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Analysis of the siRNA-Mediated Gene Silencing Process Targeting Three Homologous Genes Controlling Soybean Seed Oil Quality.

Lu S, Yin X, Spollen W, Zhang N, Xu D, Schoelz J, Bilyeu K, Zhang ZJ - PLoS ONE (2015)

Bottom Line: 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.

View Article: PubMed Central - PubMed

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.

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.

No MeSH data available.


Related in: MedlinePlus

5’RACE on GmFAD3A and GmFAD3C mRNAs in T5 RNAi lines.Arrows indicate the inferred cleavage sites and numbers above represent the fractions of cloned 5’ RACE PCR products terminating at this position. Degradation sites detected with high frequency are highlighted in red, and those present across the three RNAi lines are highlighted with asterisks. (A) Summary of the 5’ RACE analysis performed on the 318-bp region of GmFAD3A mRNA. (B) and (C) Summary of the 5’ RACE analysis performed on the corresponding region of GmFAD3C mRNA.
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pone.0129010.g008: 5’RACE on GmFAD3A and GmFAD3C mRNAs in T5 RNAi lines.Arrows indicate the inferred cleavage sites and numbers above represent the fractions of cloned 5’ RACE PCR products terminating at this position. Degradation sites detected with high frequency are highlighted in red, and those present across the three RNAi lines are highlighted with asterisks. (A) Summary of the 5’ RACE analysis performed on the 318-bp region of GmFAD3A mRNA. (B) and (C) Summary of the 5’ RACE analysis performed on the corresponding region of GmFAD3C mRNA.

Mentions: Fig 8 shows inferred cleavage sites as detected by 5’ RACE, with the fraction of cloned 5’ RACE PCR products terminating at that position. Similar to small RNA distribution patterns, cleavage sites on FAD3A mRNA were not evenly distributed along the 318-bp sequence (Fig 8A). Most of the inferred cleavage sites were located within the last fifty nucleotides in all three lines, indicating that this region is more prone to be cleaved by siRNAs. A total of 4 cleavage sites were conserved among the three RNAi lines, all of which exhibited relatively higher cleavage frequency than other non-conserved positions. The two major cleavage sites detected at 297–298 and 301–302 nucleotide positions together represented more than 50% (13/23) and 40% (10/25) cleavage events in S-24-4D and S-24-13, respectively. In contrast, only one predominant cleavage site at 274–275 nucleotides was identified in S-24-15, accounting for 35% of total sequenced cleavage products. Since 5’ RACE was performed on products with a range of sizes, cleavage sites were also detected in the WT due to natural mRNA degradation. For example, all cleavage events identified in S-24-4D were within the 318 region, whereas 8 out of 28 clones sequenced in WT were located outside of the 318-bp, indicating that cleavage products detected in WT could be a result of mRNA degradation in the transcriptome. Furthermore, the cleavage site at the 274–275 nucleotide position shared by the three RNAi lines and WT might be the position where degradation of the FAD3A transcript is most likely to occur.


Analysis of the siRNA-Mediated Gene Silencing Process Targeting Three Homologous Genes Controlling Soybean Seed Oil Quality.

Lu S, Yin X, Spollen W, Zhang N, Xu D, Schoelz J, Bilyeu K, Zhang ZJ - PLoS ONE (2015)

5’RACE on GmFAD3A and GmFAD3C mRNAs in T5 RNAi lines.Arrows indicate the inferred cleavage sites and numbers above represent the fractions of cloned 5’ RACE PCR products terminating at this position. Degradation sites detected with high frequency are highlighted in red, and those present across the three RNAi lines are highlighted with asterisks. (A) Summary of the 5’ RACE analysis performed on the 318-bp region of GmFAD3A mRNA. (B) and (C) Summary of the 5’ RACE analysis performed on the corresponding region of GmFAD3C mRNA.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4465718&req=5

pone.0129010.g008: 5’RACE on GmFAD3A and GmFAD3C mRNAs in T5 RNAi lines.Arrows indicate the inferred cleavage sites and numbers above represent the fractions of cloned 5’ RACE PCR products terminating at this position. Degradation sites detected with high frequency are highlighted in red, and those present across the three RNAi lines are highlighted with asterisks. (A) Summary of the 5’ RACE analysis performed on the 318-bp region of GmFAD3A mRNA. (B) and (C) Summary of the 5’ RACE analysis performed on the corresponding region of GmFAD3C mRNA.
Mentions: Fig 8 shows inferred cleavage sites as detected by 5’ RACE, with the fraction of cloned 5’ RACE PCR products terminating at that position. Similar to small RNA distribution patterns, cleavage sites on FAD3A mRNA were not evenly distributed along the 318-bp sequence (Fig 8A). Most of the inferred cleavage sites were located within the last fifty nucleotides in all three lines, indicating that this region is more prone to be cleaved by siRNAs. A total of 4 cleavage sites were conserved among the three RNAi lines, all of which exhibited relatively higher cleavage frequency than other non-conserved positions. The two major cleavage sites detected at 297–298 and 301–302 nucleotide positions together represented more than 50% (13/23) and 40% (10/25) cleavage events in S-24-4D and S-24-13, respectively. In contrast, only one predominant cleavage site at 274–275 nucleotides was identified in S-24-15, accounting for 35% of total sequenced cleavage products. Since 5’ RACE was performed on products with a range of sizes, cleavage sites were also detected in the WT due to natural mRNA degradation. For example, all cleavage events identified in S-24-4D were within the 318 region, whereas 8 out of 28 clones sequenced in WT were located outside of the 318-bp, indicating that cleavage products detected in WT could be a result of mRNA degradation in the transcriptome. Furthermore, the cleavage site at the 274–275 nucleotide position shared by the three RNAi lines and WT might be the position where degradation of the FAD3A transcript is most likely to occur.

Bottom Line: 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.

View Article: PubMed Central - PubMed

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.

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.

No MeSH data available.


Related in: MedlinePlus