调表达,650个TDFs的表达受到抑制。亲和组合与非亲和组合分别回收215个和300个TDFs,经克隆、测序和聚类分析后,亲和组合unigenes为186个,非亲和组合unigenes为255个。利用GO分类方法分别对两个组合的unigenes进行功能注释,两个组合的ESTs均分为14大类,包括代谢、能量、细胞生长、转录、蛋白质合成、蛋白质储藏与运输、转运子、胞内运输、细胞结构、信号转导、疾病与防御、转座、功能未知以及没有匹配的序列。数据分析发现,亲和与非亲和组合中分别有60%和56%的ESTs功能未知或者为No hits;两个组合中,差异表达的基因以基础代谢、信号及抗病类为主,表明小麦与条锈菌互作时,基础代谢以及信号传导等相关基因转录活跃,寄主通过加快自身的代谢以及积累更多的营养物质,以满足自身及小麦条锈菌专性寄生的需要,同时也会启动一系列信号途径响应病原菌的侵染。2.分别从亲和及非亲和文库中挑选部分基因进行qRT-PCR分析,以验证cDNA-AFLP技术的准确性。研究结果表明,基因的表达趋势与cDNA-AFLP表达谱基本一致,并且大部分基因均受条锈菌诱导表达。说明利用cDNA-AFLP分析小麦与条锈菌互作的基因表达谱及分离差异表达的基因是可行的。3.以非亲和差异表达TDFs文库中的unigenes为Qurey方,用BLASTN搜索亲和互作数据库DB-TDFs进行比对分析。结果发现,两个组合中共有161条ESTs序列相似性很高,94条非亲和组合中的ESTs序列在亲和组合中没有匹配或相似性很低,它们编码的产物涉及基础代谢、信号与抗病反应及蛋白质代谢等各功能分类。对两个组合中相似性较高及没有匹配的部分ESTs片段进行了qRT-PCR分析,结果表明:大部分基因在亲和互作及非亲和互作中均受诱导表达,但在两个组合中表达的强度和启动表达的时间有所不同。由qRT-PCR分析的结果并结合cDNA-AFLP的表达谱可推测,在小麦与条锈菌互作体系中,基因表达的种类在亲和与非亲和互作中基本一致,基因表达的强度与时间的不同可能是引致寄主植物抗病性变化的因素之一。4.利用PCR筛选cDNA文库方法,获得了5个激酶类基因,分别暂命名为TaRLK（2393 bp）,TaCIPK（1993 bp）,TaLRR-RLK（2272 bp）,TaSTK（1831 bp）,TaCDPK（1441 bp）。它们分别编码受体激酶（684 aa）,CBL互作蛋白激酶（439 aa）,LRR-受体激酶（605 aa）,丝氨酸/苏氨酸激酶（433 aa）和钙依赖蛋白激酶（342 aa）;InterProScan分析表明,它们所编码的蛋白具有蛋白激酶的活性位点,属于激酶类基因,且TaRLK,TaCIPK,TaSTK三个蛋白的磷酸化作用需依赖ATP,TaCIPK具有该蛋白家族特有的NAF基序,NAF编码25个氨基酸,主要调控TaCIPK与钙调磷酸酶B（CBL）的相互作用;TargetP预测发现,TaRLK及TaLRR-RLK类受体激酶编码蛋白具有N端信号肽,可能是分泌蛋白质;Tmpred分析表明,TaCIPK和TaLRR-RLK编码的蛋白具有跨膜区;经Psort预测,TaRLK,TaCIPK,TaLRR-RLK蛋白分布在质膜的可能性较大,TaCDPK则可能位于细胞质,而TaSTK蛋白可能分布在过氧化物酶体或细胞核;氨基酸比对及进化树分析表明,TaRLK,TaCDPK,TaCIPK,TaLRR-RLK及TaSTK分别与小麦（Triticum aestivum,AAK20741）、水稻（Oryza sativa,BAD53576）、大麦（Hordeum vulgare,ABQ09285）、玉米（Zea mays,NP₀01147794）、大麦（H. vulgare,ABF18544）等激酶具有很高相似性;qRT-PCR分析表明,在转录水平上,TaSTK在小麦与条锈菌互作中的表达变化不显著,其余四个基因在互作早期均受条锈菌诱导表达,并且与亲和组合相比,这四个基因在非亲和互作中表达的强度较大,而且启动时间要早。结果表明,在小麦与条锈菌互作的早期,TaRLK,TaCDPK,TaCIPK及TaLRR-RLK转录产物迅速积累,可能参与了小麦对条锈菌的抗病反应,而TaSTK是否在转录后或翻译水平调控小麦与条锈菌互作需要进一步研究。5.采用BSMV-VIGS技术,对16个差异表达的激酶类基因进行功能分析。结果显示,11个发生基因沉默的小麦接种条锈菌CY23后症状表型发生了不同程度的变化（野生型接种条锈菌表现为抗病反应,表型为0;）,且组织学观察的结果与其表型变化基本一致。其中Su11_CY31₄885、Su11_CY31₅766、Su11_CY23_C304（TaCIPK）、Su11_CY23₂159（TaSTK）、Su11_CY23_C90等5个基因沉默后,过敏性坏死面积较大,仅有少量夏孢子产生,反应型为1+型;Su11_CY31₁189、Su11_CY31_C193（TaRLK）、Su11_CY31₃468、Su11_CY23₂780等4个基因的转录产物受到抑制后,枯死面积相对较小,并在褪绿斑周围产生少量夏孢子堆,反应型为2型,但对条锈菌CY23仍表现为抗病反应;而基因Su11_CY31₂485和Su11_CY31₁437的转录产物被沉默后,小麦对CY23的反应型,由0;转变为3+反应型,即由非亲和反应转变为亲和反应。以上结果表明:Su11_CY31₄885、Su11_CY31₅766、Su11_CY23_C304、Su11_CY23₂159、Su11_CY23_C90、Su11_CY31₁189、Su11_CY31_C193、Su11_CY31₃468及Su11_CY23₂780等基因参与小麦对条锈菌CY23的抗性反应,但对条锈菌的抗性贡献较小,而Su11_CY31₂485和Su11_CY31₁437两个基因在水源11对条锈菌CY23的抗病反应中起着重要作用。6.利用PCR筛选cDNA文库及RACE方法,获得了TaPR1b（862 bp）、TaPR5（810 bp）和TaPR10b（975 bp）三个病程相关蛋白基因,分别编码164、173、166个氨基酸;TMpred预测发现,TaPR1b、TaPR5、及TaPR10b蛋白均不含跨膜区。SignalIP 3.0预测TaPR1b蛋白的前24个氨基酸和TaPR5蛋白的前20个氨基酸是典型的信号肽结构,TaPR10b蛋白没有信号肽。另外,Psort预测显示,TaPR1b、TaPR5蛋白可能是胞外蛋白,而TaPR10b可能是胞质蛋白。在转录水平上,TaPR1b、TaPR5、及TaPR10b基因均受条锈菌诱导表达,且在亲和与非亲和组合中的表达变化趋势基本一致,只是在表达强度上,非亲和组合高于亲和组合。在12 hpi（Hour post inoculation）,基因转录达到第一次表达高峰,在24 hpi-72 hpi,三个基因的表达相对稳定,在120 hpi出现第二次表达高峰,且强度均高于第一次。以上结果表明TaPR1b、TaPR5、及TaPR10b基因参与了小麦与条锈菌互作体系中的基础防御反应,只是在非亲和组合中它们对条锈菌抗性的贡献要大。