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Strength, Stability, and cis-Motifs of In silico Identified Phloem-Specific Promoters in Brassica juncea (L.).

Koramutla MK, Bhatt D, Negi M, Venkatachalam P, Jain PK, Bhattacharya R - Front Plant Sci (2016)

Bottom Line: In RT-qPCR based gene-expression study promoter of Glutamine synthetase 3A (GS3A) showed multifold higher activity compared to others, across the different growth stages of B. juncea plants.Nevertheless, the consensus in output empirically suggested consistency in promoter-activity of the six B. juncea phloem- specific promoters including GS3A.The study identified suitable endogenous promoters for high level and consistent gene-expression in B. juncea phloem exudate.

View Article: PubMed Central - PubMed

Affiliation: National Research Centre on Plant Biotechnology, Indian Agricultural Research Institute Campus New Delhi, India.

ABSTRACT
Aphids, a hemipteran group of insects pose a serious threat to many of the major crop species including Brassica oilseeds. Transgenic strategies for developing aphid-resistant plant types necessitate phloem-bound expression of the insecticidal genes. A few known phloem-specific promoters, in spite of tissue-specific activity fail to confer high level gene-expression. Here, we identified seven orthologues of phloem-specific promoters in B. juncea (Indian mustard), and experimentally validated their strength of expression in phloem exudates. Significant cis-motifs, globally occurring in phloem-specific promoters showed variable distribution frequencies in these putative phloem-specific promoters of B. juncea. In RT-qPCR based gene-expression study promoter of Glutamine synthetase 3A (GS3A) showed multifold higher activity compared to others, across the different growth stages of B. juncea plants. A statistical method employing four softwares was devised for rapidly analysing stability of the promoter-activities across the plant developmental stages. Different statistical softwares ranked these B. juncea promoters differently in terms of their stability in promoter-activity. Nevertheless, the consensus in output empirically suggested consistency in promoter-activity of the six B. juncea phloem- specific promoters including GS3A. The study identified suitable endogenous promoters for high level and consistent gene-expression in B. juncea phloem exudate. The study also demonstrated a rapid method of assessing species-specific strength and stability in expression of the endogenous promoters.

No MeSH data available.


Significant cis-motifs identified by RSAT oligo-analyzer and info-gibbs. The motifs were identified by RSAT oligo-analyzer (A) and info-gibbs (B) motif discovery tool. Frequency distribution of significant motifs were searched by using FIMO program of MEME with p < 0.0001. Two motifs showing lowest expectation (E)-values and high log likelihood ratio (Avg.llr) in each case and their frequencies in the phloem-specific promoters have been shown. The X- and Y-axis show the position of nucleotides and the bits score, respectively.
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Figure 3: Significant cis-motifs identified by RSAT oligo-analyzer and info-gibbs. The motifs were identified by RSAT oligo-analyzer (A) and info-gibbs (B) motif discovery tool. Frequency distribution of significant motifs were searched by using FIMO program of MEME with p < 0.0001. Two motifs showing lowest expectation (E)-values and high log likelihood ratio (Avg.llr) in each case and their frequencies in the phloem-specific promoters have been shown. The X- and Y-axis show the position of nucleotides and the bits score, respectively.

Mentions: DNA-motifs globally associated with phloem-specific promoters were not known. Therefore, discovery of such motifs was mandatory for understanding the architecture of the putative phloem-specific promoters identified in B. juncea. For that, comprehensive set of 39 promoter sequences from diverse origin (described in Table S3) were analyzed, and the over-represented motifs in them were discovered through string based (oligo-analysis of RSAT) and position weight matrix-based (info-gibbs of RSAT, AlignACE, and MEME) motif discovery programmes (Van Helden et al., 1998; Hughes et al., 2000; Defrance and van Helden, 2009). Oligo-analysis revealed the commonly occurring over-represented motifs in all the promoter sequences. Among the identified motifs, the two with lowest expectation (E)-values were scored as significant and their frequency of occurrence per promoter has been shown in Figure 3A. The two motifs showed similarity with known plant cis-regulatory elements which are responsive to plant hormones, such as auxin and salycilic acid. Independent analysis based on info-gibbs identified a CT-rich signature motif (Figure 3B) specifically present in the promoters of phloem-specific transcripts. Info-gibbs predicted another AG-rich motif showing similarity to CTRMCAMV 35S motif which was found in -60 nt downstream of transcription start site of the CaMV 35S RNA and known to enhance gene expression driven by the CaMV 35S (Pauli et al., 2004). Analysis based on MEME predicted three significant motifs with low (E)-values that have been shown in Figure 4. Among the three, two motifs were grouped in CT/GA-rich repeat motifs specific to phloem-specific promoters. The other one showed similarity with 314MOTIFZMSBE1 which is a positive cis-element located between –314 and –295 region of maize Sbe1 promoter and required for high level as well as sugar responsive expression. The AlignACE programme was used to identify two A/T rich degenerate motifs in phloem-specific promoter sequences which are widespread in eukaryotic genomes (Figure S3). Use of multiple motif discovery programmes led to the identification of nine significant signature cis-elements specific to the phloem-specific promoters across the vascular plant species.


