Cloning and prokaryotic expression of sucrose phosphate synthase gene (SofSPSB) in sugarcane

【Objective】Cloning and prokaryotic expression of SofSPSB gene from sugarcane will lay a foundation for further study on the enzymatic characteristic and the regulation mechanism of SPS in sugarcane. 【Method】On the basis of phylogenetic analysis, the partial sequence of SofSPSB was obtained by homologous cloning. Then, the full length cDNA sequence was ob-tained by RACE method. The ORF of SofSPSB was amplified and connected to pETBlue-2 to construct the prokaryotic expres-sion vectors, which was transferred into E.coli BL21(DE3) for expression. 【Result】The cDNA sequences of ZmSPS1 from maize (Zea mays) and OsSPS1 from rice (Oryza sativa) were aligned first, then a pair of primers was designed in conserved sequence region for amplification of partial sequence of SofSPSB. With this primer pair, a 2330 bp fragment was amplified and sequenced. According to this partial sequence of SofSPSB, a 3481 bp full length cDNA sequence was obtained by 5'-RACE and 3'-RACE. The full length cDNA contained a 3225 bp open reading frame (ORF) encoding a protein of 1074 amino acids. The start codon (ATG) lied 56 bp after the transcription start site, and a 201 bp non-coding sequence with a typical polyA tail lied after stop codon (TAA) on the full length cDNA sequence (GenBank accession No. JN584485). The theoretical molecular weight (MW) and isoelectric point (PI) of the protein were 118.96 kD and 6.30, respectively. The homology of nucleotide se-quence between SofSPSB and ZmSPS1,OsSPS1 were 94.7% and 81.3%,respectively. The homology of amino acid sequence were 96.0% and 83.9%, respectively. After prokaryotic expression and purification, the fusion protein with the tag of 6×His was at-tained. 【Conclusion】Full length cDNA sequence of SofSPSB was cloned from sugarcane B family. The prokaryotic expres-sion vector of SofSPSB gene from sugarcane was constructed successfully and could be expressed in BL21(DE3) of E.coli.