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Examining the transcriptional response of overexpressing anthranilate synthase in the hairy roots of an important medicinal plant Catharanthus roseus by RNA-seq
BACKGROUND: Clinically important anti-cancer drugs vinblastine and vincristine are solely synthesized by the terpenoid indole alkaloid (TIA) pathway in Catharanthus roseus. Anthranilate synthase (AS) is a rate-limiting enzyme in the TIA pathway. The transgenic C. roseus hairy root line overexpressin...
Autores principales: | , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
BioMed Central
2016
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4859987/ https://www.ncbi.nlm.nih.gov/pubmed/27154243 http://dx.doi.org/10.1186/s12870-016-0794-4 |
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author | Sun, Jiayi Manmathan, Harish Sun, Cheng Peebles, Christie A. M. |
author_facet | Sun, Jiayi Manmathan, Harish Sun, Cheng Peebles, Christie A. M. |
author_sort | Sun, Jiayi |
collection | PubMed |
description | BACKGROUND: Clinically important anti-cancer drugs vinblastine and vincristine are solely synthesized by the terpenoid indole alkaloid (TIA) pathway in Catharanthus roseus. Anthranilate synthase (AS) is a rate-limiting enzyme in the TIA pathway. The transgenic C. roseus hairy root line overexpressing a feedback insensitive ASα subunit under the control of an inducible promoter and the ASβ subunit constitutively was previously created for the overproduction of TIAs. However, both increases and decreases in TIAs were detected after overexpressing ASα. Although genetic modification is targeted to one gene in the TIA pathway, it could trigger global transcriptional changes that can directly or indirectly affect TIA biosynthesis. In this study, Illumina sequencing and RT-qPCR were used to detect the transcriptional responses to overexpressing AS, which can increase understanding of the complex regulation of the TIA pathway and further inspire rational metabolic engineering for enhanced TIA production in C. roseus hairy roots. RESULTS: Overexpressing AS in C. roseus hairy roots altered the transcription of most known TIA pathway genes and regulators after 12, 24, and 48 h induction detected by RT-qPCR. Changes in the transcriptome of C. roseus hairy roots was further investigated 18 hours after ASα induction and compared to the control hairy roots using RNA-seq. A unigene set of 30,281 was obtained by de novo assembly of the sequencing reads. Comparison of the differentially expressed transcriptional profiles resulted in 2853 differentially expressed transcripts. Functional annotation of these transcripts revealed a complex and systematically transcriptome change in ASαβ hairy roots. Pathway analysis shows alterations in many pathways such as aromatic amino acid biosynthesis, jasmonic acid (JA) biosynthesis and other secondary metabolic pathways after perturbing AS. Moreover, many genes in overall stress response were differentially expressed after overexpressing ASα. CONCLUSION: The transcriptomic analysis illustrates overexpressing AS stimulates the overall stress response and affects the metabolic networks in C. roseus hairy roots. The up-regulation of endogenous JA biosynthesis pathway indicates the involvement of JA signal transduction to regulate TIA biosynthesis in ASαβ engineered roots and explained why many of the transcripts for TIA genes and regulators are seen to increase with AS overexpression. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12870-016-0794-4) contains supplementary material, which is available to authorized users. |
format | Online Article Text |
id | pubmed-4859987 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2016 |
publisher | BioMed Central |
record_format | MEDLINE/PubMed |
spelling | pubmed-48599872016-05-08 Examining the transcriptional response of overexpressing anthranilate synthase in the hairy roots of an important medicinal plant Catharanthus roseus by RNA-seq Sun, Jiayi Manmathan, Harish Sun, Cheng Peebles, Christie A. M. BMC Plant Biol Research Article BACKGROUND: Clinically important anti-cancer drugs vinblastine and vincristine are solely synthesized by the terpenoid indole alkaloid (TIA) pathway in Catharanthus roseus. Anthranilate synthase (AS) is a rate-limiting enzyme in the TIA pathway. The transgenic C. roseus hairy root line overexpressing a feedback insensitive ASα subunit under the control of an inducible promoter and the ASβ subunit constitutively was previously created for the overproduction of TIAs. However, both increases and decreases in TIAs were detected after overexpressing ASα. Although genetic modification is targeted to one gene in the TIA pathway, it could trigger global transcriptional changes that can directly or indirectly affect TIA biosynthesis. In this study, Illumina sequencing and RT-qPCR were used to detect the transcriptional responses to overexpressing AS, which can increase understanding of the complex regulation of the TIA pathway and further inspire rational metabolic engineering for enhanced TIA production in C. roseus hairy roots. RESULTS: Overexpressing AS in C. roseus hairy roots altered the transcription of most known TIA pathway genes and regulators after 12, 24, and 48 h induction detected by RT-qPCR. Changes in the transcriptome of C. roseus hairy roots was further investigated 18 hours after ASα induction and compared to the control hairy roots using RNA-seq. A unigene set of 30,281 was obtained by de novo assembly of the sequencing reads. Comparison of the differentially expressed transcriptional profiles resulted in 2853 differentially expressed transcripts. Functional annotation of these transcripts revealed a complex and systematically transcriptome change in ASαβ hairy roots. Pathway analysis shows alterations in many pathways such as aromatic amino acid biosynthesis, jasmonic acid (JA) biosynthesis and other secondary metabolic pathways after perturbing AS. Moreover, many genes in overall stress response were differentially expressed after overexpressing ASα. CONCLUSION: The transcriptomic analysis illustrates overexpressing AS stimulates the overall stress response and affects the metabolic networks in C. roseus hairy roots. The up-regulation of endogenous JA biosynthesis pathway indicates the involvement of JA signal transduction to regulate TIA biosynthesis in ASαβ engineered roots and explained why many of the transcripts for TIA genes and regulators are seen to increase with AS overexpression. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12870-016-0794-4) contains supplementary material, which is available to authorized users. BioMed Central 2016-05-06 /pmc/articles/PMC4859987/ /pubmed/27154243 http://dx.doi.org/10.1186/s12870-016-0794-4 Text en © Sun et al. 2016 Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. |
spellingShingle | Research Article Sun, Jiayi Manmathan, Harish Sun, Cheng Peebles, Christie A. M. Examining the transcriptional response of overexpressing anthranilate synthase in the hairy roots of an important medicinal plant Catharanthus roseus by RNA-seq |
title | Examining the transcriptional response of overexpressing anthranilate synthase in the hairy roots of an important medicinal plant Catharanthus roseus by RNA-seq |
title_full | Examining the transcriptional response of overexpressing anthranilate synthase in the hairy roots of an important medicinal plant Catharanthus roseus by RNA-seq |
title_fullStr | Examining the transcriptional response of overexpressing anthranilate synthase in the hairy roots of an important medicinal plant Catharanthus roseus by RNA-seq |
title_full_unstemmed | Examining the transcriptional response of overexpressing anthranilate synthase in the hairy roots of an important medicinal plant Catharanthus roseus by RNA-seq |
title_short | Examining the transcriptional response of overexpressing anthranilate synthase in the hairy roots of an important medicinal plant Catharanthus roseus by RNA-seq |
title_sort | examining the transcriptional response of overexpressing anthranilate synthase in the hairy roots of an important medicinal plant catharanthus roseus by rna-seq |
topic | Research Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4859987/ https://www.ncbi.nlm.nih.gov/pubmed/27154243 http://dx.doi.org/10.1186/s12870-016-0794-4 |
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