而在120 hpi,三个病程相关蛋白基因的表达强度高于第一次表达高峰,表明由于在此阶段大量次生菌丝会产生更多的吸器,从而引发更强烈的小麦应激反应,同时也暗示吸器在条锈菌与小麦互作中的重要作用。另外,外源SA、JA和ET处理小麦叶片后,qRT-PCR的研究结果表明,TaPR1b、TaPR5、及TaPR10b基因表达对SA和JA比较敏感,在早期即被诱导表达,并保持较高表达水平。而对ET的诱导表现不敏感。因此,它们可能主要通过SA和JA信号途径参与了小麦对条锈菌的抗性反应。洋葱表皮亚细胞定位表明,融合蛋白TaPR5-hGFP在非原生质体中高强度表达。TaPR5的免疫细胞化学标记结果显示:超薄切片经TaPR5抗体孵育和山羊抗鼠免疫球蛋白-胶体金标记后,胶体金颗粒主要沉积于寄主细胞壁上,亲和组合中寄主细胞壁上胶体金颗粒的密度远低于非亲和组合。寄主细胞质和细胞器上几乎无标记的金颗粒,病菌细胞没有金颗粒分布。表明TaPR5参与了小麦对条锈菌的抗性反应。7.通过PCR筛选cDNA文库及RACE方法克隆了TaBI-1（1095 bp）、TaDAD2（608 bp）、TaPrx（689 bp）、TaELMO（1146 bp）和TaLLS（1786 bp）等与HR相关基因的全长,并利用生物信息学方法明确了它们的分类地位;小麦与条锈菌互作过程中的表达特征分析显示,它们参与了小麦与条锈菌的互作过程;另外,外源SA和JA诱导TaPrx、TaELMO基因的表达,TaBI-1、TaDAD2和TaLLS在SA及JA诱导下,表达变化不显著,而五个基因对ET诱导均不敏感,表明它们通过不同的信号途径参与小麦对条锈菌的互作过程。利用VIGS进行功能分析的结果显示:TaBI-1和TaDAD2基因分别发生沉默后,再接种条锈菌CY23,小麦叶片过敏性坏死反应面积较对照和γ病毒接种CY23后的明显减少,且坏死斑周围有少量夏孢子产生,表明TaBI-1和TaDAD2参与了小麦对条锈菌的抗性反应,并且可能是小麦与条锈菌HR反应的负调控子。而TaLLS沉默后的小麦叶片过敏性坏死的数量明显减少,表明TaLLS可能是HR反应的正调控因子。更多还原

【Abstract】 Stripe rust, caused by Puccinia striiformis Westend. f. sp. tritici Eriks. （Pst）, is a devastating disease in wheat worldwide. The use of resistance cultivars offers the most economic and environmental friendly way to control the disease. In China, we have made great achievements on the control of wheat stripe rust through research on epidemiology and control of the disease. Systemic research on the mechanisms between wheat-Pst has been extensively conducted in the fields of histology, cytology, molecular biology, and biochemistry. However, due to special features of hexaploid wheat with large and complex genome and difficulties for transformation, and of Pst without clearly defined sexual reproduction and hard to culture on media, studies of genes involved in the wheat-Pst interactions using genetic and molecular techniques were largely limited. Therefore, deciphering the expression profiling of wheat-Pst interactions at the transcriptome level, along with identification and characterization of the resistance related genes, is of greatly importance to elucidate the molecular mechanisms of wheat-Pst interactions, which will be useful for development of genetically improved disease resistance and appropriate deployment of resistant cultivars to achieve sustainable control of the disease.In the study for my Ph.D. degree, expression profiling of wheat cultivar‘Suwon11’infected by Pst pathotypes CY31 and CY23, representing compatible and incompatible reactions, respectively, were analyzed using the cDNA-AFLP technique, followed by isolation of the differentially expressed transcript derived fragments （DE-TDFs）. Quantitative RT-PCR （qRT-PCR） analyses were used to verify the cDNA-AFLP patterns of selected important resistance-related genes. A wheat full-length cDNA library was screened using a PCR method, together with the bioinformatics characterization to identify resistance-related genes encoding kinases, pathogenesis related proteins and hypersensitive reaction induced genes. The biological functions of the identified candidate genes were determined using the BSMV-VIGS （Barley Stripe Mosaic Virus-Virus Induced Gene Silencing） approach. The follow