The Cloning Of PsbA Promoter From Chlamydomonas Reinhardtii Chloroplast And Its Activity Detection In E.Coli

Chlamydomonas is used as a model green alga in many studies. Because it's genome sequence has been sequenced, and the genetic mechanism is clear; it is a eukaryotic unicellular green algae; it is not only a autotrophic organism but also a heterotrophic organism; it contains a big cup chloroplast that can be easily transformed; it has short growth cycle and high photosynthetic efficiency, it is called"green yeaet"and usually is used as host cells to express foreign gene. The Chlamydomonas is the only organism that has been transformed successfully in nucleus, chloroplast and chondriosome. But the poor foreign gene expression level in chloroplast is a bottleneck that limits its using as bioreactor. The one of main factor affecting the foreign gene expression is promoter. The strong promoter can increase the transcription frequency and increase foreign genes expression level. The Chlamydomonas chloroplast endogenic photosystem II protein D1 promoter is usually considered as a strong promoter. In this research, the promoter above mentioned is cloned, and we hope that the promoter may be promote the foreign genes expesson in Chlamydomonas chloroplast.The contents of this study is as following:Firstly, the chlamydomonas total genome is extracted, and the PsbA primer is designed. With total DNA as templates, the PsbA promoter is cloned using the method of PCR.Secondly, the plasmid p64D-PsbA-aadA is constructed. The PsbA promoter is ligated to the spectinomycin resistance gene (aadA) upstream. The construction is transformed into E.coli DH5α. After screening on ampicillin and spectinomycin plate, the positive clone is obtained. Which proves that the PsbA promoter has the promoter activity.Finally, the plasmid psk-PsbA-gfp is constructed. The green fluorescent protein gene (gfp) is ligated to PsbA promoter downstream. After transforming E.coli DH5αand screening, the positive clone is obtained. Using the fluorescent microscope, the green fluorescence is observed in E.coli. This proves that PsbA promoter can regulate the green fluorescent protein gene expression in E.coli. The results of this research lay the foundation for further chlamydomonas chloroplast engineering study.