Efficient transcription of the larvicidal cry4Ba gene from Bacillus thuringiensis in transgenic chloroplasts of the green algal Chlamydomonas reinhardtii

• Main Authors: Thanate Juntadech, Kittisak Yokthongwattana, Sithichoke Tangphatsornruang, Yun-kiam Yap, Gerd Katzenmeier, Chanan Angsuthanasombat
• Other Authors: Mahidol University. DivisionInstitute of Molecular Biosciences
• Format: Article
• Language: English
• Published: 2015
• Subjects: Chlamydomonas reinhardtii; Chloroplast Transformation; Inverted Repeats; Bt-cry4Ba Transcript; psbA Promoter; Open Access article
• Online Access: https://repository.li.mahidol.ac.th/handle/123456789/1804
• Institution: Mahidol University

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.