杨树抗病防御相关基因克隆及功能分析

【作者】 程强；

【导师】 王明庥； 黄敏仁；

【作者基本信息】 南京林业大学 ， 林木遗传育种， 2006， 博士

【摘要】 本研究使用杨树基因组数据库JGI P．trichocarpa v1.0(http://genome.jgi-psf.org/Poptr1/Poptr1.home.html)对38个差显序列进行了重新注释。注释结果显示11个差显序列为功能未知或和抗病防御相关；进而使用实时定量RT-PCR进行验证工作，实验结果显示在抗病植株Ⅰ-69(Populus deltoides cv.’Lux’ (Ⅰ-69／55))中两个差显序列69-Ⅲ-4和69-Ⅱ-6受病原菌(Marssonina brunnea f. sp. multigermtubi)诱导剧烈上调，69-Ⅲ-4为脂肪氧化酶基因，其在病原侵染后的晚期被诱导表达上调；69-Ⅱ-6为DNA损伤修复蛋白基因，其在病原诱导早期诱导上调。在本研究中，作者从RNA的提取，内对照基因的选择和扩增效率3个方面对实时定量RT-PCR技术进行了优化，建立了一套简便可靠的实时定量RT-PCR的方法，并使用方差分析给与验证。本研究在美洲黑杨Ⅰ-69中克隆得到两个脂肪氧化酶基因PdLOX1和PdLOX2(GenBank accession no．DQ131178，DQ131179)。原核表达分析PdLOX1和PdLOX2，外源蛋白与预测分子量吻合并具有脂肪氧化酶活性。推导氨基酸序列与已知的脂肪氧化酶进行系统发生分析发现PdLOX1和PdLOX2属于脂肪氧化酶类型Ⅱ 13-LOX类群，该类群脂肪氧化酶同多种生物和生物胁迫相关。使用实时定量RT-PCR对PdLOX1和PdLOX2受杨树真菌病原，机械损伤，茉莉酸甲酯，水杨酸处理时的表达情况进行分析表明：PdLOX1和PdLOX2均受M．f．sp．Multkiermtubi诱导上调，PdLOX1受诱导能力较强；两个基因均受茉莉酸甲酯和机械损伤诱导上调；在水杨酸处理时PdLOX1和PdLOX2表达水平下降，通过正向高效液相色谱分析表明两个脂肪氧化酶的酶产物主要为13-HPOD，表现为13-LOX活性，可能参与了茉莉酸的体内合成。通过以上结果我们推测这两个脂肪氧化酶可能在杨树抵抗生物和非生物胁迫中具有重要的作用。本研究中利用杨树基因组初步注释结果和美洲黑杨受真菌病原Marssonina诱后的特异表达的差显片段69-Ⅱ-6序列，克隆了一个DRT100同源基因。原核表达分析发现其具有互补宿主菌recA突变的能力，而且其表达可受真菌病原和水杨酸诱导上调，在茉莉酸甲酯处理下其表达量下降。在本研究中，还同源克隆了一个杨树PGIP基因，其与拟南芥中的PGIP1，2基因高度相似，以上工作为阐述DRT100基因同PGIP基因功能的异同提供了基础。本研究中利用实时定量RT-PCR方法分析杨树抗黑斑病QTLs目标区段候选基因在黑斑病病原诱导下的表达情况，结果显示，一个AP2类型转录因子在抗病植株中表达上调，而在感病植物中其表达量没有明显变化。更多还原

【Abstract】 Compared with the poplar genome database JGI P. trichocarpa v1.0 (http://genome. jgi-psf. org/Poptr1/Poptr1. home. html), thirty-eight differential display sequences were annotated newly. Eleven sequences were found to be associated with plant defense or were functionally unknown. Upon examination by real-time quantitative PCR, two of 11 differential display (DD) sequences - 69-III-4 and 69-II-6 -were found to be induced intensively by the pathogen. According to the genome annotation database for the species, 69-III-4 is associated with lipoxygenase (LOX) and 69-II-6 refers to a DNA damage-repair/toleration protein. In this study, we set up a sort of simple real time RT-PCR method and analyzed the reliability using ANOVA method .In the present study, we cloned two lipoxygenase genes, PdLOX1 and PdLOX2 (GenBank accession no. DQ131178, DQ131179). from Populus deltoides cv. ’Lux’ (1-69/55). A prokaryotic expression analysis of PdLOX1 and PdLOX2 revealed that the encoded exogenous proteins were identical to the predicted molecular weights and possessed the expected lipoxygenase activities. Chromatogram analysis indicated that the two lipoxygenase mainly possess 13-LOX activity. Phylogenetic analysis of the derived amino acid sequences of known lipoxygenases revealed that PdLOX1 and PdLOX2 were members of the type 2 13-LOX family of genes. This class of lipoxygenases is known to be involved in biotic and abiotic stress. Using real-time RT-PCR, we evaluated PdLOX1 and PdLOX2 expression following exposure to a Poplar fungal pathogen (Marssonina brunnea f. sp. Multigermtubi), mechanical injury, methyl jasmonate (MeJA), or salicylic acid (SA). We report that both PdLOX1 and PdLOX2 expression levels were increased following exposure to M. brunnea f. sp. Multigermtubi, with the pathogen exerting a relatively stronger influence on PdLOX1 expression. Furthermore, expression levels of the two genes were also up-regulated by mechanical damage and exposure to MeJA. In contrast, both PdLOX1 and PdLOX2 expression was down-regulated by SA treatment. We propose that the two novel lipoxygenases may play an important role in Poplar resistance to biotic and abiotic stress. According DD sequences and poplar genome database, we cloned a DNA damage-repair/toleration, PdDRT100. A prokaryotic expression analysis of PdDRT100 revealed that complementation of E. coli reck Mutation. PdDRT100 were increased following exposure to fungal pathogen and SA. In present study, we cloned a PGIP gene homolog from Populus deltoids.The sequence was highly homologous to Arabidopsis PGIP1, 2. Candidate genes in the QTLs involved in poplar black spot disease were identified based on populus gemone database. Using real-time RT-PCR, we analyzed expression level of 19 candidate gene in I-69(resistance)更多还原