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Overexpression of an apoplastic peroxidase gene CrPrx in transgenic hairy root lines of Catharanthus roseus

  • Applied Genetics and Molecular Biotechnology
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Abstract

Peroxidases are a family of isoenzymes found in all higher plants and are known to be involved in a broad range of physiological processes. However, very little information is available concerning their role in Catharanthus roseus. The present study describes the impact of both overexpression and suppression of a peroxidase gene, CrPrx in C. roseus transgenic hairy root lines. Real-time PCR analysis in 35S-CrPrx and CrPrx-RNAi transgenic lines indicated differential transcript profile for peroxidases as well as for genes and regulators involved in MIA (monoterpenoid indole alkaloid) pathway of C. roseus. Comparative analysis revealed that MIA pathway genes showing elevated levels of expression in 35S-CrPrx transgenic lines showed a significant reduction in their transcript level in CrPrx-RNAi transgenic lines. Metabolite analysis detected higher levels of ajmalicine and serpentine accumulation in overexpressed lines. It was observed that all overexpressed transgenic lines produced more amount of H2O2. These results indicate a role of CrPrx gene in the regulation of MIA pathway genes and regulators, thus affecting the production of specific alkaloids.

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Acknowledgements

MJ and SK thank Council of Scientific and Industrial Research (CSIR), India for the award of senior research fellowships. The authors are grateful to Dr. Peter Waterhouse of CSIRO Plant Industry, Canberra, Australia, for kindly providing vectors pKANNIBAL and pART27. The work is supported by financial assistance from the core grant of National Institute of Plant Genome Research, New Delhi, India.

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Correspondence to Alok Krishna Sinha.

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Table S1

List of primers for different constructs (DOC 28 kb)

Table S2

List of common primers used in the present study (DOC 31 kb)

Table S3

List of primers used for qRT-PCR (DOC 34 kb)

Fig. S1

Southern blot analysis of 35S-CrPrx hairy root lines. Autoradiogram showing hybridization signals in all transgenic hairy root lines using GUS gene as a probe. Each lane represents 15 μg of genomic DNA digested with the EcoRI and HindIII restriction enzymes. C2-C13 is DNAs from transgenic lines. PR and HR represent the untransformed C. roseus genomic DNA and normal hairy root DNA, respectively, as controls. Numbers on the left indicate positions of the DNA ladder (GIF 114 kb)

Fig. S2

Monoterpenoid Indole Alkaloid (MIA) biosynthesis in C. roseus. Solid arrows indicate single step, whereas broken arrows represent multi-step enzymatic conversions. Genes highlighted in black box and regulators in bold are analyzed in this study for their transcript accumulation (GIF 117 kb)

Fig. S3

HPLC analyses of MIAs in C. roseus hairy root extracts from control and transgenic lines. a, b The pure standards of serpentine and ajmalicine, respectively. Con - untransformed hairy root line; C3, C6 and C8 - 35S-CrPrx hairy root lines. Alkaloids peak, 1 for serpentine and 2 for ajmalicine (GIF 264 kb)

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Jaggi, M., Kumar, S. & Sinha, A.K. Overexpression of an apoplastic peroxidase gene CrPrx in transgenic hairy root lines of Catharanthus roseus . Appl Microbiol Biotechnol 90, 1005–1016 (2011). https://doi.org/10.1007/s00253-011-3131-8

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