The full-length coding region of NobZIP1 (GenBank: MH118545) and UGDH (GenBank: KU745463) were PCR-amplified from genomic DNA of N. oceanica with primers Na07F and Na08R and UG-OEF and UG-OER, respectively. The amplified PCR products were cloned separately into vector pNa03 downstream of Hsp20 promoter by using the ClonExpress II One Step Cloning Kit (Vazyme, China). FLAG tag was fused at the C terminus of the target genes for the detection of protein expression, which yielded recombinant overexpression vectors pNa03-NobZIP1 and pNa03-UGDH that were electroporated into microalgae using a Gene Pulser Xcell electroporation system (Bio-Rad) as previously described (8). RNAi vector was constructed as described previously (39). Briefly, primer sets were designed to amplify the sense and antisense fragments from the full-length target genes, and subsequently, these fragments were cloned into the pNa03 vector in sense and antisense orientations, respectively, to silence the target gene by using the ClonExpress MultiS One Step Cloning Kit (Vazyme, China) following the supplier’s instruction. The primer sequences for silencing NobZIP1 are as follows: siNobZIPF1 and siNobZIPR1 primers were used to amplify NobZIP1 gene sequence from 2597 to 3017 bp and the primers siNobZIPF2 and siNobZIPR2 were used to amplify the inverted repeats of NobZIP1 from 2800 to 2597 bp, respectively. The primer sequences for silencing UGDH are as follows: siUGDHF1 and siUGDHR1 primers were used to amplify the UGDH sequence from 163 to 662 bp, and the primers siUGDHF2 and siUGDHR2 were used to amplify the inverted repeats of UGDH from 386 to 163 bp, respectively. The primer sequences for silencing CPS are as follows: siCPSF1 and siCPSR1 primers were used to amplify the CPS sequence from 1 to 240 bp, and the primers siCPSF2 and siCPSR2 were used to amplify the inverted repeats of CPS from 240 to 1 bp, respectively. As the CPS gene lacks the intron, we used the first intron of NobZIP1 to generate hairpin loop formation as described (39). Thereafter, these fragments were cloned into the vector pNa03 separately in sense and antisense orientations to silence the respective genes, and the resultant recombinant vectors pNa03-siNobZIP1, pNa03-siUGDH, and pNa03-siCPS were electroporated into microalgae cells using a Gene Pulser Xcell electroporation system (Bio-Rad) as described previously (8).

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