【作者】 屈淑平；

【导师】 崔崇士； 马荣才；

【作者基本信息】 东北农业大学 ， 作物遗传育种， 2003， 博士

【摘要】 软腐病是大白菜生产中的细菌性病害，因其危害性也被称为大白菜三大病害之一，由胡萝卜软腐欧文氏菌胡萝卜亚种(Erwinia carotovora subsp. carotovora)引起。该病害在前期干旱、后期多雨或灌水不当情况下，很容易在生产中大面积流行，造成严重减产，是影响大白菜优质、稳产的重要因素。由于大白菜资源中缺少抗性材料，以及对植物抗病机制认识的匮乏，软腐病抗病育种一直是蔬菜育种的重大难题。只有深入研究植物的抗病机制以及植物与病原菌的互作关系，才能从根本上解决这一问题。本研究运用抑制差减杂交(SSH)技术构建cDNA文库，应用EST技术并与生物信息学分析技术相结合，通过阐明大白菜在软腐病菌致病过程中相关基因的表达分析，从基因表达整体水平来研究大白菜抗病机制。 本实验试材为东北农业大学园艺学院大白菜育种研究室培育的“东农905”大白菜品种，该品种具有良好的抗病性；软腐欧文氏菌(Erwinia carotovora subsp. carotovora)是从大白菜软腐病感病组织中分离纯化得到的。本研究构建了一个软腐病菌侵染后6、12、24、36、48、72小时的混合SSHcDNA文库，从中随机挑取单克隆进行单向测序获得1107条长度大于100bp、质量较好的ESTs序列。与GenBank的NR数据库进行BLASTx和BLASTn比较分析表明共有1058条EST具有编码蛋白质产物能力，占全部分析的EST的95.6％，49条EST与已知蛋白质同源性较低或找不到任何同源序列。具有蛋白质编码功能的1058条EST序列中793条编码产物功能已知，265条与推测、假想或功能未知的蛋白质具有很高的同源性。与793条EST编码产物具有最高同源性的推测蛋白质序列分别来自24个物种，其中77.4％来源于拟南芥(Arabidopsis thliana)、6.9％来自甘蓝型油菜(B. napus)、3.9％来源于甘蓝(B. oleracea)、2.9％来源于芥菜(B. juncea)，只有0.76％来源于大白菜已发表序列。对上述1107条EST进行片断重叠群(contig)分析共有753个非冗余EST，其中有564个单拷贝序列(singletons)和189个重叠群。 通过对获得的信号系统相关成分分析，推测钙介导的信号传导系统、MAPK相关信号传导系统可能参与了大白菜抗软腐病过程；本研究表明水杨酸信号传导系统在结球白菜抗软腐病反应中发挥作用；在我们获得的白菜EST中，存在许多参与乙烯、茉莉酸合成途径的酶基因，推测乙烯和茉莉酸参与了白菜软腐病防卫反应的信号传导，这与拟南芥的研究相符，但各信号传导系统之间的相互作用机制还不清楚。 在白菜抗软腐病过程中，除了PR-1类病程相关蛋白在本实验的测序中没有出现外，其它4类病程相关蛋白都存在。其中内切几丁质酶和Ⅰ类β-1，3葡聚糖酶出现的频率比较高，分别是10和12次。这两种酶在白菜抗软腐病中起到了重要作用；另外在所获得的EST中存在很多非生物胁迫诱导蛋白如干旱诱导蛋白、低温诱导蛋白、冷诱导蛋白、热休克蛋白、脱水胁迫诱导蛋白等。在265个推测蛋白中有46.8％来源于生物胁迫与非生物胁迫的cDNA文库。这一结果表明，生物机体对外界的胁迫反应，存在着某些共同的机制。 在软腐病菌致病过程中白菜体内合成了大量次生代谢酶类，其中包括参与木质素生一物合成的关键酶，这几种酶的表达量相对丰富，其EST拷贝数都大于3。说明木质素在白菜的抗病防御反应中具有重要的作用。细胞色素单加氧酶P450也大量存在于白菜软腐病 SSH cDNA文库中，它们参与多种次级代谢产物的合成，在植物防卫反应中具有多种复杂的作用。其它可能参与植物抗病和防卫反应的基因还包括维生素C相关的代谢酶类，抗坏血酸过氧化物酶和单抗坏血酸还原酶。另外过氧化氢酶、谷肌甘肽S－转移酶等基因的表达频率较高，说明它们参与的细胞保护机制可能参与了白莱抗软腐病的抗病反应中，具有保护细胞免受活性氧的破坏作用。 在获得的EST中还包括一些参与细胞壁合成和修饰的酶如D－葡聚糖外切水解酶、果胶甲酯酶、木搪昔酶、木聚糖内切酶、脂转移蛋白以及伸展素等，说明细胞壁的修饰作用也是植物抵抗病原菌侵染的途径之一。更多还原

