title: 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.
authors.affiliation: Temasek Life Sciences Laboratory, National University of Singapore, Singapore. qiucx@tll.org.sg
authors.forename: Chengxiang
authors.initials: C
authors.lastname: Qiu
authors.forename: Jatinder Singh
authors.initials: JS
authors.lastname: Sangha
authors.forename: Fengshun
authors.initials: F
authors.lastname: Song
authors.forename: Zhiyun
authors.initials: Z
authors.lastname: Zhou
authors.forename: Ao
authors.initials: A
authors.lastname: Yin
authors.forename: Keyu
authors.initials: K
authors.lastname: Gu
authors.forename: Dongsheng
authors.initials: D
authors.lastname: Tian
authors.forename: Jianbo
authors.initials: J
authors.lastname: Yang
authors.forename: Zhongchao
authors.initials: Z
authors.lastname: Yin
mesh_terms: D000818:Animals; D001413:Bacillus thuringiensis / Q000235:genetics; D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D004269:DNA, Bacterial / Q000235:genetics; D004731:Endotoxins / Q000235:genetics / Q000378:metabolism*; D018506:Gene Expression Regulation, Plant; D006460:Hemolysin Proteins / Q000235:genetics / Q000378:metabolism*; D007915:Lepidoptera; D008969:Molecular Sequence Data; D012275:Oryza / Q000235:genetics* / Q000378:metabolism; D010572:Pest Control, Biological; D030821:Plants, Genetically Modified / Q000235:genetics / Q000378:metabolism; D011401:Promoter Regions, Genetic; D014170:Transformation, Genetic
chemical_list: D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004731:Endotoxins; D006460:Hemolysin Proteins; C036483:T-DNA; C030956:insecticidal crystal protein, Bacillus Thuringiensis