摘要
本论文从大豆抗大豆胞囊线虫3号生理小种的抗性生化本质入手,系统地研究了大豆根系分泌物作用、膜脂过氧化作用、光合能力、物质代谢能力、防御酶系统和几丁质酶活性等多个方面与抗性的关系;本文首次利用我国特有的抗病品种哈尔滨小黑豆(ZDD7170)与辽宁省农科院培育的主栽品种辽豆10成功配制杂交组合,通过分组分离法(Bulked Segregation Analysis,BSA)建立F_4代抗感池,研究了F_4代抗感池之间与抗性相关的差异蛋白。这些对于从根本上揭示大豆抗大豆胞囊线虫病的抗性本质具有重要意义,为培育高抗的大豆品种提供了有力的理论支持。主要研究结果如下:
1.不同抗性品种不同时期的根系分泌物对二龄幼虫的趋化性影响呈现出不同规律。总体表现为感病品种根系分泌物对二龄幼虫有较强的吸引性,而抗性品种表现不规律,在线虫容易侵入的幼苗期,某些抗病品种甚至还对二龄幼虫表现出了一定的排斥性。胞囊孵化影响的试验结果表明,感病品种的根系分泌物能明显促进胞囊的孵化,而抗病品种的根系分泌物对胞囊孵化的影响不明显。
2.不同抗性大豆品种根系分泌物中氨基酸种类和含量与大豆抗病性有一定的关系。大豆根系分泌物中氨基酸总含量随品种对大豆胞囊线虫病抗性的增加而降低,抗病品种根系分泌物中检测到的氨基酸总含量平均为26.77mg·L~(-1),而在感病品种根系分泌物中平均达到87.90mg·L~(-1)。谷氨酸、甘氨酸、丙氨酸、亮氨酸、苯丙氨酸和赖氨酸等几种氨基酸在根系分泌物中的表达量与品种的抗性呈一定的负相关,而半胱氨酸和酪氨酸在抗病品种灰皮支黑豆和小粒黑豆中有高量表达而在感病品种中却未检测到,与抗病性关系更为密切。
3.大豆胞囊线虫侵染后抗感品种细胞膜系统受破坏程度不同。感病品种细胞膜受到了严重的伤害,根部组织内MDA含量显著增加,电解质渗漏严重:大多数抗病品种中的MDA含量虽然也有少量增加但与对照相比差异不大,电解质渗漏总体上低于感病品种。
4.物质代谢与抗病性有着密切关系。接种大豆胞囊线虫后供试抗病品种根内可溶性糖含量有不同程度的提高,感病品种根内可溶性糖含量低于抗病品种,认为寄主植物可能需要适当的糖类物质作为抗病反应的激发子来抵御线虫的侵染,而根系中低糖含量更有利于大豆胞囊线虫的侵入;受大豆胞囊线虫侵染诱导后,抗病品种根内可溶性蛋白含量变化较大,有明显的升高,这一现象与大豆受侵染后诱导产生与抗性相关的蛋白有一定关系。
5.受线虫侵染后大豆根内防御相关酶系,包括苯丙氨酸解氨酶(PAL)、多酚氧化酶(PPO)、超氧化物歧化酶(SOD)、过氧化物酶(POD)和几丁质酶活性出现了明显的变化,其中PAL和SOD表现出酶活的动态变化与对大豆胞囊线虫的抗病性关系更为密切。
6.通过对大豆双向电泳实验条件的优化,确定了以TCA/Acetone法提取大豆蛋白,裂解缓冲液为9M Urea,2M Thiourea,4%CHAPS,100mM DTT,0.5%Carrier ampholyte,4%NP-40,1mM PMSF,2mM EDTA,40mM Tris-HCl,18cm pH4-7IPG StripIEF等电聚焦时间最终控制在8000V,75000vh,聚丙烯酰胺凝胶浓度为12%的双向电泳最佳条件。
7.通过分离群体分组分析法构建抗病品种哈尔滨小黑豆与辽豆10杂交后代抗感池材料,F_4代抗、感池经双向电泳各检测到近500个蛋白点,从2-DE图谱上找到28个明显差异表达的蛋白点,通过基质辅助激光解析电离飞行时间质谱技术(MALDI-TOF/TOF)获得了这28个蛋白点的肽质量指纹图谱(PMF),Mascot数据库搜索比对后,鉴定了16个蛋白质点,它们分别是cytosolic ascorbate peroxidase9(112);ribulosel,5-bisphosphatecarboxylase/oxygenase,Rubisco(87);Oxidoreductase(110);retrotransposonprotein,putative,Ty3-gypsy subclass(36);calmodulin binding protein(131);phospholipase C(243);putative proteasome 20S betal.1 subunit(92);RNA polymerase beta'subunit(356);alpha 2 actin(183);Predieted protein(55,332);hypothetical protein(62,215,298,407,415)。其中,F_4抗病池上调表达的蛋白点(QUALITY)有:87,92,112;仅在F_4感病池中表达的蛋白点(QUALITY)有:183,298,332,407,415;抗病比感病池含量上调3倍以上的蛋白点有110,131;抗病比感病池含量下调3倍以上的蛋白点有:36,55,62,215,243,356。它们分别参与了植物自身的防卫反应、能量代谢、细胞信号传导和转录调控等多个生理生化过程,这些上调或下调的蛋白都是复杂的蛋白调控网络中的一部分,在植物的抗病反应中协同起着重要作用。
The researches on the resistant biochemistry essences of soybean against soybean cyst nematode(Heterodera glycines, SCN) race 3 were systematically conducted to the relationships between resistance and the root exudations, membrane lipid peroxidation, photosynthetic capacity, material metabolism, oxidases system and chitinase. The cross was made between resistant Harbin Xiaoheidou(ZDD7170) and suceptible Liaodou 10 to SCN in this paper. Applying the F_4 separated population from the cross of Harbin Xiaoheidou and Liaodou 10 as test material, according to Bulked Segregant Analysis(BSA) to study differential proteomics. It was important for fundamentally revelation resistant mechanism to H. glycines. Meanwhile this work offered theory guiding for breeding new reistant varieties. The main results of this study were as follows:
1. The different resistant variety and different stage soybean root exudation presented the different regulation to J2. The general result was that susceptible variety had the strong attractability to the J2 of SCN , but resistant variety performed less evidently, while the effect of resistant cultivars at seeding stage even rejected J2. Cyst hatching indicated that susceptible variety root exudation promoted cyst hatching, while resistant variety performance was not obvious.
