Production of marker-free transgenic rice expressing tissue-specific Bt gene

Abstract

The hybrid Bacillus thuringiensis (Bt) δ-endotoxin gene Cry1Ab/Ac was used to develop a transgenic Bt rice (Oryza sativa L.) targeting lepidopteran insects of rice. Here, we show the production of a marker-free and tissue-specific expressing transgenic Bt rice line L24 using Agrobacterium-mediated transformation and a chemically regulated, Cre/loxP-mediated DNA recombination system. L24 carries a single copy of marker-free T-DNA that contains the Cry1Ab/Ac gene driven by a maize phosphoenolpyruvate carboxylase (PEPC) gene promoter. The marker-free T-DNA was integrated into the 3' untranslated region of rice gene Os01g0154500 on the short arm of chromosome 1. Compared to the constitutive and non-specific expression of the P (Actin1):Cry1Ab/Ac:T (Nos) gene in the control Bt rice line T51-1, the P ( Pepc ):Cry1Ab/Ac:T (Nos ) gene was detected only in the leaf and stem tissues of L24. More importantly, compared to high levels of CRY1Ab/Ac proteins accumulated in T51-1 seeds, the CRY1Ab/Ac proteins were not detectable in L24 seeds by Western blot analysis. As demonstrated by insect bioassay, L24 provided similar level of resistance to rice leaffolder (Cnaphalocrocis medinalis) as T51-1. The marker-free transgenic line L24 can be used directly in rice breeding for insect resistance to lepidopteran insects where absence of Bt toxin protein in the seed is highly desirable.

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

“…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 ( P Pepc :Cry1Ab/1Ac:T Nos ), was used to produce marker-free transgenic rice line L24 that specifically expresses the Cry1Ab/1Ac proteins in leaves and stem [34]. Theoretically, the marker ( Hpt gene)-containing loxP fragment in T-DNA region of pCCreloxPBt in transgenic plants can be removed by β -estradiol-regulated Cre/ loxP -mediated excision, which yields marker-free T-DNA in transgenic plants [34].…”

“…Theoretically, the marker ( Hpt gene)-containing loxP fragment in T-DNA region of pCCreloxPBt in transgenic plants can be removed by β -estradiol-regulated Cre/ loxP -mediated excision, which yields marker-free T-DNA in transgenic plants [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]. In this study, pCCreloxPBt was used to produce transgenic J. curcas plants via Agrobacterium -mediated transformation [35].…”

“…After β -estradiol-regulated Cre/ loxP -mediated excision of the loxP fragment, a total of twenty putative transgenic T 0 plants were obtained. The 20 T 0 plants were screened by PCR for the presence of the Cry1Ab/1Ac and Hpt genes as well as for the excision of the loxP fragment, which was detected by the amplification of P1-P4 fragment [34]. All the 20 T 0 plants carried the coding region of the Cry1Ab/1Ac gene (Table  1).…”

“…Recently we produced a marker-free transgenic rice line L24 with the P Pepc :Cry1Ab/1Ac:T Nos gene using Agrobacterium -mediated transformation and a chemically regulated, Cre/ loxP -mediated DNA recombination system [34]. The P Pepc :Cry1Ab/1Ac:T Nos gene in L24 was mainly expressed in green tissues such as leaves and stem and provided resistance to rice leaf-folder ( C. medinalis ) [34]. Here, we report the adoption of similar technology to generate a marker-free transgenic line of J. curcas that expresses Cry1Ab/1Ac proteins for resistance to A. micaceanus .…”

A novel synthetic Cry1Ab gene resists rice insect pests

“…To test whether Cry1Ab proteins produced in the transgenic plants had insecticidal activity toward the rice leaffolder, an insect bioassay was conducted using the test tube method according to Qiu et al (2010). Rice leaves at the tillering stage were cut into 10-cm-long sections.…”