4.11. Functional Analysis of CoDXS and CoDXR Genes

In order to validate the accuracy of the sequences obtained from the hybrid platform, two full-length transcripts (F01.PB17587 and F01.PB29801) encoding C. obtusifolia 1-deoxy-d-xylulose 5-phosphate synthase (CoDXS) and 1-deoxy-d-xylulose 5-phosphate reductoisomerase (CoDXR) were directly chosen for functional analysis, respectively, using bacterial complementation assay [107]. In higher plant, DXS and DXR played an important role in the MEP pathway and were involved in the biosynthesis of anthraquinone [108]. The plasmids pAC-BETA and pTrc-AtIPI were purchased from ADDGENE Corporation (USA). The pAC-BETA contained all functional genes of the β-carotene synthesis, and also retained a chloramphenicol (Chl) resistance gene. Cells of E. coli containing pAC-BETA produced and accumulated β-carotene, resulting in yellow colonies. The pTrc-AtIPI contained a Trc promoter, ampicillin resistance gene and AtIPI gene.

The ORF of CoDXS was amplified by PCR with forward primer (5′-A TAA GAA TGC GGC CGC ATG GCT CTT TGC ACA TTC TC-3′, the Not1 site is underlined and the initiation codon is shown in italics) and reverse primer (5′-CGC GGA TCC TTA TGA CAA AAC CTC TAA TGC CTC-3′, the BamH1 site is underlined and the stop codon is shown in italics). The coding region of CoDXR gene was amplified by PCR using the forward primer (5′-CG GAA TTC ATG GCT CTG AAT TTG 3′, the EcoR1 site is underlined and the initiation codon is shown in italics) and the reverse primer (5′-GA AGA TCT TCA TGC AGG AAT AGG A-3, the BglII site is underlined and the stop codon is shown in italics). The fragments were cloned into a pMD19-T vector for sequencing to confirm that no substitutions or deletions occurred. After digestion of PCR fragments and plasmid pTrc-AtIPI with corresponding endonucleases, the recombinant plasmid pTrc-CoDXS and pTrc-CoDXR were generated and subsequently verified by sequencing, respectively.

The pTrc-AtIPI digested by PstI to remove AtIPI and then ligated by T4 DNA ligase was used as control. The plasmids pTrc-CoDXS and pAC-BETA, as well as the plasmids pTrc-CoDXR and pAC-BETA, were cotransformed into E. coli Top10, respectively. The plasmids pTrc and pAC-BETA were also cotransformed and the single plasmid pAC-BETA, which was transformed into the E. coli Top10 to be used as the negative controls. Transformants were cultured on solid LB medium containing ampicillin (150 mg/L) and Chl (50 mg/L) at 37 °C in dark for 2 days.