English (United Kingdom)

https://curated-unify.zendy.io/wp-json/zendy-region/v1/featured_content/oa?rat=en

https://curated-unify.zendy.io/wp-json/zendy-region/v1/highlighted_journal/

Global: Zendy Plus annual

Presents the access of premium content as premium feature

Premium Content

Presents the keyphrase highlighting as premium feature

Keyphrase Highlighting

Presents the summarisation as premium feature

Summarisation

Insights

Presents the pdf analysis as premium feature

PDF Analysis

Presents the zaia usage as premium feature

ZAIA

Global: Zendy Tools annual

Global: Zendy Free Plan

Global: Zendy Tools monthly

Global: Zendy Plus monthly

Background   Chlamydomonas reinhardtii  is a unicellular green alga, which is a most commonly used model organism for basic research and biotechnological applications. Generation of transgenic strains, which usually requires selectable markers, is instrumental in such studies/applications. Compared to other organisms, the number of selectable markers is limited in this organism. Nourseothricin (NTC) N-acetyl transferase (NAT) has been reported as a selectable marker in a variety of organisms but not including  C. reinhardtii . Thus, we investigated whether  NAT  was useful and effective for selection of transgenic strains in  C. reinhardtii . The successful use of  NAT  would provide alterative choice for selectable markers in this organism and likely in other microalgae.    Results   C. reinhardtii  was sensitive to NTC at concentrations as low as 5 µg/ml. There was no cross-resistance to nourseothricin in strains that had been transformed with hygromycin B and/or paromomycin resistance genes. A codon-optimized  NAT  from  Streptomyces noursei  was synthesized and assembled into different expression vectors followed by transformation into  Chlamydomonas . Around 500 transformants could be obtained by using 50 ng DNA on selection with 10 µg/ml NTC. The transformants exhibited normal growth rate and were stable at least for 10 months on conditions even without selection. We successfully tested that  NAT  could be used as a selectable marker for ectopic expression of  IFT54- HA in strains with paromomycin and hygromycin B resistance markers. We further showed that the selection rate for  IFT54- HA positive clones was greatly increased by fusing  IFT54- HA to  NAT  and processing with the FMDV 2A peptide.    Conclusions  This work represents the first demonstration of stable expression of  NAT  in the nuclear genome of  C. reinhardtii  and provides evidence that  NAT  can be used as an effective selectable marker for transgenic strains. It provides alterative choice for selectable markers in  C. reinhardtii .  NAT  is compatible with paromomycin and hygromycin B resistance genes, which allows for multiple selections.

Nourseothricin N-acetyl transferase (NAT), a new selectable marker for nuclear gene expression in Chlamydomonas