most important results were obtained: 1. Suwon11 had compatible and incompatible reactions when infected with CY31 and CY23, respectively. Leaf samples were collected at each time point of 12, 18, 24, 36, 48, 72, 96, 120, 144, and 168 h after inoculation with Pst to extract total RNA and synthesize cDNA using reverse transcription. cDNA from each of the samples were used to determine differential expression profiling through cDNA-AFLP analyses. A total of 54,912 and 52,992 DE-TDFs from the compatible and incompatible interactions were detected, respectively, using 64 AFLP primer combinations, of which 33 produced reliable polymorphic bands. Expression profiling of 2,306 DE-TDFs in the compatible interaction showed differentially expressed （accounting for 4.2% of the whole compatible profiling）, of which 1,340 DE-TDFs were up-regulated and 966 down-regulated. In the incompatible interaction, 2,437 DE-TDFs were differentially expressed （accounting for 4.6% of the whole incompatible profiling）, of which 1,787 DE-TDFs were up-regulated and 650 down-regulated. From the characterized DE-TDF, 186 from the compatible interaction and 255 from the incompatible interactions were successfully cloned, sequenced, and assembled. A large portion of the DE-TDFs with unclear classification or no hits accounted for more than 60% in the compatible and 56% in the incompatible interaction. The majority of the DE-TDFs with function identified in each interaction were mainly involved in basal metabolism, signal transduction, and disease/defense groups. The results suggest that the host strengthens its metabolism and accumulates more nutrients to fulfill the wheat-Pst interactions. Additionally, based on their functional classification from both of the interactions, 47 genes were selected for further qRT-PCR analyses, along with their expression patterns in the mock-inoculated samples. The expression patterns revealed by qRT-PCR analyses were basically consistent with the cDNA-AFLP patterns. Because the expression changes of the genes in mock-inoculated samples were not significant, the identified DE-TDFs were most likely pathogen-induced genes.2. To compare genes in the compatible interactions with those in the incompatible interaction, BLASTN analyses were conducted to search against the database of DB-TDFs from the compatible interaction using the unigenes from the incompatible interaction as the query. The results showed that the 161 genes were shared by the two interactions. These genes were predicted to encode proteins involved in energy and metabolism, signal transduction and disease/defense, and protein metabolism. In contrast, 94 genes were unique in the incompatible interaction, which were also in the above mentioned categories. The results of qRT-PCR analysis to compare the expression patterns of selected shared and unique genes showed that most of the genes were induced in both of the incompatible and compatible interaction, but with different expression levels and at different time points between the two