Cloning And Analysising Of Fusion Expression Of ACO Gene From Lily

In the previous study, the further study was to illuminate the ACO sequence, nucleotides similarity and protein similarity with ACO from other plants, protein expression and polycloned antibody.1.Cloning and analysis of ACO cDNA from Asiatic hybrid'Polyanna'petal.The fuLl length cDNA sequence of ACO was using RACE and specific primier was temporarily designated as LACO. LACO cDNA was 1152 bp in length.Within the full-length cDNA, a clear open reading frame (ORF) was 954 nucleotides,coding 318 amino acids. The sequence was submitted to GenBank under accession number EU296623. The resuLts of homologous analysis in GenBank demonstrated that the sequence had 71%-80% identity on the nucleotide sequence and 70%-87% identity on the deduced amino acid sequence.2.Cloning and analysis of ACO DNA from Asiatic hybrid'Polyanna'petal.Genomic DNA fragment of LACO was also cloned using one pair primers of LACO open reading frame. The LACO DNA was 972bp in length, and no intron.3.construction PGEX-ACO vector, expression of LACO gene in E.coli, purification of LACO fusion protein and antibody preparationRecombinant prokaryotic expression vector was successfuLly constructed and ACO has a complete open reading frame in pGEX-4T-1 after detecting by sequencing. Then the PGEX-ACO construct was transformed into E. coli BL21 for protein induction with 0.4 mM IPTG for 5 h at 20℃, 25℃, 28℃, 30℃, 33℃and 37℃, respectively. Fution protein couLd be expressed at all temperatures. After selected the optimal expression condition, the content of soluable protein increased. We have obtained high purity fusion protein after the soluable protein was purified by the GST affinity resin. After injecting rabbit, the polycloned antibody was obtained.