| Phytophthora root and stem rot of soybean（Glycine max （L.） Merril）, caused byPhytophthora sojae Kaufmann&Gerdemann, is a destructive disease which is a serious threat tosoybean production all over the world. The yield loss of soybean due to this disease was up to25%～50%, even100%, so it is one of devastating diseases for soybean. In China, Phytophthorasojae was reported in Heilongjiang, Anhui, Shandong, Henan, Jiangsu, Zhejiang, Fujian, Xinjiangand et al. Heilongjiang province is the main soybean production area in China where the incidenceof soybean root rot has been more and more serious in recent years.So far, the most effective approach to reduce losses caused by P. sojae is the use of geneticresistance in soybean. However, the variability of P. sojae is very high, and resistant cultivars willlose their resistance with the new race population increasing and toxicity changing.Disease-resistant genetic engineering is one of the most useful ways to solve this difficult problem.With the completion of soybean genome and the development of molecular biology, soybeandisease resistant mechanism has made a great progress.In the long-term evolution process, to resist pathogens infectation, plants gradually formed aseries of complex and effective protection mechanism and defense network. Resistant genes wereselected with SSH and cDNA genechip using “Suinong10” which was highly resistant toPhytophthora sojae race1as test materials, and it was homology with pathogenesis-related proteinfrom several plants and it was found to be up-regulated highly. Therefore, it is very significantlyimportant to rich soybean PR10and PRP genes genetic theory and practice resistance geneengineering breeding by analyzing the related resistant function of PR10and PRP genes.The objective of this study was to analyze the related resistant function of PR10and PRPgenes, which will provide theoretical basis for obtaining resistance genes and cultiving resistantcultivars.The main results were as following:1. PR10and PRP were constructed into prokaryotic expressing vector pET-29b(+). The PR10and PRP protein were successfully expressed at37℃,0.5mM IPTG in Eschericha coli and thefull-length of His-PR10protein and His-PRP was17.14kDa and30kDa, respectively. As theexpressed proteins were purified to homogeneity, and the dialytically renatured PR10and PRPproteins with nuclease activity could degradation against G.max leaf total RNA and they could inhibit hyphal growth of P. sojae race1when the proteins concentration reached to25μg and35μg.The bacteriostatic effect enhance with the increasing concentrations of protein.2. GFP fusion expression vector of pCAMBIA1302-PR10and pCAMBIA1302-PRP wereconstructed and transformed into onion epithelial cell by way of Agrobacterium tumefaciensmediation. PR10and PRP genes were found in the cytoplasm with confocal laser microscope.3. A total of six PPT transgenic tobacco resistant plants were selected by PCR amplification，and were inoculated with Phytophthora nicatianae and its resistance improved compared with thatof control.4. PR10and PRP genes were transformed into susceptible soybean Dongnong50viaAgrobacterium mediation using cotyledonary nodes, respectively. A total of115regeneratedsoybean plants were obtained, and14T1plants were positive examined by PCR. Southern blot andReal-time PCR indicated PR10and PRP genes had been transformed into soybean gernome.Resistance of transgenic Dongnong50inoculated with P. sojae race1improved compared withthat of control. |
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