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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-qPCR based analysis of expression-stability of the B. juncea phloem-specific promoters. (A) Mean Ct values of the cognate-transcripts analyzed by RT-qPCR in phloem-cDNA samples collected at different growth stages of B. juncea plants. (B–E) Ranking of the B. juncea phloem-specific promoters in terms of expression-stability measured by four Excel based statistical methods, BestKeeper (B), deltaCt method (C), geNorm (D), and NormFinder (E) and plotted in increasing manner from left to right.
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Figure 6: RT-qPCR based analysis of expression-stability of the B. juncea phloem-specific promoters. (A) Mean Ct values of the cognate-transcripts analyzed by RT-qPCR in phloem-cDNA samples collected at different growth stages of B. juncea plants. (B–E) Ranking of the B. juncea phloem-specific promoters in terms of expression-stability measured by four Excel based statistical methods, BestKeeper (B), deltaCt method (C), geNorm (D), and NormFinder (E) and plotted in increasing manner from left to right.

Mentions: Promoter-activity of the in silico identified B. juncea promoters at different growth stages of the plant were analyzed by assessing their cognate-transcript levels at the vegetative, bud initiation and flowering stage. RT-qPCR analysis of the cognate-transcripts captured at different growth stages empirically showed likely influence of plant developmental stages on the activity of these promoters (Figure 6A). Based on mean Ct values, GS3A showed highest average transcript levels, followed by GLP13 and PP2, across the developmental stages of B. juncea plants. Independent analysis of the cognate-transcript levels within the vegetative and reproductive stage also showed the highest relative expression of GS3A at both the growth-stages. However, in general the transcript levels of all the cognate genes were consistently lower during the reproductive stage compared to their levels at the vegetative stage, and also the expressions were further reduced as the plants grew toward maturity.


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-qPCR based analysis of expression-stability of the B. juncea phloem-specific promoters. (A) Mean Ct values of the cognate-transcripts analyzed by RT-qPCR in phloem-cDNA samples collected at different growth stages of B. juncea plants. (B–E) Ranking of the B. juncea phloem-specific promoters in terms of expression-stability measured by four Excel based statistical methods, BestKeeper (B), deltaCt method (C), geNorm (D), and NormFinder (E) and plotted in increasing manner from left to right.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 6: RT-qPCR based analysis of expression-stability of the B. juncea phloem-specific promoters. (A) Mean Ct values of the cognate-transcripts analyzed by RT-qPCR in phloem-cDNA samples collected at different growth stages of B. juncea plants. (B–E) Ranking of the B. juncea phloem-specific promoters in terms of expression-stability measured by four Excel based statistical methods, BestKeeper (B), deltaCt method (C), geNorm (D), and NormFinder (E) and plotted in increasing manner from left to right.
Mentions: Promoter-activity of the in silico identified B. juncea promoters at different growth stages of the plant were analyzed by assessing their cognate-transcript levels at the vegetative, bud initiation and flowering stage. RT-qPCR analysis of the cognate-transcripts captured at different growth stages empirically showed likely influence of plant developmental stages on the activity of these promoters (Figure 6A). Based on mean Ct values, GS3A showed highest average transcript levels, followed by GLP13 and PP2, across the developmental stages of B. juncea plants. Independent analysis of the cognate-transcript levels within the vegetative and reproductive stage also showed the highest relative expression of GS3A at both the growth-stages. However, in general the transcript levels of all the cognate genes were consistently lower during the reproductive stage compared to their levels at the vegetative stage, and also the expressions were further reduced as the plants grew toward maturity.

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.