Stable chloroplast transformation of immature scutella and inflorescences in wheat （Triticum aestivum L.）  

作　　者：Cuiju Cui FeiSong Yi Tan Xuan Zhou Wen Zhao Fengyun Ma Yunyi Liu Javeed Hussain Yuesheng Wang Guangxiao Yang Guangyuan He 

机构地区：[1]China-UK HUST-RRes Genetic Engineering and Genomics Joint Laboratory, The Genetic Engineering International Cooperation Base ofMinistry of Science and Technology, the Key Laboratory of Molecular Biophysics of Ministry of Education, College of Life Science andTechnology, Huazhong University of Science & Technology, Wuhan 430074, China

出　　处：《Acta Biochimica et Biophysica Sinica》2011年第4期284-291,共8页生物化学与生物物理学报（英文版）

基　　金：This work was supported by grants from the Genetically Modified New Varieties of Major Projects of China （2008ZX08002-004; 2009ZX08002-013B; 2009DFB30340） and National Natural Science Foundation of China （30871524）.

摘　　要：Chloroplast transformation in wheat was achieved by bombardment of scutella from immature embryos and immature infiorescences, respectively. A wheat chloroplast sitespecific expression vector, pBAGNRK, was constructed by placing an expression cassette containing neomycin phos- photransferase II （nptII） and green fluorescent protein （gfp） as selection and reporter genes, respectively, in the intergenic spacer between atpB and rbcL of wheat chloroplast genome. Integration of gfp gene in the plastome was identified by polymerase chain reaction （PCR） analysis and Southern blotting using gfp gene as a probe. Expression of GFP protein was examined by western blot. Three positive transformants were obtained and the Southern blot of partial fragment of atpB and rbcL （targeting site） probes verified that one of them was homoplasmic. Stable expression of GFP fluorescence was confirmed by confocal microscopy in the leaf tissues from T1 progeny seedlings. PCR analysis of g~ gene also confirmed the inheritance of transgene in the T1 progeny. These results strengthen the feasibility of wheat chloroplast transformation and also give a novel method for the introduction of important agro- nomic traits in wheat through chloroplast transformation.Chloroplast transformation in wheat was achieved by bombardment of scutella from immature embryos and immature infiorescences, respectively. A wheat chloroplast sitespecific expression vector, pBAGNRK, was constructed by placing an expression cassette containing neomycin phos- photransferase II （nptII） and green fluorescent protein （gfp） as selection and reporter genes, respectively, in the intergenic spacer between atpB and rbcL of wheat chloroplast genome. Integration of gfp gene in the plastome was identified by polymerase chain reaction （PCR） analysis and Southern blotting using gfp gene as a probe. Expression of GFP protein was examined by western blot. Three positive transformants were obtained and the Southern blot of partial fragment of atpB and rbcL （targeting site） probes verified that one of them was homoplasmic. Stable expression of GFP fluorescence was confirmed by confocal microscopy in the leaf tissues from T1 progeny seedlings. PCR analysis of g~ gene also confirmed the inheritance of transgene in the T1 progeny. These results strengthen the feasibility of wheat chloroplast transformation and also give a novel method for the introduction of important agro- nomic traits in wheat through chloroplast transformation.

关 键 词：green fluorescent protein （gfp） site-specificintegration WHEAT 

分 类 号：Q943.2[生物学—植物学] Q785