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Identified of a novel cis -element regulating the alternative splicing of LcDREB2

View Article: PubMed Central - PubMed

ABSTRACT

Alternative splicing (AS) is an important gene regulation mechanism in plants. Despite the widespread use of AS in plant gene expression regulation, the identification of the cis-elements involved in the AS mechanism is rarely reported in plants. To explore the regulation mechanism of the AS of LcDREB2, a DREB2 ortholog from Sheepgrass (Leymus chinensis), the genomic sequences of LcDREB2 and its homologs in Poaceae were aligned, and six mutations were introduced in the conserved sequence of LcDREB2. By analyzing the distinct transcript patterns of the LcDREB2 mutants in transgenic Oryza sativa, a novel cis-element that affected the AS of LcDREB2 was identified as Exonic Splicing Enhancer 1 (ESE1). In addition, five serine-arginine rich (SR) proteins were confirmed to interact with ESE1 by electrophoretic mobility shift assay (EMSA). To further explore the expression regulation mechanism of the DREB subfamily, phylogenetic analysis of DREB2 paralogous genes was performed. The results strongly supported the hypothesis that AS is conserved in Poaceae plants and that it is an evolutionary strategy for the regulation of the functional expression of genes. The findings and methods of our study will promote a substantial step forward in understanding of the plant AS regulation mechanism.

No MeSH data available.


Semi-quantitative and real-time PCR results of LcDREB2 mutants in transgenic O. sativa.(a) The transcript patterns of the wild-type exon 3 and all 6 mutants in transgenic O. sativa were analyzed by reverse transcription PCR and amplification. The M1, M2 and M3 mutants altered the transcript pattern compared with the wild type, resulting in decreased expression of the major transcript LcDREB2b and increased expression of LcDREB2a. The M4, M5 and M6 mutants had no effect on the AS pattern of LcDREB2. (b) The quantity of the three transcripts in transgenic O. sativa were detected by real-time PCR. The relative quantity of three transcripts in the wild type and six mutants were compared. The purple, blue and yellow represent LcDREB2a, LcDREB2b and LcDREB2c, respectively.
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f3: Semi-quantitative and real-time PCR results of LcDREB2 mutants in transgenic O. sativa.(a) The transcript patterns of the wild-type exon 3 and all 6 mutants in transgenic O. sativa were analyzed by reverse transcription PCR and amplification. The M1, M2 and M3 mutants altered the transcript pattern compared with the wild type, resulting in decreased expression of the major transcript LcDREB2b and increased expression of LcDREB2a. The M4, M5 and M6 mutants had no effect on the AS pattern of LcDREB2. (b) The quantity of the three transcripts in transgenic O. sativa were detected by real-time PCR. The relative quantity of three transcripts in the wild type and six mutants were compared. The purple, blue and yellow represent LcDREB2a, LcDREB2b and LcDREB2c, respectively.

Mentions: Semi-quantitative PCR was used to characterize the effects of these six mutants. Compared with the wild-type, the AS pattern of LcDREB2 with the mutations was altered. The M1, M2 and M3 mutations led to a change in the ratio of the three transcripts: LcDREB2a became the major product among the three transcripts, while the expression of LcDREB2b was sharply decreased (Fig. 3a). The M4, M5 and M6 mutations had no obvious changes (Fig. 3a). To further characterize the results and quantify the transcripts of the six different mutants, real-time quantitative PCR was performed. The results in Fig. 3b showed that the M1 mutant only produced LcDREB2a, while the other two transcripts were barely detected. The M2 and M3 mutants produced all three transcripts, but LcDREB2a was increased substantially, while the primary major transcript of LcDREB2b was decreased. Therefore, according to these mutation results, a novel cis-element was confirmed in exon 3 of LcDREB2. According to the effect of this cis-element, it is an exonic splicing enhancer (ESE) and was named as ESE1.