interactions. Therefore, we proposed that differentially expressed genes with different functional categories were most similar in two interactions, and the expression level or time of these genes might determine the resistance phenotype.3. Five kinase-encoding genes were obtained using the PCR-based method of screening the full-length cDNA library. These genes were TaRLK （2,393 bp）, TaCIPK （1,993 bp）, TaLRR-RLK （2,272 bp）, TaSTK （1,831 bp）, and TaCDPK （1,441 bp）, encoding receptor protein kinase, CBL interacting protein kinase, LRR-receptor protein kinase, Ser/Thr protein kinase, and Ca2+ dependent protein kinase, respectively. The results of the InterProScan analyses characterized the five genes to encode kinases. TaRLK, TaCIPK, and TaSIK proteins were found to have an ATP-dependent phosphorylation feature. Kinases-like TaRLK and TaLRR-RLK contained N-terminus signal peptides, which might be helpful for secretion. TaCIPK and TaLRR-RLK each contained a trans-membrane domain. TaCIPK had a typical NAF motif of its protein family, encoding 25 amino acids in responsible of regulating the TaCIPK and CBL interaction. The analysis of multiple alignment and phylogenetic tree demonstrated that TaRLK, TaCDPK, TaCIPK, TaLRR-RLK, TaSTK were highly similar to kinase genes in wheat （AAK20741）, rice （BAD53576）, barley （ABQ09285 and ABF18544）, and maize （NP₀01147794）. Expression patterns revealed by the qRT-PCR analysis indicated that these genes, except for TaSTK, were induced with high levels of expression at the earlier time points during the incompatible interaction. The rapid accumulation of these genes in the early wheat-PST interaction might be a sign of implication to wheat defense response to Pst for these genes.4. Sixteen kinase-encoding DE-TDFs were selected for further functional characterization using the BSMV-VIGS technique. Positive silencing for each of the gene was detected using qRT-PCR. Of the 16 genes, 11 showed phenotypic changes on the plants with the genes silenced. Further histological observations supported the results. Plants with silenced genes including Su11_CY31₄885, Su11_CY31₅766, Su11_CY23_C304, Su11_CY23₂159, and Su11_CY23_C90 showed larger area of hypersensitive cell death with slight amount of sporulation （infection type 1+）. Wheat plants with silenced Su11_CY31₁189, Su11_CY31_C193, Su11_CY31₃468, or Su11_CY23₂780 gene showed little sporulation around the small area of hypersensitive cell death, indicating a resistant reaction （infection type 2）. Plants with silenced Su11_CY31₂485 or Su11_CY31₁437 gene showed a compatible reaction （infection type 3+） when being challenged with CYR23. The results indicated that Su11_CY31₄885, Su11_CY31₅766, Su11_CY23_C304, Su11_CY23₂159, Su11_CY23_C90, Su11_CY31₁189, Su11_CY31_C193, Su11_CY31₃468, and Su11_CY23₂780 are involved in wheat defense response against CYR23 with low-level effects, while Su11_CY31₂485 and Su11_CY31₁437 play a more important role in the defense response. In addition, compared with control plants without virus inoculation, plants inoculated