【作者】 陈亮；

【导师】 张增艳；

【作者基本信息】 中国农业科学院 ， 生化与分子生物学， 2010， 博士

【摘要】 由腐生型真菌禾谷丝核菌(Rhizoctonia cerealis)引起的小麦纹枯病,已成为我国最严重的真菌病害之一。小麦纹枯病抗性遗传基础研究薄弱,易于育种利用的抗纹枯病小麦种质资源匮乏,迫切需要揭示抗纹枯病作用机制、发掘抗纹枯病有效基因。大量的研究已表明ERF转录因子在植物防御反应中起着重要的调控作用。本课题组在前期工作中,从抗多种病害的小麦近缘野生种中间偃麦草(Thinopyrum intermedium)中,分离到一个受纹枯病菌诱导的乙烯反应因子(ERF)基因TiERF1,并将其转入小麦品种扬麦12号中。本研究对TiERF1转录因子的特性与功能进行了更深入的研究。Southern杂交分析表明TiERF1基因在中间偃麦草基因组中至少含有两个拷贝。酵母单杂交试验结果表明,其编码的TiERF1蛋白能够在酵母体内与GCC-box顺式作用元件结合,并激活下游报告基因的表达,当预测的转录激活域缺失后,TiERF1的转录激活活性基本丧失。对转TiERF1基因小麦进行的PCR扩增和Southern杂交分析表明：TiERF1基因已成功整合入转基因小麦基因组中,并能够稳定遗传。定量RT-PCR分析结果表明,TiERF1基因能够在稳定遗传的转基因小麦株系中过量表达,并且一些乙烯/茉莉酸抗病信号途径上的病程相关基因几丁质酶和β-1,3-葡聚糖酶基因等的表达水平也显著提高。纹枯病抗性鉴定结果表明,与未转基因的小麦受体扬麦12号和TiERF1基因表达沉默的小麦株系相比,TiERF1基因的过量表达使转基因小麦对纹枯病的抗性得到提高。可以初步推测：TiERF1基因的转录调控机理是通过结合一些ET/JA信号转导途径中防御相关基因启动子上GCC-box顺式作用元件,激活这些基因的转录表达,从而介导寄主对纹枯病菌的防御反应。本研究对于揭示小麦抗纹枯病作用机制、改良小麦品种的纹枯病抗性非常重要。小麦黄矮病是由蚜虫介导的大麦黄矮病毒(BYDV)侵染引起的小麦主要病毒病害之一。至今,小麦属内尚未发现有效的抗黄矮病基因。小麦的近缘野生种中间偃麦草7X(7Ai#1)染色体长臂上的抗黄矮病基因Bdv2,高抗BYDV多个株系。本课题组已将携带Bdv2的中间偃麦草7Ai#1染色体长臂片段导入普通小麦基因组,选育出抗黄矮病的小麦-中间偃麦草易位系YW642等。由于该易位的外源染色体片段可能携带不利性状,因此分离7Ai#1L染色体上的抗黄矮病重要基因,对于深入研究抗黄矮病作用分子机制,开展抗黄矮病小麦基因工程育种十分重要。本课题组在前期研究中利用比较作图等策略,筛选出一些定位于含Bdv2的中间偃麦草染色体7Ai#1L区的基因表达序列EST-PCR标记。其中一条特异序列与蛋白激酶具有较高的同源性。本研究以上述含Bdv2的7Ai#1L区特异的蛋白激酶基因序列做为起始序列,开展了以下研究,取得良好进展。(1)首先利用瞬时的病毒诱导的基因沉默技术使抗黄矮病的小麦易位系YW642中该蛋白激酶基因表达沉默,结果此材料对黄矮病表现感病,表明该蛋白激酶基因是中间偃麦草染色体7Ai#1L区上的抗黄矮病重要基因。(2)利用RT-PCR和RACE方法,从接种饲BYDV蚜虫的中间偃麦草中,分离到该基因的全长序列。序列分析得知,该基因编码一个催化丝氨酸/苏氨酸和酪氨酸磷酸化的双底物特异性蛋白激酶,因此,命名该基因为TiDPK1 (Thinopyrum intermedium Dual-specificity Protein Kinase 1)。TiDPK1基因的编码产物具有类受体蛋白激酶(RLK)的结构特征,即含有一个胞外结构域、一个跨膜结构域及一个位于细胞质的蛋白激酶催化结构域。(3)通过自磷酸化试验证实TiDPK1蛋白的激酶催化结构域具有较强的自磷酸化活性。(4)通过基因枪转化洋葱表皮细胞瞬时表达试验表明TiDPK1-GFP融合蛋白集中分布于细胞膜。(5)Southern杂交分析表明TiDPK1基因定位于含Bdv2的中间偃麦草染色体7Ai#1L区,但在小麦与中间偃麦草基因组中都有同源拷贝,说明该基因是基因家族的一个成员。(6)对TiDPK1基因的表达特性分析结果表明：该基因仅在含Bdv2的抗黄矮病材料中表达,受BYDV诱导而上调表达,并且在接种BYDV的12h达到表达高峰；TiDPK1基因的表达在未接种BYDV的YW642幼苗期的各组织中无明显差异,但在接种BYDV约40天的YW642成株期的组织中具有表达优势,其中以叶片表达量最高,这似乎与抗黄矮病的功能相关,成株期时小麦黄矮病主要在叶部发病,TiDPK1基因的转录本优势地出现在叶片中,而小麦茎秆韧皮部可能是BYDV传输的主要部位,因此茎秆中TiDPK1基因表达量也较根和幼穗中高。此外,外源激素油菜素内酯和水杨酸处理可以明显提高TiDPK1基因的表达量。(7)利用酵母双杂交系统验证了TiDPK1蛋白激酶的胞外结构域与BYDV外壳蛋白的相互作用。(8)利用转基因技术从功能获得方面(使TiDPK1基因在感黄矮病小麦中表达)和功能缺失方面(利用双链RNA干扰技术敲减TiDPK1基因在抗黄矮病小麦中的转录本)开展该基因的功能互补研究。结果表明,受体小麦中8601对黄矮病高度敏感,TiDPK1基因表达的转基因小麦获得了对黄矮病的抗性,与携带Bdv2的抗黄矮病对照小麦易位系YW642对黄矮病的抗性程度相当；另一方面,以携带Bdv2的抗黄矮病小麦易位系YW642做受体,获得的TiDPK1基因沉默的转基因小麦对黄矮病表现高度敏感,与感黄矮病对照中8601相当,充分说明TiDPK1是中间偃麦草7Ai#1L染色体上的抗黄矮病重要基因。更多还原

【Abstract】 In recent years, the production of wheat in China has been seriously affected by wheat sharp eyespot caused by a soilborne fungus Rhizoctonia cerealis. Because the genetics of wheat resistance to sharp eyespot is not fully understood, and progress of the corresponding traditional breeding is slowly, it is urgent to unravel the wheat defense mechanisms against R. cerealis. Some ethylene response factor (ERF) transcription factors in plants have been evidenced to play important roles in regulating defense response to various pathogens. In this study, an ERF gene