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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.


RT-PCR analysis for the cognate-transcripts of the phloem-specific promoters. Total RNA was isolated from phloem exudates collected from 45 days old B. juncea leaves and assayed for the cognate-transcripts of the in silico identified B. juncea phloem-specific promoters by RT-PCR and RT-qPCR. (A) RT-PCR amplification of RbcS, Lhca2 and UBC9 in phloem exudates (P), and leaf tissues (L) along with non-template (NT) control. (B) RT-PCR amplification of the cognate-transcripts in phloem exudates. (C) RT-qPCR based analysis of the cognate-transcript levels in phloem exudates.
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Figure 2: RT-PCR analysis for the cognate-transcripts of the phloem-specific promoters. Total RNA was isolated from phloem exudates collected from 45 days old B. juncea leaves and assayed for the cognate-transcripts of the in silico identified B. juncea phloem-specific promoters by RT-PCR and RT-qPCR. (A) RT-PCR amplification of RbcS, Lhca2 and UBC9 in phloem exudates (P), and leaf tissues (L) along with non-template (NT) control. (B) RT-PCR amplification of the cognate-transcripts in phloem exudates. (C) RT-qPCR based analysis of the cognate-transcript levels in phloem exudates.

Mentions: RT-PCR based detection of the cognate mRNAs in the phloem exudates of B. juncea and their RT-qPCR based quantitative analysis validated activity of the in silico identified promoters and their relative strength, respectively in the phloem exudates of B. juncea. Limited availability of sequence information on B. juncea genome in public domain database led us to retrieve the cognate gene sequences from other species of Brassica. For each of the target transcripts, PCR and RT-qPCR conditions were optimized so that the gene specific primer-pairs amplified single PCR product of desired size and single peak in melt curve analysis (Figure S1). The amplicons were further validated through sequencing. The standard curve analysis for RT-qPCR efficiency revealed a linear regression R2 of all the primer-pairs ranging between 0.999 and 1.000 and the efficiency ranging from 93 to 105% (Figure S2). Phloem exudates were collected from excised petioles of B. juncea plants. The RNAs isolated from the phloem exudates were assayed for the presence of any non-vascular cellular contamination. For that, the RNAs were analyzed for the presence of RbcS and Lhca2 specific amplification in RT-PCR. RbcS and Lhca2 encode two highly abundant photosynthetic proteins rubisco small subunit and chlorophyll a/b binding protein, respectively. RbcS and Lhca2 gene expression is excluded from vascular cells (Sawchuk et al., 2008) and therefore, their mRNAs, despite showing different dynamics during leaf development, are likely to be completely absent in phloem exudates (Buhtz et al., 2008). No amplification of either RbcS or Lhca2 transcripts in RT-PCR unambiguously indicated purity of the phloem exudates (Figure 2A). On the other hand, specific amplification of the cognate genes in the same cDNA sample indicated phloem-bound activity of the seven orthologous B. juncea promoters (Figure 2B). The relative transcript levels of the cognate genes were estimated in RT-qPCR analysis and expressed as multifold ratio of their normalized level to the least abundant transcript level of GAS1 (Figure 2C). The results empirically showed significant variation among the transcript levels indicating significant differences in strength of the promoters. Amongst the variability, GS3A showed the highest transcript level followed by PP2, GLP13 and SULTR2. However, quantitative expressions of PP2, GLP13 and SULTR2 were statistically similar. Co-amplification of UBC9 in the exudate-cDNA indicated integrity and optimum level of cDNA in the reactions.


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)

RT-PCR analysis for the cognate-transcripts of the phloem-specific promoters. Total RNA was isolated from phloem exudates collected from 45 days old B. juncea leaves and assayed for the cognate-transcripts of the in silico identified B. juncea phloem-specific promoters by RT-PCR and RT-qPCR. (A) RT-PCR amplification of RbcS, Lhca2 and UBC9 in phloem exudates (P), and leaf tissues (L) along with non-template (NT) control. (B) RT-PCR amplification of the cognate-transcripts in phloem exudates. (C) RT-qPCR based analysis of the cognate-transcript levels in phloem exudates.
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Figure 2: RT-PCR analysis for the cognate-transcripts of the phloem-specific promoters. Total RNA was isolated from phloem exudates collected from 45 days old B. juncea leaves and assayed for the cognate-transcripts of the in silico identified B. juncea phloem-specific promoters by RT-PCR and RT-qPCR. (A) RT-PCR amplification of RbcS, Lhca2 and UBC9 in phloem exudates (P), and leaf tissues (L) along with non-template (NT) control. (B) RT-PCR amplification of the cognate-transcripts in phloem exudates. (C) RT-qPCR based analysis of the cognate-transcript levels in phloem exudates.
Mentions: RT-PCR based detection of the cognate mRNAs in the phloem exudates of B. juncea and their RT-qPCR based quantitative analysis validated activity of the in silico identified promoters and their relative strength, respectively in the phloem exudates of B. juncea. Limited availability of sequence information on B. juncea genome in public domain database led us to retrieve the cognate gene sequences from other species of Brassica. For each of the target transcripts, PCR and RT-qPCR conditions were optimized so that the gene specific primer-pairs amplified single PCR product of desired size and single peak in melt curve analysis (Figure S1). The amplicons were further validated through sequencing. The standard curve analysis for RT-qPCR efficiency revealed a linear regression R2 of all the primer-pairs ranging between 0.999 and 1.000 and the efficiency ranging from 93 to 105% (Figure S2). Phloem exudates were collected from excised petioles of B. juncea plants. The RNAs isolated from the phloem exudates were assayed for the presence of any non-vascular cellular contamination. For that, the RNAs were analyzed for the presence of RbcS and Lhca2 specific amplification in RT-PCR. RbcS and Lhca2 encode two highly abundant photosynthetic proteins rubisco small subunit and chlorophyll a/b binding protein, respectively. RbcS and Lhca2 gene expression is excluded from vascular cells (Sawchuk et al., 2008) and therefore, their mRNAs, despite showing different dynamics during leaf development, are likely to be completely absent in phloem exudates (Buhtz et al., 2008). No amplification of either RbcS or Lhca2 transcripts in RT-PCR unambiguously indicated purity of the phloem exudates (Figure 2A). On the other hand, specific amplification of the cognate genes in the same cDNA sample indicated phloem-bound activity of the seven orthologous B. juncea promoters (Figure 2B). The relative transcript levels of the cognate genes were estimated in RT-qPCR analysis and expressed as multifold ratio of their normalized level to the least abundant transcript level of GAS1 (Figure 2C). The results empirically showed significant variation among the transcript levels indicating significant differences in strength of the promoters. Amongst the variability, GS3A showed the highest transcript level followed by PP2, GLP13 and SULTR2. However, quantitative expressions of PP2, GLP13 and SULTR2 were statistically similar. Co-amplification of UBC9 in the exudate-cDNA indicated integrity and optimum level of cDNA in the reactions.

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.