Differential Proteomic Analysis Of Soybean Leaves Inoculated With Soybean Mosaic Virus

Soybean [Glycine max (L.) Merr.] is one of the most important sources of vegetable protein and edible oil world-widely. Soybean mosaic virus (SMV) is one of the most prevalent and important plant viral pathogens and pathogenic on some soybean crops or cultivars worldwide. Infection by SMV may result in severe yield losses and strain quality reduction. Due to outbreaks of SMV disease and its destructivity, many scholars have great interest in some fields that broadly involve the classification, distribution and prevalence of SMV strains, viral infection and host symptoms, structure and functions of viral genome, physiological and biochemical resistance of hosts, resistance inheritance and resistant cultivar breeding, and so on. However, little is known about the molecular basis of soybean defense mechanism against this pathogen. But isolation of SMV resistance gene and research of the resistance mechanism are greatly helpful to crop resistant breeding.1> Differential proteomic analysis of soybean leaves inoculated with soybean mosaic virus.To evaluate the adverse effects of SMV inoculation quantitatively, we performed the relative electrolyte leakage assay. The results show that the control and inoculated with vaccination of susceptible isolate of soybean leaves is very similar to each other. Only at 48h, when inoculated leaves with susceptible isolates did not return to the non-inoculated values. But control and inoculated leaves with resistant isolate have returned to the initial values. Overall inoculated leaves with resistant isolate show more obvious difference, and at 4h and 24h show the most obvious difference especially. This may be due to induction of 4h and 24h time point of the two soybean mosaic disease of soybean in response to the strong reaction to the stress, the two time points of disease-resistant soybean response may become more active.We assay the number of differentially expressed proteins in different timepoints (2h,4h,8h,12h,24h,48h) between SMV infection and control (infected with phosphate buffer). And the 3-10NL 17 cm IPG strips with silver staining method consistently yielded highly resolved 2-DE gels were used for further study. For each sample, at least triplicate gels were performed. Typically more than 1,200-1,400 protein spots were reproducibly detected by PDQuest 7.3 software on each silver-stained gels.All of the 25 spots with an increasing or decreasing trend after inoculation were chosen for identification. Nineteen sports were identified. The proteins identified were classified into nine categories similar to Bevan (Bevan et al.,1998) and Holmes-Davis (Holmes-Davis et al.,2005). And these proteins are proposed to be involved in protein degradation, defense signal transfer, reactive oxygen, cell wall reinforcement, energy and metabolism regulation.Quantitative RT-PCR analysis of RNA expression was performed as a paralleled experiment in contrast with 2-DE. We analyzed mRNA expression of all of the fifteen genes except two hypothetical proteins, and get thirteen results of them. Our present result suggests that the correlation between mRNA and protein level was very weak. Only calreticulin and NADPH-specific isocitrate dehydrogenase show the same regulative modal both in protein and mRNA level. We performed another quantitative RT-PCR analysis all of them in different timepoints after Inoculated with JN17, and they show different change trend.2、GmEN1 and GmEN2 were single-strand-specific nuclease gene.We performed tertiary protein structure prediction^ subcellular localization、quantitative RT-PCR、 transgenic analysis, in order to preliminary assay the relationship between GmEN1、 GmEN2 and SMV resistance for soybean.We predicted the tertiary protein structure of GmEN1 和 GmEN2 protein online (www.swissmodel.expasy.org). Both of them predicted basis on the model of single-strand-specific nuclease gene P1. There are only two different places in the tertiary protein structure of GmENl and GmEN2.With transient expression system in onion epidermal cell, we found GmENl was localized into nucleus, and GmEN2 was localized into plasma membrane.In order to assay the relationship between the expression of GmEN1、GmEN2 and SMV resistance for soybean at the mRNA level, we performed quantitative RT-PCR analysis of them in different timepoints after inoculated with JN17. GmEN1 show two peaks occured at 4h and 24h post inoculation with SMV, when show a dramatic sharp increase in Oh-4h and 8h-24h. But the responsion to SMV for GmEN2 was more slowly. There was no change before 4h after inoculated with SMV, but decreased in 8h-48h.To further address function of GmENl and GmEN2, full-length GmENl and GmEN2 sequence was cloned into plant binary vector pMDC83, creating pMDC83- GmENl and pMDC83-GmEN2 used for tobacco transformation. By agrobacterium mediated mediated transformation, pMDC83- GmEN1 and pMDC83- GmEN2 transgenic tobacco plant were generated, and validated them by PCR and qPCR.The 5mM salicylic acid (SA) stress test show that transgenic tobacco grown significantly better than WT, as evidenced WT seedlings 15 days later in processing the majority of yellow to stop growing. But transgenic tobacco is not affected in growing, plant size and color. Maybe transgenic tobacco has higher tolerance to salicylic acid than WT.