【作者】 曾兴权；

【导师】 吉万全；

【作者基本信息】 西北农林科技大学 ， 作物遗传育种， 2010， 博士

【摘要】 小麦(Triticum aestivum L.)是世界上栽培面积最广且最重要的粮食作物之一,其产量直接关系世界的粮食安全。由条形柄锈菌(Puccinia striiformis f.sp.tritici)引起的条锈病是世界及中国影响小麦生产的重要病害之一,发掘、研究、利用蕴藏于小麦后备种质资源的抗条锈病资源以及研究抗条锈病种质的抗性分子机理对于小麦抗条锈病研究和育种具有重要的理论意义和实践价值。本研究以西藏半野生小麦(T. aestivum ssp. tibetanum Shao)、西藏小麦地方品种和普通小麦-奥地利黑麦(Secale cereale L.)衍生系为材料,对其进行条锈病抗性鉴定,评价种质资源的遗传多样性、对筛选的抗条锈病种质进行分子细胞遗传的鉴定;以筛选的小麦-黑麦免疫条锈病材料NR1121为主要研究对象,利用抑制消减杂交方法构建条锈菌侵染小麦苗期叶片的SSH-cDNA文库,应用生物信息学方法对获得抗性相关基因表达的EST序列进行比对,分析抗性相关基因表达谱,筛选抗性相关新基因;采用半定量RT-PCR与实时荧光定量PCR分析新基因的表达模式,克隆抗性新基因;利用大麦条斑病毒(BSMV)构建新基因的病毒诱导的基因沉默(VIGS)载体进行RNAi表达分析,验证新基因的功能。研究结果如下:1.以136份西藏半野生小麦、119份西藏地方小麦品种和70份小麦-黑麦衍生系为材料,根据对条锈菌小种条中32(CY32)的抗性鉴定结果,筛选出苗期与成株期均高抗条锈病的西藏半野生小麦2份(隆子折达7,隆子折达10)以及小麦-奥地利黑麦衍生系NR1121。利用内含子切接点引物(Intron-splice junction primers,ISJ)和长随机引物的PCR分子标记技术分析遗传多样性。结果表明,在26个ISJ引物和300个引物组合中,33(11%)个引物或组合的PCR产物具有多态性。在西藏半野生小麦中共扩增出333条稳定清晰的条带,243(72.97%)条具有多态性条带;在西藏地方小麦品种中共扩增出316条稳定清晰的条带,197(62.34%)条具有多态性条带。西藏半野生小麦的遗传多样性高于西藏地方小麦品种;多态性丰富的ISJ标记较好的反应了西藏半野生小麦与西藏地方小麦品种之间遗传差异。2.采用细胞学、C-分带、GISH、SSR、SCAR、STS和A-PAGE等方法对小麦-黑麦免疫条锈病的衍生系NR1121进行了鉴定。NR1121和奥地利黑麦对CY32免疫,其普通小麦亲本陕麦611和携带Yr9的洛夫林10、洛夫林13、秦麦9号、丰抗8号、陕229和偃师9号均高感。NR1121形态学和细胞学稳定,2n=42=21Ⅱ;C-分带表明NR1121有一对奥地利黑麦的1R染色体;以奥地利黑麦总基因组DNA为探针的原位杂交结果显示,NR1121含有2条奥地利黑麦染色体;SCAR(AF1和AF4引物对以及SCM-9)标记和STS(ω-sec-p3/ω-sec-p4)标记表明,NR1121携带黑麦1R的遗传物质;A-PAGE结果显示,NR112在ω区的Gli-B1位点具有黑麦特征带,说明NR1121含有黑麦1RS遗传物质。鉴定结果表明,NR1121是一个免疫条锈菌小种CY32的小麦-黑麦1R二体异代换系,它携带的抗条锈病基因来源于奥地利黑麦,该基因可能是一个不同于Yr9的新抗条锈病基因。3.采用SSH技术构建了CY32侵染NR1121苗期叶片(非亲和互作)SSH-cDNA文库。在正交文库中随机挑取350个阳性克隆、测序,获得96条高质量EST序列,GenBank序列号为GO254150～GO254231、GR884577～GR884584和GT270714～GT270718。利用NCBI的BLASTX分析96条EST序列表明,78(81.2%)条EST找到同源性>90%的蛋白,其中已知蛋白功能的EST序列50条,主要涉及代谢(12.8%)、能量(9.0%)、信号转导(6.4%)、抗病防御(16.7%)、转运(5.1%)、蛋白质合成加工与储藏(5.1%)、转录(5.1%)、细胞结构(2.6%)和免疫(1.3%);未知功能的EST占35.9%。发现了与衰老联系蛋白、泛素蛋白连接酶2(Uubiquitin protein ligase 2, UPL2)、成熟酶K、膜转运蛋白YKT61和腺苷甲硫氨酸脱羧酶(S-adenosylmethionine decarboxylase, SAMDC)、丝氨酸/苏氨酸蛋白激酶SNT7(Serine/threonine protein kinase SNT7,S/PKSNT7)基因一致性高的EST序列。经文库比对,得到条锈病抗性相关蛋白22个,其中与抗病信号传导相关的蛋白5个,过敏性坏死反应(HR)体系表达蛋白1个,系统获得性抗性(SAR)体系病程相关蛋白13个以及SAR体系诱导防卫蛋白3个。4.反交文库中随机挑取150个阳性克隆、测序,获得69条高质量EST序列,GenBank序列号为GR884585～GR884652。69条EST分析表明,57(82.6%)条EST找到同源性>90%的蛋白,已知蛋白功能的EST序列28条。主要涉及代谢(12.3%)、能量(14.0%)、信号转导(7.0%)、抗病防御(1.8%)、转运(5.3%)、蛋白质合成加工与储藏(5.7%)、细胞结构(3.8%)和免役(1.9%);未知功能的EST占48.2%。经文库比对,得到条锈病抗性相关蛋白9个,其中与抗病信号传导相关的蛋白4个,HR体系表达蛋白1个,SAR体系病程相关蛋白3个,SAR体系诱导防卫蛋白1个。5.以酰基辅酶A合成酶(Acyl-coenzyme A synthetase,AcsA)和谷胱甘肽硫转移酶(Glutathione-S-transferase,GST)、脂转移蛋白(Lipid transfer protein,LTP2)和细胞色素P450(Cytochrome P450,CP450)、UPL2、S/PKSNT7、丝氨酸羧甲基转移酶(Serine hydroxy methyl transferas,SHMT)和SAMDC等8个候选基因为研究对象,利用半定量RT-PCR和实时荧光定量PCR分析了条锈菌侵染后它们在陕麦611(亲和反应)与NR1121(非亲和反应)中的表达模式。半定量RT-PCR与实时荧光定量PCR两种分析候选基因表达模式的结果基本一致;但是在条锈菌侵染后的非亲和反应与亲和反应中的它们表达时间和表达量有较大差异。