by wild-type BSMV virus showed larger area of hypersensitive cell death after CYR23 inoculation, without sporulation, therefore indicating that virus may induce defense response as well.5. Three wheat pathogenesis-related （Pr） protein genes, TaPR1b （862 bp）, TaPR5 （810 bp）, and TaPR10b （975 bp）, were successfully identified by screening the cDNA library and using the RACE technique. The encoded proteins of TaPR1b, TaPR5 and TaPR10b consisted of 164, 172, 166 amino acids, respectively. The TaPR1b and TaPR5 proteins were found to have a signal peptide within its N-terminal residues lacking of transmembrane, suggesting the putatively secretary function, while TaPR10b did not have a transmembrane or signal peptide structure. TaPR1b, TaPR5 and TaPR10b transcripts were induced upon Pst inoculation, with slightly higher transcription levels in the incompatible reaction than the compatible reaction, indicating that TaPR1b, TaPR5, and TaPR10b may be involved in wheat against the stripe rust fungus. Additionally, they were sensitive to SA and JA, and therefore, may play a role in resistance to Pst in the signal pathway of SA and JA. Transient expression analyses using gun-mediated bombardment method revealed that TaPR5 proteins were located in the epidermal cell walls. Immunocytochemical localization of TaPR5 in wheat-Pst interactions showed more gold particles were deposited over host cell wall in incompatible reaction, indicating that TaPR5 may take part in resistance to Pst.6. Five wheat genes, TaBI-1 （1,095 bp）, TaDAD （608 bp）, TaPrx （689 bp）, TaELMO （1,146 bp）, and TaLLS （1783 bp）, were successfully cloned by screening the cDNA library and using the RACE technique. These genes were identified using a series of bioinformatical methods. To further characterize their functions, TaDAD2, TaBI-1, and TaLLS were silenced with the BSMV-based VIGS system. The results showed that conspicuous hypersensitive response （HR） was elicited by CYR23 on leaves pre-infected with BSMV:γor BSMV:TaDAD2as and BSMV:TaBI-1as, as well as mock-inoculated plants. However, limited fungal sporulation around the necrotic spots was observed only on leaves infected with BSMV:TaDAD2as and BSMV:TaBI-1as by 10 day post inoculation. Therefore, knocking down TaDAD2 or TaBI-1 expression reduced fungal-induced cell death and allowed limited fungal growth and uredium development, which indicate that TaDAD2 may function as a suppressor of cell death in the early stages of wheat-stripe rust fungus interaction. Additionally, necrotic cell areas were significantly smaller compared with BSMV:γand mock-inoculated treatments when TaLLS was knocked down. These results suggested that TaLLS was a positive regulator of plant cell death in wheat-Pst interaction.In conclusion, the results of this study have elucidated molecular mechanisms and advanced our understanding of wheat-Pst interactions. The genes identified to be related to resistance could be used to improve stripe rust resistance in wheat cultivars through transformation or genetic manipulation.更多还原