Designing, construction and transformation of silencing constructs for F3´H and F3´5´H genes in grapevine cv. Shiraz

Document Type : Full Paper

Authors

1 Post Graduate Student and Professor, University College of Agriculture and Natural Resources, University of Tehran, Karaj, Iran

2 . Post Graduate Student and Professor, University College of Agriculture and Natural Resources, University of Tehran, Karaj, Iran

3 . Associate Professor, National Institute of Genetic Engineering and Biotechnology (NIGEB), Tehran, Iran

4 . Researcher and Professor in CSIRO Plant Industries, Adelaide, Australia

Abstract

abstract
 In order to study F3´5´H and F3´H function and their intraction in the grapevine flavonoids biosynthetic pathway, designing and constraction of suitable constructs encoding ihpRNA was performed and transgenic lines containing silencing construct of F3´H and simultaneous silencing construct of F3´5´H and F3´H were created. For this purpose, fragments were selected from both genes and inserted into pHANNIBAL vector as inverted repeats. Using binary vector system, each construct inserted into p27 mod GFP 4a vector and were used for Agrobacterium transformation. Embryogenic calli, drived from anther culture, were co-cultivated with Agrobacterium for transformation. Expression of GFP gene was detected in transgenic embryogenic calli, 5 days after inoculation using microscope. Finally, 42 and 34 lines were generated from single (F3´H) and simultaneous silencing (F3´5´H and F3´H) cultures, respectively. PCR analysis confirmed 36 single (F3´H) silencing line and 27 simultaneous silencing (F3´5´H and F3´H) lines, showing high efficacy of regeneration and transformation methods.

Keywords

References

  1.  