Identified of a novel cis -element regulating the alternative splicing of LcDREB2
Semi-quantitative and real-time PCR results of LcDREB2 mutants in transgenic O. sativa.(a) The transcript patterns of the wild-type exon 3 and all 6 mutants in transgenic O. sativa were analyzed by reverse transcription PCR and amplification. The M1, M2 and M3 mutants altered the transcript pattern compared with the wild type, resulting in decreased expression of the major transcript LcDREB2b and increased expression of LcDREB2a. The M4, M5 and M6 mutants had no effect on the AS pattern of LcDREB2. (b) The quantity of the three transcripts in transgenic O. sativa were detected by real-time PCR. The relative quantity of three transcripts in the wild type and six mutants were compared. The purple, blue and yellow represent LcDREB2a, LcDREB2b and LcDREB2c, respectively.
© Copyright Policy - open-access
Related In: Results  -  Collection

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f3: Semi-quantitative and real-time PCR results of LcDREB2 mutants in transgenic O. sativa.(a) The transcript patterns of the wild-type exon 3 and all 6 mutants in transgenic O. sativa were analyzed by reverse transcription PCR and amplification. The M1, M2 and M3 mutants altered the transcript pattern compared with the wild type, resulting in decreased expression of the major transcript LcDREB2b and increased expression of LcDREB2a. The M4, M5 and M6 mutants had no effect on the AS pattern of LcDREB2. (b) The quantity of the three transcripts in transgenic O. sativa were detected by real-time PCR. The relative quantity of three transcripts in the wild type and six mutants were compared. The purple, blue and yellow represent LcDREB2a, LcDREB2b and LcDREB2c, respectively.
Mentions: Semi-quantitative PCR was used to characterize the effects of these six mutants. Compared with the wild-type, the AS pattern of LcDREB2 with the mutations was altered. The M1, M2 and M3 mutations led to a change in the ratio of the three transcripts: LcDREB2a became the major product among the three transcripts, while the expression of LcDREB2b was sharply decreased (Fig. 3a). The M4, M5 and M6 mutations had no obvious changes (Fig. 3a). To further characterize the results and quantify the transcripts of the six different mutants, real-time quantitative PCR was performed. The results in Fig. 3b showed that the M1 mutant only produced LcDREB2a, while the other two transcripts were barely detected. The M2 and M3 mutants produced all three transcripts, but LcDREB2a was increased substantially, while the primary major transcript of LcDREB2b was decreased. Therefore, according to these mutation results, a novel cis-element was confirmed in exon 3 of LcDREB2. According to the effect of this cis-element, it is an exonic splicing enhancer (ESE) and was named as ESE1.

View Article: PubMed Central - PubMed

ABSTRACT

Alternative splicing (AS) is an important gene regulation mechanism in plants. Despite the widespread use of AS in plant gene expression regulation, the identification of the cis-elements involved in the AS mechanism is rarely reported in plants. To explore the regulation mechanism of the AS of LcDREB2, a DREB2 ortholog from Sheepgrass (Leymus chinensis), the genomic sequences of LcDREB2 and its homologs in Poaceae were aligned, and six mutations were introduced in the conserved sequence of LcDREB2. By analyzing the distinct transcript patterns of the LcDREB2 mutants in transgenic Oryza sativa, a novel cis-element that affected the AS of LcDREB2 was identified as Exonic Splicing Enhancer 1 (ESE1). In addition, five serine-arginine rich (SR) proteins were confirmed to interact with ESE1 by electrophoretic mobility shift assay (EMSA). To further explore the expression regulation mechanism of the DREB subfamily, phylogenetic analysis of DREB2 paralogous genes was performed. The results strongly supported the hypothesis that AS is conserved in Poaceae plants and that it is an evolutionary strategy for the regulation of the functional expression of genes. The findings and methods of our study will promote a substantial step forward in understanding of the plant AS regulation mechanism.

No MeSH data available.