条锈菌侵染后,AcsA在非亲和反应与亲和反应中均于24hpi(hours post inoculation)上调表达至最高水平,仅仅是表达量存在差异。GST、LTP2、CP450、UPL2、S/PKSNT7、SHMT和SAMDC基因在两种组合中的表达量和表达模式不同,在非亲和反应中均在24 hpi或48hpi上调表达较高水平;而在亲和反应中延迟至72hpi或更晚上调表达、或者呈下调表达(UPL2、SAMDC)。正是由于转运与抗病信号转导类和病害防御类等7个候选基因在关键的24 hpi至48 hpi特异上调表达(非亲和反应),参与了小麦的抗条锈病反应。结果表明条锈菌侵染后24 hpi至48 hpi是小麦苗期(非亲和反应)抗性相关基因表达的关键时期,可能是构建垂直抗CY32小麦材料SSH-cDNA文库的最佳时期。6.利用电子克隆、RACE和RT-PCR方法相结合,分别克隆了新的小麦腺苷甲硫氨酸脱羧酶(TaSAMDC2,GU016570)和丝氨酸/苏氨酸蛋白激酶SNT7基因(TaSNT7,GU574209)。TaSAMDC2基因cDNA序列全长2003 bp,5′非翻译区区域和一个带有Poly(A)的3′非翻译区区域分别长553和283 bp;该基因的开放阅读框为1167 bp,编码388个氨基酸,编码的氨基酸序列包含酶原剪切位点和PEST结构域。TaSAMDC2的基因组序列全长2539 bp,位于5′UTR存在526 bp的内含子序列,内含子的剪切位点均符合真核生物GT-AG规则。同源序列分析表明,TaSAMDC 2与来自大麦、水稻(Oryza sativa L.)、玉米(Zea mays L.)、一粒小麦(T. monococcum L.)4种植物SAMDC蛋白的相似性分别为95.0%、85.0%、80.0%和80.0%。TaSNT7基因cDNA序列与基因组序列均为1555 bp,TaSNT7基因的开放阅读框为1035 bp,编码344个氨基酸的多肽。同源序列分析表明,TaSNT7基因与水稻、玉米、茄属(Solanum lycopersicum)和烟草(Nicotiana tabacum)同源性分别为84.0%、84.0%、25.0%和25.0%。利用中国春缺体-四体系,将TaSNT7基因定位在小麦1D染色体上。7.利用VIGS体系分析了TaSNT7和TaSAMDC 2基因的功能,明确它们在小麦抗条锈病病程中的作用。对BSMV转染抗病植株后的转录物进行实时荧光定量PCR检测发现,BSMV-TaSNT7和BSMV-TaSAMDC2转染的小麦中检测到较低基因的转录物,表明TaSNT7基因和TaSAMDC2基因发生了沉默和部分沉默;小麦植株表型结果初步证实了TaSNT7基因参与了小麦的抗条锈病反应。更多还原

【Abstract】 Common wheat(Triticum aestivum L.)is the most widely grown food crop. Its production directly affects food security in the world. Stripe rust, caused by the obligate biotroph fungus Puccinia striiformis f. sp. Tritici Eriks (Pst), is one of the most destructive disease in common wheat production worldwide. Therefore, it is urgent to identify new stripe rust resistance germplasm and genes, broaden the spectrum of disease-resistant varieties, and study gene expression profiling and pattern for dissecting the regulatory mechanism and transcriptional networks that underlie phenotypic responses.T. aestivum ssp. tibetanum Shao, Tibetan wheat landraces (T. aestivum L.) and common wheat-Austrila rye (Secale cereale L.) derivatives as materials, their stripe rust resistance and germplsm genetic diversity were analyzed. Molecular cytogenetic identification of screened germplasm wheat- S. cereale derivative NR1121 immune to stripe rust was done. A SSH(Suppression subtractive hybridization)-cDNA library was constructed with the seedling leaves from wheat germplasm NR1121 infected by Chinese Pst race CY32. Resistance-related ESTs from SSH-cDNA library were anlyzed by bioinformatics methods, and resistance-related gene expression profiles was obtained, and then resistance-related novel genes were screened. The expression pattern of novel genes were analyzed by the semi-quantitative RT-PCR and real-time PCR, and the novel genes were cloned. The function of novel genes was verified by VIGS (Virus induced gene silencing) system constructed with BSMV (Barley stripe mosaic virus). The main results are as follows:1. Based on the identification results