2011 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.
Proteomic and transcriptomic analyses are currently 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. Proteins and Ribo Nucleic Acid were extracted using Trizol from the 9 time points (4 biological replicates per time point). Trizol is an approach enabling to extract RNA and proteins from the same tissue. Therefore, identical biological material will be used to compare transcriptomic and proteomic data. 1D-PAGE-LC-Q-TOF approach was initiated for proteome analysis. Proteins from the first biological replicate of Rag2 and rag2 lines 0, 2, 8, 24, 36, 48, 72 and 96 hours after aphid infestation were separated by 1D gel electrophoresis, cut into 12 bands and digested with trypsin. Tryptic peptides were analyzed on an LTQ-Orbitrap.
A transcriptomic approach (RNA-seq) was initiated using Illumina® technology. A high throughput RNA-seq approach was used to examine mRNA expression in Rag2 and rag2 soybean leaves 0, 4h, 8h, 24h and 48h (2 biological replicates per time point) after aphid infestation.
This research relates to objective 2A (Characterize and map aphid resistance genes in three soybean plant introductions) of the parent project.
The progress is monitored by monthly telephone conversation between the Co-PI and the authorized departmentalal officer's designated representative (ADODR) and quarterly written progress reports from Co-PI to ADODR. Also, face to face meetings of the Co-PI and ADODR at soybean breeder’s workshop once a year.