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Marker-free transgenic rice expressing the vegetative insecticidal protein (Vip) of Bacillus thuringiensis shows broad insecticidal properties

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Abstract

Main conclusion

Genetically engineered rice lines with broad insecticidal properties against major lepidopteran pests were generated using a synthetic, truncated form of vegetative insecticidal protein (Syn vip3BR) from Bacillus thuringiensis. The selectable marker gene and the redundant transgene(s) were eliminated through Cre/ lox mediated recombination and genetic segregation to make consumer friendly Bt -rice.

For sustainable resistance against lepidopteran insect pests, chloroplast targeted synthetic version of bioactive core component of a vegetative insecticidal protein (Syn vip3BR) of Bacillus thuringiensis was expressed in rice under the control of green-tissue specific ribulose-1,5-bisphosphate carboxylase/oxygenase small subunit gene promoter. The transgenic plants (in Oryza sativa indica Swarna cultivar) showed high insect mortality rate in vitro against major rice pests, yellow stem borer (Scirpophaga incertulas), rice leaf folder (Cnaphalocrocis medinalis) and rice horn caterpillar (Melanitis leda ismene) in T1 generation, indicating insecticidal potency of Syn vip3BR. Under field conditions, the T1 plants showed considerable resistance against leaf folders and stem borers. The expression cassette (vip-lox-hpt-lox) as well as another vector with chimeric cre recombinase gene under constitutive rice ubiquitin1 gene promoter was designed for the elimination of selectable marker hygromycin phosphotransferase (hptII) gene. Crossing experiments were performed between T1 plants with single insertion site of vip-lox-hpt-lox T-DNA and one T1 plant with moderate expression of cre recombinase with linked bialaphos resistance (syn bar) gene. Marker gene excision was achieved in hybrids with up to 41.18 % recombination efficiency. Insect resistant transgenic lines, devoid of selectable marker and redundant transgene(s) (hptII + cre-syn bar), were established in subsequent generation through genetic segregation.

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Acknowledgments

The authors extend their sincere appreciation for Partha Das and Sona Dogra for their assistance in generating the transgenic rice lines related to this study, Sudarshan Maity for maintenance and rearing of the genetic lines under outdoor cultural conditions. pX6-GFP plasmid DNA was a generous gift from Dr. N-H Chua, Rockefeller University, USA. Financial assistance from National Agricultural Innovation Project, Indian Council of Agricultural Research (NAIP/ICAR) in terms of grant support to Advanced Laboratory for Plant Genetic Engineering and fellowship to SP is thankfully acknowledged. Finally, the authors extend their sincere gratitude to the distinguished editor and the anonymous reviewers, whose incisive comments have helped immensely to improve the clarity of the manuscript.

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  1. Advanced Laboratory for Plant Genetic Engineering, Indian Institute of Technology Kharagpur, Kharagpur, 721302, India

    Subrata Pradhan, Anirban Chakraborty, Narattam Sikdar, Saikat Chakraborty, Jagannath Bhattacharyya, Joy Mitra, Anulina Manna & Soumitra Kumar Sen

  2. Department of Agricultural and Food Engineering, Indian Institute of Technology Kharagpur, Kharagpur, 721302, India

    Snehasish Dutta Gupta

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425_2016_2535_MOESM1_ESM.jpg

Suppl. Fig. S1 Synthetic vip3BR sequence with chloroplast targeting signal (syn vip3BR) green portion: chloroplast targeting signal to target the gene product into chloroplast blue portion: 5′ portion of rubisco gene to allow proper processing of the signal peptide purple portion: codon optimized synthetic vip3BR gene (JPEG 7094 kb)

425_2016_2535_MOESM2_ESM.jpg

Suppl. Fig. S2 a Schematic map of syn vip3BR gene cassette carrying hptII marker gene, flanked by two direct-repeat (indicated by black arrows) of loxP sequences (pLHRV). Oligos used for cloning loxP containing hptII gene are indicated by red arrowheads. Blue arrowheads indicate locations of oligos used for PCR screening of syn vip3BR gene. Probe A indicates the radiolabeled probe used for Southern blot of plant lines carrying syn vip3BR gene b cre recombinase gene cassette, under Os Ubi1 promoter with syn bar as selection marker (pBUC). Violet arrowheads indicate oligos used for cloning of syn bar gene in place of hptII gene. Green arrowheads indicate oligos to screen plant lines for cre gene. Probe B indicates radiolabeled probe used for Southern blot of plant lines carrying cre gene (JPEG 2686 kb)

425_2016_2535_MOESM3_ESM.jpg

Suppl. Fig. S3 Spatial expression profile (leaf vs grain tissue) of the syn vip3BR gene in T1 rice lines a Normalized, relative expression level of syn vip3BR gene in T1 transgenic rice lines by quantitative RT-PCR analysis. Results are expressed as mean ± SD, for three biological replicates b Immunoblot of Syn vip3BR protein (Upper panel). Actin is used as loading control (Lower panel) (JPEG 2253 kb)

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Suppl. Fig. S4 Molecular analysis of marker gene excision a, b, c Southern blot analysis of seven hptII negative T1F1 hybrid plants probed with syn vip3BR, cre and hptII gene specific probes, respectively. Lane PC represents in c PCR amplified and gel purified hptII gene, loaded in the amount 0.1 ng. Lane UC represents HindIII digested genomic DNA from untransformed control plants. Approximate molecular weight markers are indicated (JPEG 3386 kb)

Supplementary Table 1 List of oligonucleotides used in the study (DOC 44 kb)

425_2016_2535_MOESM6_ESM.doc

Supplementary Table 2 Inheritance of the hptII gene in the T1 progeny of pLHRV containing transgenic rice plants (DOC 29 kb)

425_2016_2535_MOESM7_ESM.doc

Supplementary Table 3 Inheritance of the syn bar gene in the T1 progeny of pBUC containing transgenic rice plants (DOC 28 kb)

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Pradhan, S., Chakraborty, A., Sikdar, N. et al. Marker-free transgenic rice expressing the vegetative insecticidal protein (Vip) of Bacillus thuringiensis shows broad insecticidal properties. Planta 244, 789–804 (2016). https://doi.org/10.1007/s00425-016-2535-1

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