resistant to Pst isolation CY32 at seedling and adult, two T. Tibetanum Shao accessions(Longzizheda7and Longzizheda10) highly resistant to Pst and wheat- S. cereale derivative NR1121 immune to Pst were screened from 136 T. Tibetanum Shao accessions, 119 Tibetan wheat landraces and 70 wheat- S. cereale derivatives. The genetic diversities of 136 T. Tibetanum Shao accessions and 119 Tibetan wheat landraces were detected by using PCR method with ISJ (intron-splice junction) primers and long random primers. The results indicated 33(11%) primers or primer combinations out of 26 ISJ primers and 300 primer combinations could yield polymorphic bands. 333 good bands were produced from T. Tibetanum Shao accessions and 243(72.97%) bands were polymorphic. 316 good bands were produced from Tibetan wheat landraces and 197(72.97%) bands were polymorphic. The genetic diversities of T. Tibetanum Shao accessions were more than that of Tibetan wheat landraces. The genetic difference between T. Tibetanum Shao and Tibetan wheat landraces were identified by high polymorphic ISJ markers.2. S.cereal chromation in NR1121 was detected by chromosome karyotyping, C-banding, GISH (genomic in situ hybridization), SSR, SCAR, STS and A-PAGE. NR1121 and Austrian rye were immune to Pst isolate CY32 and Shaanmai 611 and six wheat cultivars (Lovrin 10,Lovrin 13,Qingmai No.9,Fengkang No.8,Shaan229 and Yanshi No. 9) with Yr9 were highly susceptible to CY32. NR1121 was stable in cytology with chromosome numbers 2n=42=21Ⅱ. Giemsa C-banding result showed that one pair of rye 1R chromosomes was detected in NR1121. GISH results with Austrian rye genomic DNA as a probe showed that two chromosomes of NR1121 was transferred from S.cereal. SCAR (primer combination AF1 and AF4, and primer SCM-9) and STS (primer combinationω-sec-p3 andω-sec-p4) markers indicated that NR1121 possessed chromatin of rye 1R chromosome. A-PAGE results indicated that Gli-B1 inωregion of NR1121 had the specific band of Austrian rye 1R short chromosome arm. All the results demonstrated that NR1121 was a wheat-S. cereale 1R disomic substitution line and immune to CY32. The resistant gene in NR1121 was derived from Austrian rye and probably had a new gene differing from Yr9.3. A SSH-cDNA library was constructed with the seedling leaves from NR1121 infected by Chinese Pst race CY32. From the forward library, 350 positive clones were randomly selected for plasmid extraction and sequencing, and 96 ESTs (expressed sequence tags) were obtained after removing repeated and redundancy sequences and submitted to GenBank. Accession numbers of GenBank were from GO254150 to GO254231, GR884577 to GR884584 and GT270714 to GT270718. BlastX alignment revealed that 78 of 96 ESTs predicted genes had more than 90% similarity to proteins and 50 ESTs with