from a wheat relative Thinopyrum intermedium, TiERF1, was characterized further, transgenic wheat lines expressing TiERF1 were developed, and the resistance of the transgenic wheat lines against R. cerealis was investigated. Southern blotting analysis indicated that at least two copies of the TiERF1 gene exist in the Th. intermedium genome. Yeast one-hybrid assay indicated that the activation domain of TiERFl is essential for activating the transcript of the reporter gene with the GCC-box cis-element. The TiERF1 gene was introduced into a Chinese wheat cultivar, Yangmail2, by biolistic bombardment. Results of PCR and Southern blotting analyses indicated that TiERF1 was successfully integrated into the genome of the transgenic wheat, where it can be passed down. Quantitative reverse transcriptional-PCR analysis demonstrated that TiERF1 could be overexpressed in the stable transgenic plants, in which the transcript levels of wheat pathogenesis-related (PR) genes primarily in the ethylene-/jasmonic acid-dependent signal pathway, such as a chitinase gene and aβ-1,3-glucanase gene, were increased dramatically. Disease tests indicated that the overexpression of TiERF1 conferred enhanced resistance to sharp eyespot in the transgenic wheat lines compared with the wild-type and silenced TiERF1 plants. These results suggested that the overexpression of TiERF1 enhances resistance to sharp eyespot in transgenic wheat lines by activating PR genes primarily in the ET/JA-dependent pathway.Barley yellow dwarf virus, which was spreaded by aphids, can cause one of the most serious virus diseases of small-grain cereals, i.e. wheat, worldwide. Up to now, there is no effective resistance gene in Triticum. Thinopyrum intermedium, a wheat relative, shows a high level of resistance to BYDV. It possesses Bdv2 resistance gene which locates on the long arm of 7X(7Ai#1) chromosome of Th. intermedium and has high resistance to several isolates of BYDV. Through biotechnology and distant crossing methods, the 7Ai#1 long arm fragment of Th. intermedium that possesses Bdv2 has been introduced into wheat, and developed some wheat-Th. intermedium translocation lines with BYDV resistance, for example YW642. However, the alien translocation chromosome may possess disadvantageous characteristics. It is necessary to isolate BYDV resistance gene in 7Ai#1L for unraveling BYDV resistance mechanism and carrying out engineering breeding for disease resistance. In our previous study, we isolated 14 novel genetic expression sequences specific to the Bdv2 region in wheat-Th. intermedium T7DS.7DL-7Ai#1L translocation lines YW642 based on comparing genomics and chromosome mapping. Out of the expression sequences, one has homology with a rice protein kinase. This study was carried out with the gene sequence of that protein kinase as the initiation sequence, and obtained the following results:(1) Knocking down the transcript of the protein kinase gene in BYDV-resistant wheat-Th. intermedium