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| Cloning and Sequence Analysis of Sucrose Phosphate Synthase Genomic DNA and 5'-Flanking Sequence from Sugarcane(Saccharum spp.) |
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Abstract Sugarcane (Saccharum spp.) is a commercial important sugar crop in South China. In order to achieve the breeding aim of increasing sugar yield, it is essential to focus on the principal rate limiting steps in sugarcane sucrose accumulation processes. Sucrose phosphate synthase (SPS) is considered a key rate-limiting enzyme in the pathway of sucrose synthetization and carbon partitioning processes. In this study, two different genomic DNA framents(GenBank accession No. EU278617 and EU278618) which encoding Sugarcane SPSⅢ protein were cloned by long?distance?PCR(LD-PCR). DNA Exon-intron?structure analysis suggested that both sequences contained twelve introns, thirteen exons, and the open reading frame encoded a protein of 964 amino acids. Subsequently, the 5'-flanking sequence (GenBank accession No. KC422670) of SPSⅢ was aslo isolated from sugarcane genome DNA using Adaptor-ligation PCR method. This novel sequence contained the important DNA putative cis-acting elements as well as TATA box. To identify promoter activtity of SPSⅢ, we generated five different vectors which contained different length of the 5'-flanking fragment fused to the the β-glucuronidase (GUS) reporter gene . Transient expression experimen was carried out by the method of particle bombardment. Histochemical activity of b-glucuronidase was observed in callus. The gene expression results confirmed its promoter activtity. This stuty will provide theory basis for further study on the gene structure and biological function of SPSⅢ via gene clone and promoter analysis.
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Received: 14 May 2013
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