Cloning of Sucrose Phosphate Synthase Gene (SofSPSA) from Sugarcane and Construction of Its Sense and Antisense Plant Expression Vector
Sucrose phosphate synthase (hereinafter as SPS) is the key enzyme that controls the sucrose biosynthesis in plants. By searching NCBI GenBank, 77 SPS genes identified to date from plants were obtained, in which 43 were in full length sequences. By phylogenetic analysis of the 43 full length sequences, the SPS in plants were classed into four families, named A, B, C and D family, respectively, and there were several SPS genes in the same plant belonging to different families. By alignment of the four genes, SofSPSII from sugarcane (Saccharum offici-narum), OsSPS4 from rice (Oryza sativa), LpSPS from ryegrass (Lolium perenne) and BoSPS from bamboo(Bam-busa oldhamii) had very close phylogenetic relation. A pair of primers were designed in conserved sequence region to amplify partial sequence of SofSPSA. With the primers, a 2 553 bp fragment was amplified and sequenced. According to this partial sequence of SofSPSA, a 3 906 bp full length cDNA sequence was obtained by 5'-RACE and 3'-RACE. The full length cDNA contained a 3 180 bp open reading frame (ORF) encoding a protein of 1 060 amino acids. The theoretical molecular weight and isoelectric point (PI) of the protein were 118.4 kD and 6.09, respectively. The start codon (ATG) lies 359 bp after the transcription start site, and a 364 bp non-coding sequence with a typical polyA tail lies after stop codon (TAA) on the full length cDNA sequence (GenBank accession No. HM854011). The amino acid sequense of SojSPSA showed 99.3%, 91.8%, 91.6% and 91.9% similarity, and 99.2%, 87.9%, 87.5% and 87.8% identify to that of SoJSPSlI, OsSPS4, LpSPS and BoSPS, respectively. Finally, sense and antisense SofSPSA gene ORFs were amplified and connected to pBI121 to construct the plant expression vectors, pBI-SofSPSA and pBI-anti-SofSPS4, respectively. This work will lay a foundation for further study on the biological function of SPS from sugarcane and variety impovement by transfer of sugarcane SPS gene.
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维普
