【作者】 周新刚；

【导师】 吴凤芝；

【作者基本信息】 东北农业大学 ， 植物学， 2009， 硕士

【摘要】 黄瓜(Cucumis sativus L.)是一种世界性蔬菜,同时作为我国设施园艺栽培的第一大蔬菜作物,在蔬菜产业中地位均十分重要。由尖孢镰刀菌(Fusarium oxysporum)引起的黄瓜枯萎病是影响黄瓜产量和品质三大病害之一。随着黄瓜生产规模化、专业化、产量化的不断发展,在保持目前生产方式的前提下,黄瓜枯萎病等土传病害的危害日益严重,已经成为制约黄瓜持续高效生产的重要因素之一,生产上迫切需要解决枯萎病危害的问题。目前黄瓜枯萎病主要通过嫁接和施加农药的方式来防治,但分别存在费力和存在污染等缺点。深入研究黄瓜的抗病机制以及黄瓜与尖孢镰刀菌的互作关系,才可能建立经济有效的防治黄瓜枯萎病的方法。为了研究黄瓜接种尖孢镰刀菌后抗病相关基因的表达情况,本研究运用抑制消减杂交技术构建了cDNA文库,从分子水平来研究黄瓜的抗枯萎病机制。为进一步深入开展黄瓜抗枯萎病的抗病机制研究及利用抗性资源进行黄瓜抗病品种选育奠定基础。本试验主要试验结果如下:1.以黄瓜抗枯萎病品种中农13为材料,构建了黄瓜幼苗接种尖孢镰刀菌后12、36、72、120、144小时的抑制消减杂交cDNA文库,保存了1400个克隆。经菌落PCR和斑点杂交筛选后,随机挑选206个阳性克隆进行测序,获得188条高质量EST序列,序列聚类后共获得161个Unigenes,其中包括17个Contigs和144个Singlets。2.去除其中的真菌序列后,通过与GenBank数据库进行BLAST比较分析,发现有171条ESTs (96.6 %)可以找到已知的同源序列,6条ESTs (3.5 %)没有显著匹配结果,推测它们可能是新的抗病相关基因,或变异度较高的cDNA非编码区。其中有112条ESTs功能已知(63.3 %),65条功能未知(36.7 %)。功能已知的ESTs所编码的蛋白涉及黄瓜的能量代谢、信号转导、转录调控及抗病防卫等多方面。就BLAST对比同源性最高的基因的来源看,大部分来源于黄瓜、拟南芥、玉米、蓖麻、西瓜、南瓜、苜蓿和水稻等。3.通过对功能已知的基因分析,推测编码DnaJ、泛肽连接酶、WD-repeat蛋白、丝氨酸/苏氨酸激酶、锚蛋白重复序列、CBF4转录因子、锌指蛋白、NAC结构域等蛋白的基因是黄瓜经尖孢镰刀菌诱导后表达的细胞信号传递和转录调控的基因,可能在黄瓜抗病防卫反应基因的表达调控中起到重要作用;过氧化物酶、2-Cys peroxiredoxin、细胞色素P450、cupin超家族、Clp蛋白酶、半胱氨酸蛋白酶、Hsp70、金属硫蛋白等基因可能参与了黄瓜的主要抗病过程;在尖孢镰刀菌侵染过程中也出现了次生代谢相关基因,如编码肉桂酸-4-羟化酶、纤维素合成酶、肉桂酰辅酶A还原酶、玉米黄素环氧化酶等的基因。另外在所获得的ESTs中存在部分编码非生物胁迫诱导蛋白(如热休克蛋白、脱水响应蛋白、类金属硫蛋白等)的基因,说明生物机体对外界的胁迫反应存在某种协同机制。在获得的ESTs中还包括一些光合作用和能量代谢相关基因,说明这些基础代谢基因也与黄瓜的抗病机制相关。4.通过Gene Ontology方法对所得基因在分子功能、细胞组成及生物过程三个方面进行了功能分类和分析。结果表明经尖孢镰刀菌侵染后黄瓜在细胞组份及生物代谢中发生了明显的变化。生物过程方面,与代谢过程和细胞过程相关的基因得以大量表达,细胞的生理代谢和刺激反应加强;分子功能方面,许多具有绑定功能和催化功能的分子大量表达;同时细胞、细胞组分及细胞器等相关基因也大量表达,而这些基因的表达可能与黄瓜的枯萎病抗性相关。更多还原

