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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.


Conserved sequences and mutations of LcDREB2 exon 3 and RNA probes designed based on the exon 3 sequence.(a) The genomic sequences of LcDREB2, HvDRF1, PjDREB2, TdDRF1, TuDREB2, AtDREB2, AsDREB2, BdDREB2, SbDREB2, OsDREB2B and ZmDREB2 were aligned. The red pillars represent highly conserved sequences, and the green pillars represent sequences that are not conserved. The sequences on exon 3 were highly conserved. (b) Mutations were carried out on the conserved sequences of exon 3. All the mutations and the wild-type sequence were aligned by DNAMAN 5.2. The location and sequences are shown in the alignment pattern. (c) The RNA probes E3-1 and E3-2 were located on exon 3 of LcDREB2. The purple-highlighted sequences reflect the RNA probe locations, and the sequences are listed below with RNA bases.
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f2: Conserved sequences and mutations of LcDREB2 exon 3 and RNA probes designed based on the exon 3 sequence.(a) The genomic sequences of LcDREB2, HvDRF1, PjDREB2, TdDRF1, TuDREB2, AtDREB2, AsDREB2, BdDREB2, SbDREB2, OsDREB2B and ZmDREB2 were aligned. The red pillars represent highly conserved sequences, and the green pillars represent sequences that are not conserved. The sequences on exon 3 were highly conserved. (b) Mutations were carried out on the conserved sequences of exon 3. All the mutations and the wild-type sequence were aligned by DNAMAN 5.2. The location and sequences are shown in the alignment pattern. (c) The RNA probes E3-1 and E3-2 were located on exon 3 of LcDREB2. The purple-highlighted sequences reflect the RNA probe locations, and the sequences are listed below with RNA bases.

Mentions: To isolate the site of cis-elements that are critical for the AS of LcDREB2, we aligned the genomic sequences of LcDREB2 and its orthologous genes from 10 other gramineous plants (see Supplementary Fig. S1). Because the second and third exons are different between the three transcripts of LcDREB2, and a highly conserved motif on exon 3 was screened through sequence alignment (Fig. 2a), six mutations (from M1 to M6) were designed and introduced into the conserved sequence of exon 3 (Fig. 2b). Nearly the entire sequence of exon 3 was mutated in the M1 mutation. The 5′ and 3′ ends of exon 3 were mutated in the M2 and M3 mutations, respectively. The other three mutations were deletions that were evenly distributed in exon 3 (Fig. 2b). Two RNA probes, E3-1 and E3-2, were designed on the alignment of the exon 3 sequence. The location and corresponding sequences of these probes on exon 3 were shown in Fig. 2c.


Identified of a novel cis -element regulating the alternative splicing of LcDREB2
Conserved sequences and mutations of LcDREB2 exon 3 and RNA probes designed based on the exon 3 sequence.(a) The genomic sequences of LcDREB2, HvDRF1, PjDREB2, TdDRF1, TuDREB2, AtDREB2, AsDREB2, BdDREB2, SbDREB2, OsDREB2B and ZmDREB2 were aligned. The red pillars represent highly conserved sequences, and the green pillars represent sequences that are not conserved. The sequences on exon 3 were highly conserved. (b) Mutations were carried out on the conserved sequences of exon 3. All the mutations and the wild-type sequence were aligned by DNAMAN 5.2. The location and sequences are shown in the alignment pattern. (c) The RNA probes E3-1 and E3-2 were located on exon 3 of LcDREB2. The purple-highlighted sequences reflect the RNA probe locations, and the sequences are listed below with RNA bases.
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Related In: Results  -  Collection

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f2: Conserved sequences and mutations of LcDREB2 exon 3 and RNA probes designed based on the exon 3 sequence.(a) The genomic sequences of LcDREB2, HvDRF1, PjDREB2, TdDRF1, TuDREB2, AtDREB2, AsDREB2, BdDREB2, SbDREB2, OsDREB2B and ZmDREB2 were aligned. The red pillars represent highly conserved sequences, and the green pillars represent sequences that are not conserved. The sequences on exon 3 were highly conserved. (b) Mutations were carried out on the conserved sequences of exon 3. All the mutations and the wild-type sequence were aligned by DNAMAN 5.2. The location and sequences are shown in the alignment pattern. (c) The RNA probes E3-1 and E3-2 were located on exon 3 of LcDREB2. The purple-highlighted sequences reflect the RNA probe locations, and the sequences are listed below with RNA bases.
Mentions: To isolate the site of cis-elements that are critical for the AS of LcDREB2, we aligned the genomic sequences of LcDREB2 and its orthologous genes from 10 other gramineous plants (see Supplementary Fig. S1). Because the second and third exons are different between the three transcripts of LcDREB2, and a highly conserved motif on exon 3 was screened through sequence alignment (Fig. 2a), six mutations (from M1 to M6) were designed and introduced into the conserved sequence of exon 3 (Fig. 2b). Nearly the entire sequence of exon 3 was mutated in the M1 mutation. The 5′ and 3′ ends of exon 3 were mutated in the M2 and M3 mutations, respectively. The other three mutations were deletions that were evenly distributed in exon 3 (Fig. 2b). Two RNA probes, E3-1 and E3-2, were designed on the alignment of the exon 3 sequence. The location and corresponding sequences of these probes on exon 3 were shown in Fig. 2c.

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.