known function were involved in metabolism (12.8%), energy (9.0%), disease/defense (16.7%), signal transduction (6.4%), transportation (5.1%), protein synthesis, destination and storage (5.1%), transcription (5.1%), cell growth/division (2.6%), and the immune system (1.3%). The function of 35.9% ESTs were not known. ESTs with a high similarity to sequences of senescence-related proteins, UPL2 (Ubiquitin-protein ligase 2), maturase K, membrane transporter YKT61, SAMDC (S-adenosylmethionine decarboxylase), and S/PKSNT7 (Serine/threonine protein kinase SNT7) were discovered. There were 22 ESTs associated with stripe rust resistance, including 5 ESTs for signal transduction, 1 for HR (Hypersensitive necrosis reaction) system, and 16 for SAR (Systemic acquired resistance) system, respectively. 4. A total of 150 positive clones were randomly chosen from the inverse SSH-cDNA library and sequenced. 69 ESTs were obtained after removing repeated and redundancy sequences and submitted to GenBank. Accession numbers of GenBank were from GR884585 to GR884652. BlastX alignment revealed that 57(82.6%) of 69 ESTs predicted genes had 90% similarity to known genes and ESTs with known function were involved in metabolism (12.3%), energy (14.0%), disease/defense (1.8%), signal transduction (7.0%), transportation (5.3%), protein synthesis, destination and storage (5.17%), cell growth/division (3.8%), and the immune system (1.9%). The function of 48.2% ESTs was not known. There were 9 ESTs associated with stripe rust resistance, including 4 ESTs for signal transduction, 1 for HR system, and 4 for SAR system, respectively.5. The expression pattern of eight genes, which may participate in energy metabolism, disease resistance and defense, transportation, and signal transduction, were analyzed in Shaanmai 611 (Compatible interaction) and NR1121 (Incompatible interaction) by the semi-quantitative RT-PCR and real-time PCR. These genes were AcsA (Acyl-coenzyme A synthetase), GST ( Glutathione-S-transferase ) , LTP2 (Lipid transfer protein), CP450 (Cytochrome P450), UPL2, S/PKSNT7, SHMT (Serine hydroxy methyl transferas) and SAMDC. The results from semi-quantitative RT-PCR and real-time PCR of eight predicated genes were consistent with each other. The expression time and level of these genes were obviously different between Shaanmai 611 and NR1121. The genes AcsA was up-regulated to their highest expression level at 24 hpi(hours post inoculation) after Pst infection in Shaanmai 611 and NR1121, but the expression level was different. The expression level and time of the genes GST, LTP2, CP450, UPL2, S/PKSNT7, SHMT and SAMDC were different in between Shaanmai 611 and NR1121. These six genes were up-regulated to their highest expression level at 24 hpi or 48 hpi after Pst infection in NR1121, but