2. Free amino acids from these soybean cultivars root exudates were detected using an Automatic Amino Acid Analyzer and their relationships of the resistance against H. glycines were studied. Data indicated that the kinds and contents of amino acids were different in these root exudates. Total amino acid contents decreased with increasing degree of resistance capability in these soybean varieties. Total amino acid contents of resistant variety was 26.77mg·L~(-1), of susceptible variety was 87.90mg·L~(-1). A significant negative relationship was detected between the disease resistance and the ratios of content of Glutamic acid, Glycine, Alanine, Leucine, Lysine to Phenylalanine. The ability of resistant to SCN was highly positively correlated with the ratios of content of Tyrosine and Cysteine.
3. Inoculated by SCN, the cell membrane of susceptible variety had serious destruction. MDA content in roots significantly increased. The cell membrane electrolyte leakage increased. But of resistant variety had no significantly changes, electrolyte leakage was lower than those of susceptible variety.
4. The study on the relationships between the metabolism and resistance to H.glycines indicated that the soluble sugar content of resistant varieties root was increased in different degree after inoculating by SCN, the soluble sugar content of susceptible varieties root was relatively lower than CK. Host plants need carbohydrate to resist SCN. Low sugar content in soybean roots was beneficial to nematode invasion. Moreover, infected and inducted by nematode, the soluble protein content in the roots of resistant varieties obviously increased, maybe soybean infected by SCN were induced to express resistant protein
5. Defensive enzymes were stimulated after infected by SCN J2. The dynamic changes of defensive enzymes, include phenylalanine ammonialyase(PAL), polyphenoloxidase(PPO), superoxide dismutase(SOD) , peroxidase(POD) and Chitinase were studied. The results showed that PAL and SOD had closely related to resistance to H.glycines.
6. Soybean protein was extracted with TCA/acetone, then resolubilized in lysis buffer(9 M Urea, 2M Thiourea, 4%CHAPS, 100 mM DTT, 0.5%Carrier ampholyte, 4%NP-40, 1mM PMSF, 2mM EDTA, 40mM Tris-HCl), 18cm pH4-7IPG Strip Isoelectric focusing(IEF)was performed at 20℃under 75000vh conditions. SDS-PAGE was performed in 12% polyacrylamide gels.
7. Differential proteomics were studied by the method of Bulked Segregant Analysis (BSA). Two-dimensional gel electrophoresis (2-DE) were adopted to identify proteins from soybean that the plants were differentially expressed in F4. Nearly 500 protein spots were detected. Furthermore, analysis with MALDI-TOF-MASS identification Peptide Mass Fingerprinting of 28 protein spots. The 16 protein spots were obtained by Mascot database searching and intercomparison preliminary identification. The proteins were respectively CytosolicAscorbatePeroxidase(112),Oxidoreductase(110),Ribulosel ,5-bisphosphateCarboxyl ase/Oxygenase,Rubisco(87),RetrotransposonProtein,Putative,Ty3-gypsySubclass(36),Calmod ulinBindingProtein(131),PhospholipaseC(243), Putative Proteasome 20S beta1.1 Subunit(92), RNA Polymerase beta' subunit(356), andAlpha 2 Actin(183), Predieted Protein(55, 332) and Hypothetical Protein(62, 215, 298, 407, 415). Protein spots 87, 92 and 112 were identified in resitant pool , protein spots 83, 298, 332 ,407and 415 were identified in sensitive pool. Three-fold upper protein spots were110 and 131. Three-fold lower protein spots were36, 55, 62, 215, 243 and 356. All of those up- or down-regulated proteins were probably parts of network of resistance or development-related proteins had close connection with plant defense responses, energy metabolism, cell signal transduction and transcriptional regulation.
引文
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