Genetic transformation of the freshwater diatom Cyclotella meneghiniana via bacterial conjugation

Abstract

Diatoms, the most species-rich algae, produce the main primary productivity of marine and freshwater ecosystems. Though, genetic transformation has been established in a variety of marine diatoms, genetic modification of freshwater diatoms is still difficult to achieve. Centric diatom Cyclotella is a major genus of freshwater diatoms, and C. meneghiniana is the most well-known and intensively studied species in this genus. In this study, episomal plasmids for C. meneghiniana were constructed, and endogenous promoters of fucoxanthin chlorophyll a/c-binding protein 3 gene (Fcp3) or ribosomal protein L14 gene (RL14) were used to drive the expression of blasticidin-S deaminase gene (bsr), enhanced green fluorescent protein gene (eGFP) and β-glucuronidase gene (GUS). The plasmids were introduced into algal cells by bacterial conjugation, and transformants were obtained by screening on solid plates containing 0.2 μg mL⁻¹ blasticidin-S with the transformation efficiency of 9–58 transformants per 10⁶ cells. PCR analysis verified the transfer of the plasmid sequences in the cells, and the fluorescence detection and staining analysis demonstrated that eGFP and GUS proteins were expressed in the cytoplasm, indicating the successful and stable expression of exogenous genes in C. meneghiniana through bacterial conjugation.