title: Analysis of the siRNA-Mediated Gene Silencing Process Targeting Three Homologous Genes Controlling Soybean Seed Oil Quality.
abstract: In the past decade, RNA silencing has gained significant attention because of its success in genomic scale research and also in the genetic improvement of crop plants. However, little is known about the molecular basis of siRNA processing in association with its target transcript. To reveal this process for improving hpRNA-mediated gene silencing in crop plants, the soybean GmFAD3 gene family was chosen as a test model. We analyzed RNAi mutant soybean lines in which three members of the GmFAD3 gene family were silenced. The silencing levels of FAD3A, FAD3B and FAD3C were correlated with the degrees of sequence homology between the inverted repeat of hpRNA and the 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 while much lower levels of siRNAs were found outside of the target region. siRNAs generated from the 318-bp inverted repeat were found to be diced much more frequently at stem sequences close to the loop and associated with the inferred cleavage sites on the target transcripts, manifesting "hot spots". The top candidate hpRNA-derived siRNA share certain sequence features with mature miRNA. This is the first comprehensive and detailed study revealing the siRNA-mediated gene silencing mechanism in crop plants using gene family GmFAD3 as a test model.
authors.affiliation: Plant Transformation Core Facility, University of Missouri, Columbia, MO, United States of America; Division of Plant Sciences, University of Missouri, Columbia, MO, United States of America.
authors.forename: Sha
authors.initials: S
authors.lastname: Lu
authors.affiliation: Plant Transformation Core Facility, University of Missouri, Columbia, MO, United States of America; Division of Plant Sciences, University of Missouri, Columbia, MO, United States of America.
authors.forename: Xiaoyan
authors.initials: X
authors.lastname: Yin
authors.affiliation: Bioinformatics Core Facility, University of Missouri, Columbia, MO, United States of America.
authors.forename: William
authors.initials: W
authors.lastname: Spollen
authors.affiliation: Department of Computer Sciences and Informatics Institute, University of Missouri, Columbia, MO, United States of America.
authors.forename: Ning
authors.initials: N
authors.lastname: Zhang
authors.affiliation: Department of Computer Sciences and Informatics Institute, University of Missouri, Columbia, MO, United States of America.
authors.forename: Dong
authors.initials: D
authors.lastname: Xu
authors.affiliation: Division of Plant Sciences, University of Missouri, Columbia, MO, United States of America.
authors.forename: James
authors.initials: J
authors.lastname: Schoelz
authors.affiliation: Division of Plant Sciences, University of Missouri, Columbia, MO, United States of America; USDA-ARS, University of Missouri, Columbia, MO, United States of America.
authors.forename: Kristin
authors.initials: K
authors.lastname: Bilyeu
authors.affiliation: Plant Transformation Core Facility, University of Missouri, Columbia, MO, United States of America; Division of Plant Sciences, University of Missouri, Columbia, MO, United States of America.
authors.forename: Zhanyuan J
authors.initials: ZJ
authors.lastname: Zhang
mesh_terms: D019175:DNA Methylation; D044943:Fatty Acid Desaturases / Q000235:genetics* / Q000378:metabolism; D020868:Gene Silencing; D005810:Multigene Family; D010940:Plant Proteins / Q000235:genetics* / Q000378:metabolism; D034741:RNA, Small Interfering / Q000378:metabolism*; D012689:Sequence Homology, Nucleic Acid; D013024:Soybean Oil / Q000235:genetics / Q000592:standards*; D013025:Glycine max / Q000737:chemistry / Q000201:enzymology / Q000235:genetics*
chemical_list: D010940:Plant Proteins; D034741:RNA, Small Interfering; D013024:Soybean Oil; D044943:Fatty Acid Desaturases