2010 Annual Report
1a.Objectives (from AD-416)
Identify candidate proteins and genes that are responsible for aphid resistance of Rag2 gene.
1b.Approach (from AD-416)
Proteomics and transcriptomics research on soybean aphid infested and control tissue samples from BC4 near siogenic lines (NILs) with and without the Rag2 gene will be perfomed to identify the proteins and transcriptomes that may express differentially between NILs. Samples will be collected after 0, 2, 4, 8, 24, 48, and 72 hours after infestation with aphids. The proteins and transcriptomes that are unique to the resistant lines will be used as the candidates for further research.
The soybean aphid, a plant sap sucking insect, has become an important soybean pest in the USA and infestation of soybean by this insect can lead to significant yield losses. The Rag2 gene of soybean, providing resistance to soybean aphid biotypes I (IL) and II (OH), was identified by researchers in Ohio. A proteomic analysis is currently being performed on BC4 near isogenic lines (NILs) with the Rag2 gene for resistance or rag2 for susceptibility to the soybean aphid to unravel mechanisms of aphid resistance. Soybeans were grown in an environment controlled greenhouse to the V1 (first trifolitate) stage and infested with 20 adult soybean aphids of biotype II. Leaves were collected in four replicates at 0, 2, 4, 8, 24, 36, 48, 72 and 96 hours after infestation. The biological material was produced at the University of Ohio and then shipped on dry ice to the University of Missouri-Columbia for proteomic analysis. Proteins and RNA from 4 biological replicates of Rag2 and rag2 NILs 48h after aphid infestation were extracted using Trizol method, enabling us to compare transcriptomic and proteomic data coming from identical biological material. Proteomic analysis is being performed for the first time. Proteins (50 µg) were separated by 1D gel electrophoresis and stained by colloidal Coomassie blue. Each line of the gel was divided into 12 bands and digested by Trypsin. Tryptic peptides were analyzed on an Agilent 6520 Quadruplole-Time of Flight-Mass spectrometer using a 43 mm chip. Fourteen hundred and eighty proteins were detected in at least 3 biological replicates of Rag2 or rag2 NILs. Statistical analysis identified 112 proteins differentially regulated between NILs 48h after aphid infestation. The cutoffs of fold change ratio used were 1.8 for the upregulated proteins and 0.55 for the downregulated proteins. The p-value cutoff used was p<0.05. Efforts are continuing to analyze samples from other time points. Proteins and RNA were extracted from other time points including 24h, 36h and 96h after infestation. Trypsin digestion was performed on proteins extracted from infested leaves 24h after aphid infestation. However, corresponding tryptic peptides have not been successfully analyzed by MS/MS due to technical issues on the LC system of the Q-TOF. The ultimate goal is to identify proteins involved in soybean aphid resistance conferred by the Rag2 gene.
Progress on this project is monitored by monthly telephone conversations between the Co-PI and the ADODR and quarterly written progress reports from the Co-PI to the ADODR. Face to face meetings between the Co-PI and the ADODR take place at the soybean breeder's workshop once a year.