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Applied Microbiology and Biotechnology

, Volume 90, Issue 3, pp 1005–1016 | Cite as

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

  • Monika Jaggi
  • Santosh Kumar
  • Alok Krishna Sinha
Applied Genetics and Molecular Biotechnology

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.

Keywords

Agrobacterium rhizogenes Catharanthus roseus Monoterpenoid indole alkaloids Peroxidases Transgenic hairy roots 

Notes

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.

Supplementary material

253_2011_3131_MOESM1_ESM.doc (28 kb)
Table S1 List of primers for different constructs (DOC 28 kb)
253_2011_3131_MOESM2_ESM.doc (32 kb)
Table S2 List of common primers used in the present study (DOC 31 kb)
253_2011_3131_MOESM3_ESM.doc (34 kb)
Table S3 List of primers used for qRT-PCR (DOC 34 kb)
253_2011_3131_Fig5_ESM.gif (114 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)

253_2011_3131_MOESM4_ESM.tif (258 kb)
High resolution image (TIFF 258 kb)
253_2011_3131_Fig6_ESM.gif (117 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)

253_2011_3131_MOESM5_ESM.tif (76 kb)
High resolution image (TIFF 75 kb)
253_2011_3131_Fig7_ESM.gif (265 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)

253_2011_3131_MOESM6_ESM.tif (209 kb)
High resolution image (TIFF 209 kb)

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Copyright information

© Springer-Verlag 2011

Authors and Affiliations

  • Monika Jaggi
    • 1
  • Santosh Kumar
    • 1
    • 2
  • Alok Krishna Sinha
    • 1
  1. 1.National Institute of Plant Genome ResearchNew DelhiIndia
  2. 2.University of KentuckyLexingtonUSA

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