Transcription of the larvicidal cry4Ba gene from Bacillus thuringinesis in the green algal Chlamydomonas reinhardtii chloroplast

• Main Author: Thanate Juntadech
• Other Authors: Chanan Angsuthanasombat
• Language: English
• Published: Mahidol University. Mahidol University Library and Knowledge Center 2023
• Subjects: Chlamydomonas reinhardtii; Inverted Repeat Sequences; Transformation, Genetic
• Online Access: https://repository.li.mahidol.ac.th/handle/123456789/89524
• Institution: Mahidol University

The well-known single cell green alga Chlamydomonas reinhardtii has been recognized as a potential platform for expression of recombinant proteins due to its potential for large scale production as well as low maintenance costs. In this study, transcription of the 3.4-kb mosquito-larvicidal cry4Ba gene from Bacillus thuringiensis subsp. israelensis (Bti) in transgenic chloroplasts of C. reinhardtii under control of the promoter and 5'-UTR of photosynthetic psbA gene was achieved. Inverted repeats in a chloroplast genome of the host strain with deleted photosynthetic psbA genes were selected as recombination targets. Three transformant lines showing stable and site-specific integration of intact cry4Ba and psbA genes into C. reinhardtii chloroplast genomes were obtained by means of dual-phenotypic screening via exhibition of resistance to spectinomycin and restoration of the photosynthetic activity. Achievement in co-transcription of recombinant cry4Ba and psbA transgenes in all stable lines revealed by RT-PCR and Northern blot analyses demonstrates the sufficiency of this system's transcription machinery, offering further innovation for Bt-insecticidal protein production.