translocation line YW642 by virus-induced gene silencing compromised the Bdv2-mediated resistance to BYDV. This result revealed that the protein kinase gene was an important BYDV-resistant gene in the 7Ai#1L region. (2) The full-length cDNA containing complete ORF, which encodes a dual-specificity phosphatase potentially capable of acting on substrate Ser/Thr and Tyr residues, was isolated from Th. intermedium leave cDNA by RACE and RT-PCR, and temporarily named as TiDPK1 (Thinopyrum intermedium Dual-specificity Protein Kinase 1). TiDPK1 contains three distinctive domains of receptor-like kinase (RLK):(a) an extracellular domain; (b) a membrane-spanning domain; and (c) a protein kinase domain that contains all of the 11 subdomains conserved among protein kinases. (3) The protein kinase domain of recombinant TiDPK1 showed strong autophosphorylation. (4)The fusion protein of TiDPK1-green fluorescent protein was targeted to cytomembrane, suggesting that TiDPK1 may contribute to signal transduction. (5) Southern hybridization analysis revealed that TiDPK1 was located in the region of 7Ai#1L that possesses the BYDV resistance gene Bdv2. Additionally, homologous copies were both present in the genomes of wheat and Th. intermedium. This result revealed that TiDPK1 was a member of a small multigene family. (6) Q-RT-PCR analysis showed that TiDPK1 was expressed predominantly in Bdv2-harboring materials and upregulated by BYDV infection in a time-dependent manner. The activation of TiDPK1 mRNA expression is obvious at 12 to 24h post BYDV infection. At the young seedling untreated, the expression of TiDPK1 did not show tissue-specific. At 40 days post BYDV infection, at the adult stage, TiDPK1 transcript was tissue-specific, the transcript level was high in leaves (the disease mainly occurred tissues), moderate in stems (BYDV transmitted tissues), and low in immature spikes and roots, implying that TiDPK1 expression may correspond to its resistant function. Additionally, the transcript level of TiDPK1 in the seedlings was significantly induced by treatment for 12 h with exogenous BR and SA hormones. (7) The yeast two-hybrid system was used to investigate the interaction of TiDPK1 with the coat protein or RdRp of BYDV. The results showed that TiDPK1 could interact with BYDV coat protein but not with RdRp. (8) The function of TiDPK1 gene was studied through functional complementary experiments by means of loss-of-function and gain-of-function. Comparing to the transgenic receptor of susceptible wheat Zhong8601, transgenic wheat lines expressing TiDPK1 exhibited good resistance to BYDV, in which the resistance was similar to the gene donor YW642. However, transgenic wheat plants silencing TiDPK1 expression by RNA interfering broke Bdv2 mediated-resistance in the recipient-wheat YW642. Based on these findings, TiDPK1 should be an important gene during the host resistance to BYDV, and may facilitate the wheat breeding with BYDV resistance.更多还原