    1. Boase, M.R., Lewis, D.H., Davies, K.M., Marshall, G.B., Patel, D., Schwinn, K.E. & Deroles, S.C. (2010). Isolation and antisense suppression of flavonoid 3', 5'-hydroxylase modifies flower pigments and colour in cyclamen. BMC Plant Biology, 10, 107-118.
    2. Castellarin, S.D., Di Gaspero, G., Marconi, R., Nonis, A., Peterlunger, E., Paillard, S., Adam-Blondon, A.F. & Testolin, R. (2006). Colour variation in red grapevines (Vitis vinifera L.): genomic organisation, expression of flavonoid 3'-hydroxylase, flavonoid 3',5'-hydroxylase genes and related metabolite profiling of red cyanidin-/blue delphinidin-based anthocyanins in berry skin. BMC Genomics, 7, 12-12.
    3. Davies, K.M., Schwinn, K.E., Deroles, S.C., Manson, D.G., Lewis, D.H., Bloor, S.J. & Bradley, J.M. (2003). Enhancing anthocyanin production by altering competition for substrate between flavonol synthase and dihydroflavonol 4-reductase. Euphytica, 131, 259-268.
    4. Fitzgerald, A., van Kan, J.A.L. & Plummer, K.M. (2004). Simultaneous silencing of multiple genes in the apple scab fungus, Venturia inaequalis, by expression of RNA with chimeric inverted repeats. Fungal Genetics and Biology, 41, 963-971.
    5. Franks, T., He, D.G. & Thomas, M.R. (1998). Regeneration of transgenic Vitis vinifera L. Sultana plants: genotypic and phenotypic analysis. Molecular Breeding, 4, 321-333.
    6. Garfinkel, D.J. & Nester, E.W. (1980). Agrobacterium tumefaciens mutants affected in crown gall tumorigenesis and octopine catabolism. Journal of Bacteriology, 144, 732-743.
    7. Gleave, A.P. (1992). A versatile binary vector system with a T-DNA organizational structure conducive to efficient integration of cloned DNA into the plant genome. Plant Molecular Biology, 20, 1203-1207.
    8. Helliwell, C. & Waterhouse, P. (2003). Constructs and methods for high-throughput gene silencing in plants. Methods, 30, 289-295.
    9. Helliwell, C.A., Wesley, S.V., Wielopolska, A.J. & Waterhouse, P.M. (2002). High-throughput vectors for efficient gene silencing in plants. Functional Plant Biology, 29(10), 1217-1225. 
    10. Hoffmann, T., Kalinowski, G. & Schwab, W. (2006). RNAi-induced silencing of gene expression in strawberry fruit (Fragaria x ananassa) by agroinfiltration: a rapid assay for gene function analysis. Plant Journal, 48(5), 818-826.
    11. Hooykaas, P.J.J. & Schilperoort, R.A. (1992). Agrobacterium and plant genetic engineering. Plant Molecular Biology, 19, 15-38.
    12. Iocco, P., Franks, T. & Thomas, M.R. (2001). Genetic transformation of major wine grape cultivars of Vitis vinifera L. Transgenic Research, 10, 105-112.
    13. Jeong, S. T., Goto-Yamamoto, N., Hashizume, K. & Esaka, M. (2006). Expression of the flavonoid 3′-hydroxylase and flavonoid 3′, 5′-hydroxylase genes and flavonoid composition in grape (Vitis vinifera). Plant Science, 170, 61-69.
    14. Mahajan, M., Ahuja, P.S. & Yadav, S.K. (2011). Post-Transcriptional Silencing of Flavonol Synthase mRNA in Tobacco Leads to Fruits with Arrested Seed Set. PLoS ONE, 6(12), e28315.
    15. Murashige, T. & Skoog, F. (1962). A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiologia Plantarum, 15, 473-497.
    16. Nakatsuka, T., Abe, Y., Kakizaki, Y., Yamamura, S. & Nishihara, M. (2007). Production of red-flowered plants by genetic engineering of multiple flavonoid biosynthetic genes. Plant Cell Reports, 26, 1951-1959.
    17. Newton, D.J. & Goussard, P.G. (1990). The ontogeny of somatic embryos from in vitro cultured grapevine anthers. South African Journal for Enology and Viticulture, 11(2), 70-75.
    18. Scherer, L.J. & Rossi, J.J. (2003). Approaches for the sequence-specific knockdown of mRNA. Nature Biotechnology, 21(12), 1457-65.
    19. Schwab, W., Hoffmann, T., Kalinowski, G. & Preu, B.A. (2011). Functional genomics in strawberry fruit through RNAi-mediated silencing. Genes, Genomes and Genomics, 5, 91-101.
    20. Subramanian, S., Graham, M.Y., Yu, O. & Graham, T.L. (2005) RNA interference of soybean isoflavone synthase genes leads to silencing in tissue distal to the transformation site and to enhanced susceptibility to Phytophthora sojae. Plant Physiology, 137, 1345-1353.
    21. Thomas, M. R., Matsumoto, S. & Cain P. (1993). Repetitive DNA of grapevine: classes present and sequences suitable for cultivar identification. Theoretical and Applied Genetics, 86, (2-3), 173-180.
    22. Torregrosa, L. (1998) A simple and efficient method to obtain stable embryogenic cultures from anthers of Vitis vinifera L. Vitis, 37, 91-92.
    23. Torregrosa, L., Iocco, P. & Thomas, M.R. (2002). Influence of Agrobacterium strain, culture medium, and cultivar on the transformation efficiency of Vitis vinifera L. American Journal of Enology and Viticulture, 53, 183-190.
    24. Wang, M. B. & Waterhouse, P.M. (2000). High-efficiency silencing of a b-glucuronidase gene in rice is correlated with repetitive transgene structure but independent of DNA methylation. Plant Molecular Biology, 43, 67-82.
    25. Wasson, A. P., Pellerone, F. I. & Mathesius, U. (2006). Silencing the flavonoid pathway in Medicago truncatula inhibits root nodule formation and prevents auxin transport regulation by Rhizobia. Plant Cell, 18, 1617-1629.
    26. Wesley, V.S., Helliwell, C.A., Smith, N.A., Wang, M.B., Rouse, D.T., Liu, Q., Gooding, P.S., Singh, S.P., Abbott, D., Stoutjesdijk, P.A., Robinson, S.P., Gleave, A.P., Green, A.G. & Waterhouse, P. M. (2001). Construct design for efficient, effective and high-throughput gene silencing in plants. The Plant Journal, 27, 581-590.

     

     

     

Files

History

  • Receive Date: 27 April 2014
  • Revise Date: 24 May 2014
  • Accept Date: 24 May 2014

Share

How to cite

Statistics