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A Modified MultiSite Gateway Cloning Strategy for Consolidation of Genes in Plants

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Abstract

The genome information is offering opportunities to manipulate genes, polygenic characters and multiple traits in plants. Although a number of approaches have been developed to manipulate traits in plants, technical hurdles make the process difficult. Gene cloning vectors that facilitate the fusion, overexpression or down regulation of genes in plant cells are being used with various degree of success. In this study, we modified gateway MultiSite cloning vectors and developed a hybrid cloning strategy which combines advantages of both traditional cloning and gateway recombination cloning. We developed Gateway entry (pGATE) vectors containing attL sites flanking multiple cloning sites and plant expression vector (pKM12GW) with specific recombination sites carrying different plant and bacterial selection markers. We constructed a plant expression vector carrying a reporter gene (GUS), two Bt cry genes in a predetermined pattern by a single round of LR recombination reaction after restriction endonuclease-mediated cloning of target genes into pGATE vectors. All the three transgenes were co-expressed in Arabidopsis as evidenced by gene expression, histochemical assay and insect bioassay. The pGATE vectors can be used as simple cloning vectors as there are rare restriction endonuclease sites inserted in the vector. The modified multisite vector system developed is ideal for stacking genes and pathway engineering in plants.

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Acknowledgments

This study is supported by the research grants from the Indian Council of Agricultural Research, Department of Biotechnology and DST-FIST Government of India. Authors would like to thank to Dr. Rakesh Tuli, National Agri-Food Biotechnology Institute, Mohali, India and Dr. P. Anandakumar, National Research Centre for Plant Biotechnology, IARI, New Delhi 110012, India, for providing cry1Ec and cry1Aabc genes used in this study. Dr. A.R.V Kumar, Department of Entomology, University of Agricultural Sciences, GKVK, Bangalore, India provided DBM insect larvae for insect bioassay.

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Authors and Affiliations

  1. Department of Crop Physiology, University of Agricultural Sciences, GKVK Campus, Bangalore, 560065, India

    Ramu S. Vemanna, Babitha K. Chandrashekar, H. M. Hanumantha Rao, Shailesh K. Sathyanarayanagupta, Karaba N. Nataraja & M. Udayakumar

  2. Department of Microbiology & Biotechnology, Bangalore University, Jnanabharathi Campus, Bangalore, 560056, India

    K. S. Sarangi

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  1. Ramu S. Vemanna
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  2. Babitha K. Chandrashekar
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  6. Karaba N. Nataraja
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Correspondence to Karaba N. Nataraja.

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Vemanna, R.S., Chandrashekar, B.K., Hanumantha Rao, H.M. et al. A Modified MultiSite Gateway Cloning Strategy for Consolidation of Genes in Plants. Mol Biotechnol 53, 129–138 (2013). https://doi.org/10.1007/s12033-012-9499-6

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