Production of marker-free transgenic rice expressing tissue-specific Bt gene.
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.
Qiu
Chengxiang
C
Temasek Life Sciences Laboratory, National University of Singapore, Singapore. qiucx@tll.org.sg
Sangha
Jatinder Singh
JS
Song
Fengshun
F
Zhou
Zhiyun
Z
Yin
Ao
A
Gu
Keyu
K
Tian
Dongsheng
D
Yang
Jianbo
J
Yin
Zhongchao
Z
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
D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004731:Endotoxins; D006460:Hemolysin Proteins; C036483:T-DNA; C030956:insecticidal crystal protein, Bacillus Thuringiensis