| Soybean cyst nematode (SCN, Heterodera glycines Ichinohe) is one of the serious soil-borne diseases of soybean production in the world currently. Selecting and using resistant varieties is the most economical and effective measures of preventing and combating the SCN. In order to expand the use of resistant resources, Wuzhaiheidou (ZDD2255) that is resistant to multiple physiological races 3 of SCN was chosen as a high resistance germplasm resource in this experiment. Wuzhaiheidou (ZDD2255), resistant to multiple physiological races 3 of SCN was used to make a combination with Hefeng 35 for the first time.With analyzing the resistant inheritance of the combinantion, the relationship between physiological and biochemical quotas of the performance of plant photosynthesis, root activity, metabolism, cell membrane permeability, defense enzyme activity, the content of secondary metabolites and hormones, and Wuzhaiheidou resistance to races 3 of SCN was studied systematically. To reveal the mechanism of Wuzhaiheidou resistance to races 3 of SCN from the molecular mechanisms, the transcriptome and quantitative proteomics of resistant and susceptible varieties and resistant and susceptible pool sample of hybrids F5 were studied by using the high-throughput of new generation sequencing technology RNA-Seq and iTRAQ respectively. The different genes and proteins in the resistant and susceptible samples were also analyzed. The main research results show as follows:1. Wuzhaiheidou (ZDD2255) and Hefeng 35 formulate hybrid combinations was used for the first time to research the resistant inheritance, it is initially cleared that, the inheritance resistance of Wuzhaiheidou to races 3 of SCN is controlled by a couple recessive gene under Hefeng 35 genetic background.2. The characteristics of Wuzhaiheidou resistant to the infection of races 3 of SCN and inhibit the development and reproduction of races 3 of SCN is convinced. Compared with the susceptible variety Hefeng 35, the resistance of Wuzhaiheidou root shows a low infection rate, and a low natural hatching rate of cyst that grow on the root of the resistant variety, and the auxo action that the root secretion have for hatching the eggs of races 3 of SCN is significantly less than susceptible cultivars. Wuzhaiheidou can inhibit J3 and J4 to the development of mature females, the ratio of male to female was 1:4.13, while the Hefeng 35 the ratio of male to female was 10.56:1. Under the races 3 of SCN stress, photosynthetic characteristics, chlorophyll content and root activity of resistance and susceptible varieties decrease, but the decline of the Wuzhaiheidou is less than that of Hefeng 35. The cell membrane permeability, content of soluble sugar and soluble protein increase, the increase of cell membrane permeability of Wuzhaiheidou is less than that of susceptible varieties, and soluble sugar and soluble protein of Wuzhaiheidou is greater than that of Hefeng 35.3. Endogenous hormones of soybean root inoculated the races 3 of SCN in different periods was measured by ELISA, and the role of endogenous hormones of the soybean root in resisting soybean cyst nematode was made clear for the first time. Under the the stress of races 3 of SCN, endogenous auxin (IAA) and abscisic acid (ABA) of the disease-resistant varieties and susceptible cultivars root increased with different degrees compared with that of the control plants; the increases of IAA of Wuzhaiheidou are greater than that of the susceptible cultivars, and IAA in soybean root has a large effect on the races 3 of SCN infection in early stage. The amount of ABA of WuZhaiheidou increased less than that of Hefeng 35. The content of cytokinins (ZR) and gibberellin (GA) decrease after races 3 of SCN infection, and the content of ZR and GA in Wuzhaiheidou root decreased less than that in Hefeng 35.4. Under the stress of the races 3 of SCN, defense enzymes and secondary metabolites in the root are involved in the biochemical reaction of anti nematode. Polyphenol oxidase (PPO), phenylalanine ammonia lyase (PAL), and chitinase activity in Wuzhaiheidou roots are highly expressed in the process of the resistance to the races 3 of SCN, and the dynamic changes of PAL, PPO and chitinase are more closely related with its resistance to the races 3 of SCN, the activities of superoxide dismutase (SOD), peroxidase (POD) vary in different stages. Under the stress of the races 3 of SCN, the content of total phenols and flavonoids in resistant and susceptible cultivars root