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

Author:
Lu, Sha, Yin, Xiaoyan, Spollen, William, Zhang, Ning, Xu, Dong, Schoelz, James, Bilyeu, Kristin, Zhang, Zhanyuan J.
Source:
Plos One 2015 v.10 no.6 pp. e0129010-
ISSN:
1932-6203
Subject:
RNA interference, crops, genetic improvement, high-throughput nucleotide sequencing, models, phenotype, seed oils, sequence homology, small interfering RNA, soybeans, technology, transgenes
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”.