Strength, Stability, and cis-Motifs of In silico Identified Phloem-Specific Promoters in Brassica juncea (L.).

Koramutla MK, Bhatt D, Negi M, Venkatachalam P, Jain PK, Bhattacharya R - Front Plant Sci (2016)

Significant cis-motifs identified by RSAT oligo-analyzer and info-gibbs. The motifs were identified by RSAT oligo-analyzer (A) and info-gibbs (B) motif discovery tool. Frequency distribution of significant motifs were searched by using FIMO program of MEME with p < 0.0001. Two motifs showing lowest expectation (E)-values and high log likelihood ratio (Avg.llr) in each case and their frequencies in the phloem-specific promoters have been shown. The X- and Y-axis show the position of nucleotides and the bits score, respectively.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4834444&req=5

Figure 3: Significant cis-motifs identified by RSAT oligo-analyzer and info-gibbs. The motifs were identified by RSAT oligo-analyzer (A) and info-gibbs (B) motif discovery tool. Frequency distribution of significant motifs were searched by using FIMO program of MEME with p < 0.0001. Two motifs showing lowest expectation (E)-values and high log likelihood ratio (Avg.llr) in each case and their frequencies in the phloem-specific promoters have been shown. The X- and Y-axis show the position of nucleotides and the bits score, respectively.
Mentions: DNA-motifs globally associated with phloem-specific promoters were not known. Therefore, discovery of such motifs was mandatory for understanding the architecture of the putative phloem-specific promoters identified in B. juncea. For that, comprehensive set of 39 promoter sequences from diverse origin (described in Table S3) were analyzed, and the over-represented motifs in them were discovered through string based (oligo-analysis of RSAT) and position weight matrix-based (info-gibbs of RSAT, AlignACE, and MEME) motif discovery programmes (Van Helden et al., 1998; Hughes et al., 2000; Defrance and van Helden, 2009). Oligo-analysis revealed the commonly occurring over-represented motifs in all the promoter sequences. Among the identified motifs, the two with lowest expectation (E)-values were scored as significant and their frequency of occurrence per promoter has been shown in Figure 3A. The two motifs showed similarity with known plant cis-regulatory elements which are responsive to plant hormones, such as auxin and salycilic acid. Independent analysis based on info-gibbs identified a CT-rich signature motif (Figure 3B) specifically present in the promoters of phloem-specific transcripts. Info-gibbs predicted another AG-rich motif showing similarity to CTRMCAMV 35S motif which was found in -60 nt downstream of transcription start site of the CaMV 35S RNA and known to enhance gene expression driven by the CaMV 35S (Pauli et al., 2004). Analysis based on MEME predicted three significant motifs with low (E)-values that have been shown in Figure 4. Among the three, two motifs were grouped in CT/GA-rich repeat motifs specific to phloem-specific promoters. The other one showed similarity with 314MOTIFZMSBE1 which is a positive cis-element located between –314 and –295 region of maize Sbe1 promoter and required for high level as well as sugar responsive expression. The AlignACE programme was used to identify two A/T rich degenerate motifs in phloem-specific promoter sequences which are widespread in eukaryotic genomes (Figure S3). Use of multiple motif discovery programmes led to the identification of nine significant signature cis-elements specific to the phloem-specific promoters across the vascular plant species.

Bottom Line: In RT-qPCR based gene-expression study promoter of Glutamine synthetase 3A (GS3A) showed multifold higher activity compared to others, across the different growth stages of B. juncea plants.Nevertheless, the consensus in output empirically suggested consistency in promoter-activity of the six B. juncea phloem- specific promoters including GS3A.The study identified suitable endogenous promoters for high level and consistent gene-expression in B. juncea phloem exudate.

View Article: PubMed Central - PubMed

Affiliation: National Research Centre on Plant Biotechnology, Indian Agricultural Research Institute Campus New Delhi, India.

ABSTRACT
Aphids, a hemipteran group of insects pose a serious threat to many of the major crop species including Brassica oilseeds. Transgenic strategies for developing aphid-resistant plant types necessitate phloem-bound expression of the insecticidal genes. A few known phloem-specific promoters, in spite of tissue-specific activity fail to confer high level gene-expression. Here, we identified seven orthologues of phloem-specific promoters in B. juncea (Indian mustard), and experimentally validated their strength of expression in phloem exudates. Significant cis-motifs, globally occurring in phloem-specific promoters showed variable distribution frequencies in these putative phloem-specific promoters of B. juncea. In RT-qPCR based gene-expression study promoter of Glutamine synthetase 3A (GS3A) showed multifold higher activity compared to others, across the different growth stages of B. juncea plants. A statistical method employing four softwares was devised for rapidly analysing stability of the promoter-activities across the plant developmental stages. Different statistical softwares ranked these B. juncea promoters differently in terms of their stability in promoter-activity. Nevertheless, the consensus in output empirically suggested consistency in promoter-activity of the six B. juncea phloem- specific promoters including GS3A. The study identified suitable endogenous promoters for high level and consistent gene-expression in B. juncea phloem exudate. The study also demonstrated a rapid method of assessing species-specific strength and stability in expression of the endogenous promoters.

No MeSH data available.