the gene UPL2 and SAMDC were down- regulated and the other five genes up-regulated to their highest level at 72 hpi or later in Shaanmai 611. The results demonstrated that GST, LTP2, CP450, UPL2, S/PKSNT7, SHMT and SAMDC were highly induced at 24 hpi or 48 hpi after Pst infection in NR1121, suggesting they are transcriptionally activated for the host defense response. The key expression stage of resistance-related genes were from 24 hpi to 48 hpi in NR1121 seedling (Incompatible interaction) after Pst infection, which could be the optimal stage constructed seedling SSH-cDNA library of wheat resistant (Race specific) to CY32.6. Two new wheat genes, which were SAMDC gene designated as TaSAMDC2(GenBank accession number is GU016570) and S/PKSNT7 gene designated as TaSNT7(GenBank accession number is GU574209), were isolated from NR1121with in silico cloning,RT-PCR and RACE techniques. The cDNA complete sequence of TaSAMDC2 was 2003 bp in length and its 5’ untranslated region (5’-UTR) and 3’ untranslated region (3’-UTR) with Poly(A) were 553 and 283 bp , rsspectively. Open reading frame (ORF) of TaSAMDC2 gene was 1167 bp and encoded 388 amino acids containing conserved sequence with the proenzyme cleavage site and one typical conserved PEST domain of pathogenesis related protein SAMDC family. The genome DNA complete sequence of TaSAMDC2 was 2539 bp in length and its 5’-UTR contained 526bp intron with the splicing sites of GT-AT bi-nucleotidesequence. Homologous analysis found that TaSAMDC2 were 95.0%, 85.0%, 80.0% and 80.0% of sequence similarity with SAMDC proteins from barley(Hordeum vulgare L.), rice (Oryza sativa L.), maize (Zea mays L.) and wheat (T. monococcum L.), respectively. The cDNA and genome DNA complete sequence of TaSNT7 was 1555 bp in length. Open reading frame (ORF) of TaSNT7 gene was 1035 bp and encoded 388 amino acids. Homologous analysis found that TaSNT7 were 84.0%, 84.0%, 25.0% and 25.0% of sequence similarity with S/PKSNT7 proteins from rice (Oryza sativa L.), maize (Zea mays L.), solanum (Solanum lycopersicum) and tobacco (Nicotiana tabacum) , respectively. TaSNT7 was located on 1D chromosome by Chinese spring nullisomic-tetrasomic lines.7. With the established VIGS system, function analysis of two genes, TaSAMDC2 and TaSNT, were carried out in order to clarify their role during wheat in response to Pst attack. RNA was reversely transcripted into cDNA and BSMV recombination system was constructed to inoculate wheat. Real-time PCR results indicated that the low transcription products were detected in wheat inoculated by BSMV-TaSNT7 and BSMV-TaSAMDC2, which suggesting that TaSNT7 and TaSAMDC2 genes were silenced or partially silenced. Phenotype results indicated that the gene TaSNT7 played a role in stripe rust resistance pathway in wheat.更多还原