The Resistant Mechanisms Of Huipizhi Black Soybean To Race 3 Of Heterodera Glycines

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.