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Title: Analysis of the siRNA-mediated gene silencing process targeting three homologous genes controlling soybean seed oil quality

Author
item LU, SHA - University Of Missouri
item YIN, XIAOYAN - University Of Missouri
item SPOLLEN, WILLIAM - University Of Missouri
item ZHANG, NING - University Of Missouri
item XU, DONG - University Of Missouri
item SCHOELZ, JAMES - University Of Missouri
item Bilyeu, Kristin
item ZHANG, ZHANYUAN - University Of Missouri

Submitted to: PLOS ONE
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 5/4/2015
Publication Date: 6/10/2015
Publication URL: http://handle.nal.usda.gov/10113/61081
Citation: Lu, S., Yin, X., Spollen, W., Zhang, N., Xu, D., Schoelz, J., Bilyeu, K.D., Zhang, Z.J. 2015. Analysis of the siRNA-mediated gene silencing process targeting three homologous genes controlling soybean seed oil quality. PLoS One. 10(6). Available: http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0129010.

Interpretive Summary: Identifying and understanding the mechanisms to alter the experession of targeted genes in crop species is critical to develop new traits to meet the demand for more efficient production of nutritious food. In this work, the technology of RNA silencing via an introduced transgene that is made from an inverted repeat sequence of a target soybean gene separated by an intron as a spacer (hairpin structure) was studied. To better understand the mechanism of down-regulation of target genes with closely related sequences, a series of experiments were performed for the soybean low linolenic acid seed oil trait that is known to be controlled by three closely related genes. The results demonstrated different levels of gene down-regulation based on the relatedness of the target genes to the introduced transgene, the methylation status of the transgene, and the sequence preference of small interfering RNAs produced. The impact of this research is a more thorough understanding of the process of RNA silencing in a crop plant that can be applied to future research with different crop traits.

Technical Abstract: Since the discovery of RNA silencing in the nineties, the implication and potential application of this new technology have been recognized. In the past decades, RNA silencing has gained significant attention because its success in genomic scale research and also in the genetic improvement of crop plants. To improve hpRNA-mediated gene silencing in soybean, the GmFAD3 gene family was chosen as a test model. In this study, all three family members of GmFAD3 were silenced and the silencing phenotype was stably inherited. Silencing levels of FAD3A, FAD3B and FAD3C correlate to the degrees of sequence homology between the inverted repeats (IR) of hpRNA and GmFAD3 transcripts in the RNAi lines. Strikingly, transgenes in two of the three RNAi lines were heavily methylated, leading to a dramatic reduction of hpRNA-derived siRNAs. Small RNAs corresponding to the loop portion of the hairpin transcript were detected, implicating possible transitive self-silencing of the hairpin transgene. In contrast, much less RNAs were found outside of the target region, suggesting that transitivity along endogenous transcripts is prohibited by some inherent protective feature. Small RNAs encoding part of the transgene promoter as well as the bar gene coding sequences were also detected by deep sequencing. siRNAs generated from the 318-bp IR were further characterized and found to be associated with the inferred cleavage sites on the target transcripts, manifesting “hot spots”.