Inhibition of pds gene expression via the RNA interference approach in Dunaliella salina (Chlorophyta).
To investigate the potential of double-stranded RNA interferencing with gene expression in Dunaliella salina, a plasmid pBIRNAI-Dsa was constructed to express hairpin RNA (hpRNA) containing sequences homologous to phytoene desaturase gene (pds), a key gene in carotenoid biosynthesis, and transformed into D. salina by electroporation. The relative transcription level of pds in pBIRNAI-Dsa-treated cells to nontreated cells was quantitated and the gene silencing efficiency by RNAi was evaluated via real-time polymerase chain reaction (PCR). The transcriptions of pds of the pBIRNAI-Dsa-treated group changed compared to those of the control group, and the 2(-delta deltaC)(T) was lowest on the 7th day, corresponding to 0.281265-fold of the relative pds control transcript; a relatively strong gene inhibition effect was therefore deduced. The transcript of pds may be modulated in a wide range, and a reduced transcription even to 28% of the normal level may be tolerated for its survival. This study shows that dsRNA-mediated genetic interference can induce sequence-specific inhibition of gene expression and pBIRNAI-Dsa can be used for transient suppression of gene expression in D. salina. The aim of this study was to exploit dsRNA-mediated gene silencing and to provide a foundation for gene function research in D. salina.
Sun
Guohua
G
Laboratory of Marine Genetics and Breeding, College of Marine Life Sciences, Ocean University of China, Qingdao, 266003, People's Republic of China.
Zhang
Xuecheng
X
Sui
Zhenghong
Z
Mao
Yunxiang
Y
D001483:Base Sequence; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics; D000460:Chlorophyta / Q000235:genetics*; D015971:Gene Expression Regulation, Enzymologic; D008969:Molecular Sequence Data; D010088:Oxidoreductases / Q000235:genetics*; D010957:Plasmids / Q000235:genetics; D034622:RNA Interference; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013997:Time Factors
D010088:Oxidoreductases; C022398:phytoene dehydrogenase; D015500:Chloramphenicol O-Acetyltransferase