【作者】 刘大伟；

【导师】 段玉玺；

【作者基本信息】 沈阳农业大学 ， 植物病理学， 2011， 博士

【摘要】 大豆胞囊线虫病（Soybean Cyst Nematode, SCN）是大豆生产中的重要病害之一。从世界范围来看,大豆胞囊线虫病的为害和蔓延日趋加重。本论文系统地从防御酶系的活性、次生代谢物质的含量、膜脂过氧化作用、光合作用、物质代谢等角度研究了我国特有的抗病品种灰皮支黑豆（ZDD2315）抗大豆胞囊线虫3号生理小种的生理生化机制；并首次利用抗病品种灰皮支黑豆与辽宁省的主栽品种辽豆15配制杂交组合,通过分离群体分组分析法（Bulked Segregant Analysis, BSA）建立了F4代抗、感池,采用双向电泳技术和质谱分析技术,寻找与大豆胞囊线虫3号生理小种抗性相关的差异蛋白；同时利用新一代高通量测序技术,筛选抗病品种灰皮支黑豆和感病品种辽豆15在大豆胞囊线虫3号生理小种侵染前后差异表达的基因,揭示灰皮支黑豆抗大豆胞囊线虫3号生理小种的分子机制。主要研究结果如下：1.明确了抗病品种灰皮支黑豆对大豆胞囊线虫3号生理小种的抗性不具有抗侵入的作用,而具有抗发育的作用。利用冰冻切片技术研究抗、感品种灰皮支黑豆和辽豆15受大豆胞囊线虫3号生理小种侵染后根部的组织病理学反应,明确了灰皮支黑豆抗大豆胞囊线虫3号生理小种的组织结构抗性。2.明确了灰皮支黑豆抗大豆胞囊线虫3号生理小种的生化机制。抗病品种灰皮支黑豆和感病品种辽豆15受大豆胞囊线虫3号生理小种侵染后,根内多酚氧化酶（PPO）、过氧化物酶（POD）、苯丙氨酸解氨酶（PAL）、几丁质酶的活性均表现升高,并且在灰皮支黑豆中各种防御酶系的活性高峰出现的较早,根内的总酚、类黄酮含量也都表现增加,但在灰皮支黑豆中的含量后达到峰值。3.明确了灰皮支黑豆抗大豆胞囊线虫3号生理小种的生理机制。抗病品种灰皮支黑豆和感病品种辽豆15受大豆胞囊线虫3号生理小种侵染后,辽豆15的细胞膜受到了严重的伤害,根内丙二醛（MDA）的含量显著增加,电解质大量外渗,根内可溶性糖的含量与对照相比显著增加,可溶性蛋白的含量也明显增加,叶片的叶绿素含量明显降低；而灰皮支黑豆的细胞膜受损程度较轻,根内丙二醛（MDA）的含量虽有少量增加但与对照相比差异不大,电解质少量外渗,根内可溶性糖的含量低于辽豆15,可溶性蛋白的含量却高于辽豆15,叶片的叶绿素含量无明显变化。4.利用BSA法成功构建了灰皮支黑豆（（?））和辽豆15（（?））杂交后代F4抗、感池材料,通过双向电泳获得了抗、感池的2-DE图谱,在凝胶上各检测到近400个蛋白点,对差异表达的27个蛋白点进行MALDI-TOF-MS质谱鉴定,获得了这27个蛋白点的肽质量指纹图谱（PMF）,通过Mascot数据库搜索比对,共鉴定出了16个蛋白点,其中仅在抗病池特异表达的蛋白点有hypothetical protein SORBIDRAFT₀4g012541 （Spot No.7）, predicted protein （Spot No.195）, ATPase alpha subunit （Spot No.224）, homeobox-like protein （Spot No.243）;仅在感病池特异表达的蛋白点有unknown （Spot No.14）;抗病池中表达量上调2倍以上的蛋白点有unknown （Spot No.53,71,284）, hypothetical protein SORBIDRAFT₀4g012541 （Spot No.116）, trypsin inhibitor p20 （Spot No.117）, Xylem serine proteinase 1 precursor, putative （Spot No.118）, triosephosphate isomerase （Spot No.173,187）, caffeoyl coenzyme A 3-O-methyltransferase 2 （Spot No.204）;抗病池中表达量下调2倍以上的蛋白点有putative RNA polymerase III （Spot No.296）, ATP synthase beta chain （Spot No.340）.5.利用RT-PCR技术分别获得了灰皮支黑豆573bp的胰蛋白酶抑制剂基因的序列和848bp的咖啡酰辅酶A甲基转移酶（CCoAOMT）基因的序列,同时利用实时荧光定量PCR技术（Real-Time Quantitative PCR）明确了抗病品种灰皮支黑豆和感病品种辽豆15受大豆胞囊线虫3号生理小种侵染后根内胰蛋白酶抑制剂基因的变化规律：大豆胞囊线虫侵染后,胰蛋白酶抑制剂基因在灰皮支黑豆根内的相对表达量始终高于辽豆15,并且在大豆胞囊线虫侵染后25d时灰皮支黑豆根内的胰蛋白酶抑制剂基因的相对表达量达到最高值。6.利用新一代高通量测序技术对灰皮支黑豆和辽豆15受大豆胞囊线虫3号生理小种侵染后的基因表达谱进行分析,筛选出受大豆胞囊线虫3号生理小种胁迫诱导表达的差异基因,灰皮支黑豆受大豆胞囊线虫胁迫诱导上调表达的差异基因有389个,下调表达的差异基因有344个；辽豆15豆受大豆胞囊线虫胁迫诱导上调表达的差异基因有222个,下调表达的差异基因有691个。这些差异表达的基因涉及激素应答、转录因子、蛋白激酶、热激蛋白、防御酶系、PR蛋白等方面。更多还原

