Highlights

Agar concentrations for the cultivation of Chaetoceros muelleri were determined.

Appropriate antibiotics were determined for selection of Chaetoceros muelleri.

Genetic transformation in C. muelleri was established by electroporation.

GFP and GUS were efficiently expressed under the control of endogenous promoter.

Abstract

Cosmopolitan Chaetoceros is one of the largest genera of marine diatoms, which serves as a major carbon contributor in waters and is also frequently used in aquaculture. In this study, a simple, rapid and effective genetic transformation system of Chaetoceros muelleri, a suitable feed microalga, has been established by electroporation, and transformants could be observed in 4–7 days. C. muelleri demonstrated sensitivity to nourseothricin, zeocin and blasticidin-S based on the inhibitory effects of 10 antibiotics on its growth. Three corresponding resistance genes, nourseothricin acetyltransferase (nat), bleomycin-resistance (ble) and blasticidin-S deaminase (bsr) driven respectively by C. muelleri promoters of fucoxanthin chlorophyll a/c binding protein (Lhcf4p) and acetyl-CoA acetyltransferase (ACATp), were successfully and stably expressed. The transformation was verified by southern blot and the expression of enhanced green fluorescent protein (eGFP) and β-glucuronidase (GUS), two exogenous proteins. Varied transformation efficiencies were observed using the three resistance genes, which were 2900 (bsr), 1700 (ble) and 25 (nat) transformants per 108 cells. In addition, the optimum voltage intensity for the electroporation was 500 V with the presence of salmon sperm DNA (ssDNA) and 700 V with the absence of ssDNA.

Keywords

Chaetoceros muelleri

Diatom

Genetic transformation

Blasticidin-S

Zeocin

Nourseothricin

Electroporation

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