【Abstract】 The soft-rot disease caused by Erwinia carotovora subsp. carotovora (Ecc) is one of the three major diseases of Chinese cabbage. It causes severe reduction in the production of several species of plants. The understanding of the resistance mechanism is of great importance to construct plants resistant to Ecc infection. In this work, expression sequence tags (ESTs) were analyzed in the process of Ecc pathogenesis to understand the resistance mechanism at global transcriptional level.A suppression subtractive hybridization ( SSH) cDNA library was first constructed from Chinese cabbage leaves challenged with Ecc at the early stages of soft-rot disease. The Chinese cabbage cultivar Dong-Nong 905 showing medium resistance to bacterial soft-rot was used in this study. In total, 1216 cDNA clones were randomly selected and sequenced and 1107 EST sequences longer than 100 bp with good quality were then obtained. The sequence similarity analysis was done using our local BLAST software by aligning the sequences with those of Non-redundant nucleotide sequence database (NR) of GenBank, and 1058 of the ESTs’ were identified encoding putative proteins. Out of the 1058 ESTs, 793 coding for functionally known proteins, 256 for the hypothetical, putative and unknown proteins, and 49 had not match with the sequences in GenBank. The best matched protein sequences with those encoded by the 793 ESTs were from 24 plant species, most of which from Arabidopsis thaliana and Brassica species, and only 0.76% were of published sequences from Chinese cabbage.After EST analysis, we supposed that calcium-mediated signal transduction system and MAPK-mediated signal transduction system play roles in Chinese cabbage’s resistance to Envinia carotovora subsp.carotovora. Ethylene, jasminate and salicylic acid have been shown to play important roles in plant’s disease resistance. Among the ESTs we obtained, many ESTs encode enzymes involved in the biosynthesis of ethylene, jasminate and salicylic acid, suggesting they take part in the signal transduction of cabbage’s defense reaction towards Ecc, which is consistent with the results obtained in Arabidopsis thaliana. However, the interactions among various signal transduction pathways are not clear yet.In Chinese cabbage’s responses to infection of Ecc four pathogenesis-related proteins, PR2, 3, 4 and 5, except for PR-1, exist in the sequenced ESTs. Among these, ESTs of endo-chitinase and class I -1, 3 glucanase have relative high copy number, 10 and 12 copies,respectively, in the 1058 ESTs. Therefore, these two enzymes play important roles in cabbage’s resistance to Ecc. In addition, in the ESTs obtained, there are many ESTs for abiotic stress-induced proteins, such as drought induced protein, low-temperature induced protein, cold induced protein, heat shock protein, and dehydration induced protein. Among the 265 ESTs encoding putative proteins, 46.8% were homologous to those induced under biotic and non-biotic stresses. The result suggests that a possible common mechanism exist during the stress reaction of plants towards different environments.In the infection process of the soft rot bacteria, the Chinese cabbage accumulated many transcripts encoding the enzymes involved in the biosynthesis of secondary metabolites. Five different ESTs of the genes for lignin biosynthesis have been cloned and sequenced from the SSH cDNA library, and they all exist in more than three copies. This result suggested that the lignin play an important role in the defenses against Ecc. Cytochrome P450 has been reported to be involved in various secondary metabolisms and defense responses, and EST analysis indicated that it was abundantly expressed in the responses of Chinese cabbage to Ecc infection. Other genes related to disease resistance and defenses were also identified, such as those for ascorbate peroxidase and monodehydroascorbate reductase in vitamin C biosynthesis. Furthermore, catalase and glutathione S-transferase were also shown to be involved in the defensive responses.Some cDNA fragmen更多还原