Production of marker-free transgenic Jatropha curcas expressing hybrid Bacillus thuringiensis δ-endotoxin Cry1Ab/1Ac for resistance to larvae of tortrix moth (Archips micaceanus).

Keyu Gu,Zhongchao Yin,Huizhu Mao

doi:10.1186/1754-6834-7-68

Abstract

BackgroundThe potential biofuel plant Jatropha curcas L. is affected by larvae of Archips micaceanus (Walker), a moth of the family Tortricidae. The hybrid Bacillus thuringiensis (Bt) δ-endotoxin protein Cry1Ab/1Ac confers resistance to lepidopteran insects in transgenic rice.ResultsHere, we report the production of a marker-free transgenic line of J. curcas (L10) expressing Cry1Ab/1Ac using Agrobacterium-mediated transformation and a chemically regulated, Cre/loxP-mediated DNA recombination system. L10 carries a single copy of marker-free T-DNA that contains the Cry1Ab/1Ac gene under the control of a maize phosphoenolpyruvate carboxylase gene promoter (P Pepc :Cry1Ab/1Ac:T Nos ). The P Pepc :Cry1Ab/1Ac:T Nos gene was highly expressed in leaves of L10 plants. Insecticidal bioassays using leaf explants of L10 resulted in 80-100% mortality of larvae of A. micaceanus at 4 days after infestation.ConclusionThe results demonstrate that the hybrid Bt δ-endotoxin protein Cry1Ab/1Ac expressed in Jatropha curcas displays strong insecticidal activity to A. micaceanus. The marker-free transgenic J. curcas line L10 can be used for breeding of insect resistance to A. micaceanus.

Highlights

The potential biofuel plant Jatropha curcas L. is affected by larvae of Archips micaceanus (Walker), a moth of the family Tortricidae
Generation of marker-free transgenic line of J. curcas containing phosphoenolpyruvate carboxylase gene promoter (PPepc):Cry1Ab/1Ac:TNos gene The binary construct pCCreloxPBt, which carries a chemically regulated Cre/loxP system and a hybrid Cry1Ab/1Ac gene driven by maize phosphoenolpyruvate carboxylase (PEPC) gene promoter (PPepc:Cry1Ab/1Ac:TNos), was used to produce marker-free transgenic rice line L24 that expresses the Cry1Ab/1Ac proteins in leaves and stem [34]
The marker-free T-DNA is detected by PCR amplification of P1-P4 fragment (385 bp) flanking the remaining loxP site after DNA recombination, while the marker-containing T-DNAs, T-DNAs that have undergone incomplete loxP fragment excision and truncated T-DNAs are detected by PCR amplification of the P1-P2 (534 bp) fragment flanking the loxP site at the left border of T-DNA and/or the P3-P4 (460 bp) fragment flanking the loxP site adjacent to the maize PEPC gene promoter [34]

Summary

Introduction

The potential biofuel plant Jatropha curcas L. is affected by larvae of Archips micaceanus (Walker), a moth of the family Tortricidae. The hybrid Bacillus thuringiensis (Bt) δ-endotoxin protein Cry1Ab/1Ac confers resistance to lepidopteran insects in transgenic rice. The crystalline (Cry) proteins from Bacillus thuringiensis (Bt) have toxic activity against numerous insect species of the orders Lepidoptera, Diptera, Coleoptera, Hymenoptera and nematodes [1]. The Cry proteins are inactive until they get solubilized by proteases in the insect’s midgut at high pH (>9.5), releasing proteins called δ-endotoxins [2,3]. J. curcas is a drought-resistant, non-food plant that can grow in marginal lands. J. curcas seeds contain about 25 to 40% storage lipids [19]. J. curcas has emerged as a potential biofuel plant. Despite the presence of toxins such as phorbol ester and curcins in J. curcas leaves and seeds, J. curcas is still attacked by insects [20,21,22,23,24], fungi [25] and viruses [26]

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