increased, with a bigger amount of Wuzhaiheidou than that of Hefeng 35, and the content of chlorogenic acid and lignin increased abnormally which showed that phenylpropanoid metabolites had played an important role in the biochemical mechanisms of Wuzhaiheidou resistance to the races 3 of SCN. The study with exogenous and in vitro assay showed that the flavonoids extracts of Wuzhaiheidou root inhibitory effect on the hatching of the races 3 of SCN eggs and the content of protein and carbohydrate of J2, and had less effect on the activity of antioxidant enzymes of J2. Wuzhaiheidou probably interfered the sugar and protein metabolism in the races 3 of SCN to kill it.5. The high-throughput sequencing technology Illumina HiSeqTM2000 was used to make clear the differential gene expression related with resistance in the Wuzhaiheidou for the first time. By studying the transcriptome of the Wuzhaiheidou before and after infection, 1045 differential gene expressions were screened among two transcription, with 567 genes up-regulated and 478 genes down-regulated. Compared with the susceptible cultivar Hefeng 35,757 differential gene expressions were detected. Wuzhaiheidou T2 had 570 up-regulated genes and 187 down regulated genes with respect to Hefeng35 T4.Comparing the different expressed genes in KEGG database, and analyses KEGG pathway. Discover that there are totally 307 DEGs which enrich in the 14 pathways in Wuzhaiheidou before and after inoculation.. Furthermore, there is the most DEGs were enriched in phenylpropanoid biosynthetic pathway, followed by phenylalanine metabolism pathway, plant--pathogen interaction of metabolic pathwa, the flavonoid biosynthesis pathway and Stilbenoid, diarylheptanoid and gingerol biosynthesis pathway, The differential expressed genes in 5 pathways occupied relatively high ratio. Hefeng 35 totally has 28 DEGs that annotates 19 pathways, which the nitrogen metabolism pathway is the most DEGs, followed by alanine, aspartic acid and glutamic acid metabolism. Resistant and susceptible cultivars inoculated have totally 219 DEGs that annotates 56 pathways, which the most DEGs were significantly enriched in phenylpropanoid biosynthesis pathway and the expression of those genes were up-regulated; the second is enriched phenylalanine pathway, The metabolic pathways of oxidative phosphorylation has the 17 differential gene expression, which is significantly enriched, and expression is up-regulated. In plant-pathogen interaction and flavonoid biosynthesis pathway, a lot of related transcription factors of resistance and pathogenesis related protein gene were identified and showed different expression degrees before and after nematode infection.6. By using BSA to construct resistant and susceptible pool materials of F5 generation group of Wuzhaiheidou and using iTRAQ technology to study the different proteomics of the soybean root protein samples after 7d under the races 3 of SCN infection.175 differential protein spots were collected in resistant pool compared to susceptible pool, with 102 proteins up-regulated expression and 73 proteins down-regulated expression. GO analysis showed differential expression proteins in cell components can be annotated to 8 items; and differential expression proteins in biological processes clustered into 15 items; and differential expression proteins in molecular function clustered into 7 GO items. By comparing with KEGG database, it was found that related different expression proteins had involved in 16 related plant metabolic pathways, an important class of plant disease resistance related are phenylpropanoid biosynthesis and metabolism related proteins, oxidative phosphorylation metabolism related proteins, signal transduction, cell related protein amino acid metabolism related proteins, peroxisomal proteins, plant pathogen interaction proteins and so on.The result of proteomic analysis is almost identical with those of genome analysis. And combined with the analysis of physiological and biochemical indexes, the mechanism of Wuzhaiheidou resistance to the races 3 of SCN includes the anti invasion, anti development and reproduction. From gene to protein level as well as secondary metabolism substance analysis, phenylpropanoid biosynthesis and metabolism, phenylalanine metabolism, oxidative phosphorylation as well as plant and pathogen interaction pathway and related proteins play a comprehensive role in Wuzhaiheidou resistance to the races 3 of SCN. |
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