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Homologous promoter derived constitutive and chloroplast targeted expression of synthetic cry1Ac in transgenic chickpea confers resistance against Helicoverpa armigera

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Abstract

The insecticidal crystal protein derived from gram positive soil bacterium Bacillus thuringiensis plays an important role in controlling lepidopteran infestation. The present study seeks to protect chickpea plants from Helicoverpa armigera infestation by over expressing cry1Ac. Homologous Ubiquitin and RuBisCO small subunit (rbcS) promoters were used to transcribe cry1Ac in transgenic chickpea both constitutively and in a tissue specific manner through Agrobacterium mediated transformation of chickpea var. ICCV89314. Expressed Cry1Ac was specifically targeted to the chloroplast rich tissues using transit peptide sequence. After monitoring transgene integration by Southern hybridization, transgenic chickpea lines were further analyzed by western blot, ELISA and insect bioassay. Expression of cry1Ac in chickpea under the control of above two promoters conferred a high level of protection against pod borer infestation, where chloroplast targeting system was found to be more efficient in controlling this particular devastating lepidopteran pest.

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Acknowledgments

JC and SS are indebted to Indian Council of Agricultural Research (No. NFBSFARA/AB-2010(2010-11 dt. 24.01.2011) for providing financial support. PG and AS are thankful to Bose Institute for financial assistance. Dr. Dipankar Chakraborty, St. Xavier’s college, Kolkata is acknowledged for his intellectual contribution. Dr. Sujayanand G.K. of Indian Institute of Pulse Research is duly acknowledged for providing H. armigera strain. All the authors are grateful to Bose Institute for infrastructural facilities. Special thanks are extended to Mr. Sudipta Basu, Mr. Swarnava Das and Mr. Surajit Maity for their sincere efforts in the field and necessary laboratory assistance.

Author contributions

JC, SS, DB and SD conceived and designed the experiments. JC, SS and PG carried out Southern analysis, Immunohistofluorescence, ELISA and statistical analysis. JC and SS conducted all the other experiments. JC, SS, PG, AS and SD analyzed the data. JC, SS and PG drafted the manuscript. DB and SD edited the manuscript and supervised the work. SD contributed all the reagents, materials and analysis tools. All authors read and approved the final manuscript.

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Correspondence to Sampa Das.

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The authors declare that they have no conflict of interest.

Additional information

Joydeep Chakraborty and Senjuti Sen contributed equally to this work.

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Chakraborty, J., Sen, S., Ghosh, P. et al. Homologous promoter derived constitutive and chloroplast targeted expression of synthetic cry1Ac in transgenic chickpea confers resistance against Helicoverpa armigera . Plant Cell Tiss Organ Cult 125, 521–535 (2016). https://doi.org/10.1007/s11240-016-0968-7

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Keywords

  • cry1Ac
  • Chloroplast transit peptide
  • Chloroplast targeted expression
  • RuBisCO small subunit promoter
  • Transgenic chickpea
  • Ubiquitin promoter