【Abstract】 The soybean cyst nematode （SCN, Heterodera glycines Ichinohe） is the most important disease of soybean in the world. This disease was widely distributed and seriously disserved in the world. The best control method was using resistant cultivars against this disease. Huipizhi Heidou （ZDD2315） is a good resistant germplasm among the resistant materials. The research was systematically conducted from the view of the activities of defense enzymes, the contents of secondary metabolites, membrane lipid peroxidation, photosynthetic capacity and material metabolism to reveal the biochemical and physiological mechanism of Huipizhi Heidou resistant to SCN race 3. The F4 separated populations from the cross between the resistant Huipizhi Heidou and the susceptible Liaodou 15 to SCN race 3 were used as test materials by Bulked Segregant Analysis （BSA）. Two-dimensional gel electrophoresis （2-DE） and mass spectrometry （MS） were employed to separate and identify the differentially expressed proteins from soybean induced by SCN. The differentially expressed genes of the resistant Huipizhi Heidou and the susceptible Liaodou 15 infected with SCN race 3 were screened using digital gene expression （DGE） analysis. The main results were as follows:1. The resistant Huipizhi Heidou had no effect on the invading of SCN race 3, but had an effect on the development of SCN race 3. Histopathological response in roots of the resistant Huipizhi Heidou and the susceptible Liaodou 15 infected with SCN race 3 was studied by cryo-sectioning technique.2. The resistant Huipizhi Heidou and the susceptible Liaodou 15 were inoculated with eggs of SCN race 3 indoor, then the dynamic changes of defense enzymes, including polyphenol oxidase （PPO）, peroxidase （POD）, phenylalanine （PAL）, Chitinase were studied, and the contents of total phenolics and isoflavone were also determined in order to reveal the biochemical mechanism of Huipizhi Heidou resistant to SCN race 3. The results showed that the activities of PPO, POD, PAL could be increased both in Huipizhi Heidou and Liaodou 15, and the fastigium of defensive enzyme activities presented earlier in Huipizhi Heidou, the contents of total phenolics and isoflavone could also be increased in Huipizhi Heidou.3. The resistant Huipizhi Heidou and the susceptible Liaodou 15 were inoculated with eggs of SCN race 3 indoor, and the contents of MDA, chlorophyll, soluble sugar and soluble protein were mensurated in order to reveal the physiological mechanism of Huipizhi Heidou resistant to SCN race 3. The results showed that the cell membrane of Liaodou 15 had seriously destroyed, the content of MDA was significantly increased, the rate of electrolyte leakage increased, the contents of soluble sugar and soluble protein were increased compared to the uninoculated, the content of chlorophyll was decreased. But the cell membrane of Huipizhi Heidou had slightly destroyed, there was a small increase on the content of MDA and the rate of electrolyte leakage, the content of soluble sugar was less than Liaodou 15, the content of soluble protein was more than Liaodou 15, the content of chlorophyll had no significantly changes.4. The F4 separated populations from the cross between the resistant Huipizhi Heidou and the susceptible Liaodou 15 were used as test materials by bulked segregant analysis. Two-dimensional gel electrophoresis （2-DE） were adopted to separate the differentially expressed proteins from F4 induced by SCN. Nearly 400 protein spots were detected on the 2-DE gels. Of the 27 differentially expressed protein spots,16 protein spots were identified by MALDI-TOF-MS and 11 protein spots were not identified due to low scores. Hypothetical protein SORBIDRAFT₀4g012541 （denoted as No.7）, predicted protein （denoted as No. 195）, ATPase alpha subunit （denoted as No.224） and homeobox-like protein （denoted as No. 243） were newly expressed in the resistant samples; whereas unknown （denoted as No.14） was newly expressed in the susceptible samples; unknown （denoted as No.53,71 and 284）, hypothetical protein SORBIDRAFT₀4g012541 （denoted as No.116）, trypsin inhibitor p20 （denoted as No.117）, Xylem serine proteinase 1 precursor, putative （denoted as No.118）, triosephosphate isomerase （denoted as No.173 and 187） and caffeoyl coenzyme A 3-O-methyltransferase 2 （denoted as No.204） were up-expressed in the resistant samples; putative RNA polymerase III （denoted as No.296） and ATP synthase beta chain （denoted as No.340） were down-expressed in the resistant samples.5. The soybean Kunitz Trypsin Inhibitor （KTI） gene with a cDNA sequence of 573bp and CCoAOMT gene with a cDNA sequence of 848bp were obtained from Huipizhi Heidou by RT-PCR method. The expression of the KTI gene was detected in the resistant Huipizhi Heidou and the susceptible Liaodou 15 infected by SCN race 3 with the method of Real-Time Quantitative PCR, the results showed that the relative expression level of the KTI gene was higher in Huipizhi Heidou than that in Liaodou 15, and reached the top in Huipizhi Heidou at 25d after inoculating by SCN race 3.6. The differentially expressed genes of the resistant Huipizhi Heidou and the susceptible Liaodou 15 infected with SCN race 3 were screened using DGE analysis.389 genes were up-regulated and 344 genes were down-regulated in Huipizhi Heidou induced by SCN,222 genes were up-regulated and 691 genes were down-regulated in Liaodou 15 induced by SCN. These differentially expressed genes were involved in transcription factor, protein kinase, heat shock proteins, defense enzymes, PR proteins, hormone responses and so on.更多还原