The Resistant Mechanism And Different Proteomics Of Soybean Aganist Heterodera Glycines

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 F₄ 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 protein5. 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.