4.3. Plasmids for Protein Expression in Hi5 Insect Cells

The plasmids used for expression of GFP, HaCAD-GFP (GenBank: {"type":"entrez-nucleotide","attrs":{"text":"AF519180","term_id":"1126528267","term_text":"AF519180"}}AF519180) and HaABCC2-GFP (GenBank: {"type":"entrez-nucleotide","attrs":{"text":"KF479231","term_id":"594008765","term_text":"KF479231"}}KF479231) were previously constructed in our laboratory [36,37,38]. The plasmids used for expression of SlCAD-GFP (GenBank: {"type":"entrez-nucleotide","attrs":{"text":"JN687590","term_id":"386277194","term_text":"JN687590"}}JN687590) and HevCAD-GFP (GenBank: {"type":"entrez-nucleotide","attrs":{"text":"AF367362","term_id":"15149239","term_text":"AF367362"}}AF367362) were constructed using the specific primers through the gene fusion method. It was briefly introduced as follow. The inserted fragments and the vector (pie2-HaCAD-GFP or pie2-SlCAD-GFP) fragments were amplified by PCR using the different templates, and purified using the Gel Extraction Kit (Bio-tek, Winooski, VT, USA), respectively. The purified inserted fragments were mixed with the purified vector fragments and transformed into E. coli DH5α. The homologous recombination occurred between the two fragments in the bacterium and the positive clones were identified by sequencing. The various deletions in HaCAD-GFP gene were constructed using the overlap PCR method and inserted into the plasmid pie2-EGFP-N/pGFP as previously described [36,38]. It was briefly described as follow. The up-fragment and the down-fragment of target gene were amplified by PCR using the corresponding plasmids containing the target fragments as templates, respectively. The products of PCR were run on agarose gels and the up-fragment and the down-fragments were cut from gel and purified using the Gel Extraction kit (Bio-tek, Winooski, VT, USA), respectively. The full-length gene was amplified by PCR using the mixture of the up-fragment with the down-fragment as template and the specific gene primers. The full-length PCR product was purified and digested with corresponding restriction endonuclease. Finally the digested fragments were cloned into expression plasmid pie2-EGFP-N1. The plasmids for expression of the different hybrid CAD proteins between HaCAD-GFP and SlCAD-GFP were constructed using gene-fusion through recombinase method as described above [39,40]. All primers were listed in Supplemental Tables S6–S13. For plasmid purification, plasmid DNA mini Kit was from Omega Bio-teck, Inc (Winooski, VT, USA) was used.