Efficient transcription of the larvicidal <i>cry</i>4<i>Ba</i>gene from <i>Bacillus thuringiensis</i>in transgenic chloroplasts of the green algal <i>Chlamydomonas reinhardtii</i>  

作　　者：Thanate Juntadech Kittisak Yokthongwattana Sithichoke Tangphatsornruang Yun-kiam Yap Gerd Katzenmeier Chanan Angsuthanasombat 

机构地区：[1]Department of Biochemistry and Center for Excellence in Protein Structure and Function, Faculty of Science, Mahidol University, Bangkok, Thailand [2]DivisionInstitute of Molecular Biosciences, Mahidol University, Salaya Campus, Nakornpathom, Thailand [3]National Center for Genetic Engineering and Biotechnology, Thailand Science Park, Pathumthani, Thailand

出　　处：《Advances in Bioscience and Biotechnology》2012年第4期362-369,共8页生命科学与技术进展（英文）

摘　　要：Unicellular micro-alga Chlamydomonas reinhardtii has been recognized as a promising host for expressing recombinant proteins albeit its limited utility due to low levels of heterologous protein expression. Here, transcription of the 3.4-kb mosquito-larvicidal cry4Ba gene from Bacillus thuringiensis in transgenic C. reinhardtii chloroplasts under control of the promoter and 5’-untranslated region of photosynthetic psbA gene was accomplished. Inverted repeats in chloroplast genomes of the host strain with deleted endogenous psbA genes were selected as recombination targets. Two transformant lines were obtained by dual-phenotypic screening via exhibition of resistance to spectinomycin and restoration of photosynthetic activity. Stable and site-specific integration of intact cry4Ba and psbA genes into chloroplast genomes found in both transgenic lines implied homoplasmy of organelle populations. Achievement in cotranscription of cry4Ba and psbA transgenes revealed by RT-PCR and Northern blot analyses demonstrates the sufficiency of this system’s transcription machinery, offering the further innovation for insecticidal protein production.Unicellular micro-alga Chlamydomonas reinhardtii has been recognized as a promising host for expressing recombinant proteins albeit its limited utility due to low levels of heterologous protein expression. Here, transcription of the 3.4-kb mosquito-larvicidal cry4Ba gene from Bacillus thuringiensis in transgenic C. reinhardtii chloroplasts under control of the promoter and 5’-untranslated region of photosynthetic psbA gene was accomplished. Inverted repeats in chloroplast genomes of the host strain with deleted endogenous psbA genes were selected as recombination targets. Two transformant lines were obtained by dual-phenotypic screening via exhibition of resistance to spectinomycin and restoration of photosynthetic activity. Stable and site-specific integration of intact cry4Ba and psbA genes into chloroplast genomes found in both transgenic lines implied homoplasmy of organelle populations. Achievement in cotranscription of cry4Ba and psbA transgenes revealed by RT-PCR and Northern blot analyses demonstrates the sufficiency of this system’s transcription machinery, offering the further innovation for insecticidal protein production.

关 键 词：Chlamydomonas REINHARDTII Chloroplast Transformation Inverted Repeats Bt-cry4Ba Transcript PSBA Promoter 

分 类 号：R73[医药卫生—肿瘤]