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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

318-bp IR-derived siRNAs targeting FAD3B and FAD3C.(A) and (B) Small RNAs generated from the 318-bp IR matching the corresponding FAD3B and FAD3C target regions were plotted versus the average of their normalized abundance from three replications, respectively. For visual clarity, Y-axis of each diagram is adjusted according to the small RNA abundance.
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pone.0129010.g006: 318-bp IR-derived siRNAs targeting FAD3B and FAD3C.(A) and (B) Small RNAs generated from the 318-bp IR matching the corresponding FAD3B and FAD3C target regions were plotted versus the average of their normalized abundance from three replications, respectively. For visual clarity, Y-axis of each diagram is adjusted according to the small RNA abundance.

Mentions: To further interrogate the association between hpRNA-produced siRNAs and target mRNA silencing efficacy, siRNAs from the 318-bp IR of FAD3A were mapped to the same region of the other two FAD3 genes, respectively (Fig 6). As shown in Fig 6, the number of distinct siRNAs mapped to FAD3B 318-bp region was greatly reduced compared to that of FAD3A (Fig 5A), while only siRNAs around 175 nucleotides share 100% homology with the same region of FAD3C. Particularly, the total number of distinct antisense siRNAs, which are triggers of target gene silencing, decreased from 1371 in FAD3A to 49 in FAD3C (Fig 6, Table B in S1 File). Moreover, the total antisense siRNA abundance also fell from 7441.89 to 34.73 CPM, 482.92 to 2.37 CPM, 340.53 to 1.93 CPM in S-24-4D, S-24-13, S-24-15, respectively (Fig 6, Table B in S1 File). As shown previously, the 318-bp IR used to generate FAD3 siRNAs is 100% identical with GmFAD3A but only shares 96.5% and 84.3% sequence homology with GmFAD3B and GmFAD3C, respectively (Fig 1). Therefore, siRNAs generated from the 318-bp IR region contained a considerable number of mismatches especially with FAD3C, which could abort their function through the failure of target binding or transcript cleavage. As a result, much less noticeable changes in mRNA level was achieved for FAD3C than FAD3A and FAD3B (Fig 1A–1C). However, the silencing efficacy of FAD3B did not seem to be affected by the reduced amount of functional siRNAs, probably because it still shares relatively high identity with FAD3A, and the amount of functional siRNAs was sufficient to induce an efficient silencing.


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)

318-bp IR-derived siRNAs targeting FAD3B and FAD3C.(A) and (B) Small RNAs generated from the 318-bp IR matching the corresponding FAD3B and FAD3C target regions were plotted versus the average of their normalized abundance from three replications, respectively. For visual clarity, Y-axis of each diagram is adjusted according to the small RNA abundance.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0129010.g006: 318-bp IR-derived siRNAs targeting FAD3B and FAD3C.(A) and (B) Small RNAs generated from the 318-bp IR matching the corresponding FAD3B and FAD3C target regions were plotted versus the average of their normalized abundance from three replications, respectively. For visual clarity, Y-axis of each diagram is adjusted according to the small RNA abundance.
Mentions: To further interrogate the association between hpRNA-produced siRNAs and target mRNA silencing efficacy, siRNAs from the 318-bp IR of FAD3A were mapped to the same region of the other two FAD3 genes, respectively (Fig 6). As shown in Fig 6, the number of distinct siRNAs mapped to FAD3B 318-bp region was greatly reduced compared to that of FAD3A (Fig 5A), while only siRNAs around 175 nucleotides share 100% homology with the same region of FAD3C. Particularly, the total number of distinct antisense siRNAs, which are triggers of target gene silencing, decreased from 1371 in FAD3A to 49 in FAD3C (Fig 6, Table B in S1 File). Moreover, the total antisense siRNA abundance also fell from 7441.89 to 34.73 CPM, 482.92 to 2.37 CPM, 340.53 to 1.93 CPM in S-24-4D, S-24-13, S-24-15, respectively (Fig 6, Table B in S1 File). As shown previously, the 318-bp IR used to generate FAD3 siRNAs is 100% identical with GmFAD3A but only shares 96.5% and 84.3% sequence homology with GmFAD3B and GmFAD3C, respectively (Fig 1). Therefore, siRNAs generated from the 318-bp IR region contained a considerable number of mismatches especially with FAD3C, which could abort their function through the failure of target binding or transcript cleavage. As a result, much less noticeable changes in mRNA level was achieved for FAD3C than FAD3A and FAD3B (Fig 1A–1C). However, the silencing efficacy of FAD3B did not seem to be affected by the reduced amount of functional siRNAs, probably because it still shares relatively high identity with FAD3A, and the amount of functional siRNAs was sufficient to induce an efficient silencing.

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