【Abstract】 As an economically important vegetable, cucumber (Cucumis sativus L.) is one of the most popular greenhouse plants. Fusarium wilt in cucumber, caused by Fusarium oxysporum, occurs worldwidely and can cause severe yield loss. Susceptible plants can be infected in all stages of their development. The pathogen infects the roots and then blocks the uptake of water and nutrients, killing the root and above ground tissue. Fusarium wilt is one of the three most severe diseases in cucumber production. With the scale and specialization production of cucumber, soil born diseases like Fusarium wilt are becoming more and more severe under the present production mode. So it is urgent to deal with Fusarium wilt. At present, grafting and using pestiside are main courses to control Fusarium wilt in cucumber production. But they are labour consuming or pollutive. So it is necessary to elucidate the resistance mechanism of cucumber to Fusarium wilt and the mechanism of action between cucumber and Fusarium oxysporum in developing ecologically sound strategies to overcome Fusarium wilt. In our study, a suppression subtractive hybridization (SSH) cDNA library was constructed with cucumber leaves induced by Fusarium oxysporum. And expression sequence tags (ESTs) and bioinformatics were used to analyze the expression of defence relating genes. This might set foundation for the further studies of resistance mechanisms of cucumber to Fusarium wilt and disease resistance breeding. Main conclusions of this paper are as follows:1. A suppression subtractive hybridization cDNA library was constructed with Fusarium wilt resistant cucumber cultivar (Zhongnong 13). Seedling root, picked 6, 12, 24, 36, 48 and 72 h after inoculating with Fusarium oxysporum were used as tester, and seedlings dipped with water were used as driver. And 1 400 cDNA library clones were conserved. 206 positive clones were selected and sequenced, and 188 high quality ESTs were obtained. And 161 Unigenes, including 17 Contigs and 144 Singlets, were obtained after clustering.2. Similarity analysis based on BLASTx and BLASTn softwares in GenBank was performed. 171 (96.6 %) of the ESTs could find encoding putative proteins. 6 (3.5 %) of the ESTs were found with no significant similarity and they might represent new genes or high variant non-coding regions of cDNAs. Function of 112 ESTs (63.3 %) were known, 65 (36.7 %) were unknown. The best matched protein sequences with those encoded by the 170 ESTs were from many plant species, such as cucumber, Arabidopsis thaliana, maize, Ricinus communis, watermelon and rice.3. DnaJ, WD-repeat protein, serine/threonine protein kinase, ubiquitin-conjugating enzyme, ankyrin repeat-containing protein, CBF4 transcription factor, NAC domain protein and HD-ZIP protein genes were supposed to function as signal transduction and transcription regulating genes in cucumber after inoculated with Fusarium oxysporum. Peroxydase, 2-Cys peroxiredoxin, cytochrome P450, enzyme of the cupin superfamily, Clp protease, cysteine protease and Hsp70 might be involved in the main resistant course of cucumber. Secondary metabolism relating genes also appeared, such as cinnamate-4-hydroxylase, cinnamoyl-CoA reductase, zeaxanthin epoxidase and cellulose synthase genes. Some abiotic stress induced genes, including dehydration-responsive protein and metallothionein-like protein coding genes, also occurred several times, which indicated that there was crosstalk between biotic and abiotic resistance. Photosynthesis and energy metabolism relating genes were also included in acquired ESTs, indicating primary metabolites, and they might function in cucumber resistance to Fusarium wilt.4. All of the acquired ESTs were analyzed with Gene Ontology in cellular component, molecular function and biological process. Results showed that sort and number of cucumber resistance relating genes induced by Fusarium oxysporum were significantly different. Genes relating to metabolic process and cellular process existed high expression, binding and catalytic molecules increased. And these led biological regulation and response stimulus enhanced; cell, cell part and organelle relating genes also expressed extensively. And these differencially expressed genes might be closely related to cucumber resistance to Fusarium wilt.更多还原