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| Cloning and Function Verification of Glyceraldehyde- 3- phosphate dehydrogenase Gene(GAPDH) Upstream Sequence from Wheat (Triticum aestivum L.) |
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Abstract Glyceraldehyde-3-phosphate dehydrogenase(GAPDH) widely exists in various organisms and it is a key enzyme in process of glycolysis and gluconeogenesis. The genome walking method was applied with primers designed according to the 5'terminal sequence of GAPDH gene (GenBank accession No.: EF592180.1) in order to obtain its upstream sequence from wheat(Triticum aestivum L.), and a 1 071 bp upstream sequence of GAPDH was cloned. Sequence structure analysis and transcriptional regulatory elements prediction showed that the transcription start site was located at about 700 bp upstream of ATG, containing TATA-box, CAAT-box and several cis-acting elements such as MYB, MYC, WRKY, ABRE, HSE and GT-1, which were responsible to abiotic stress like ABA, drought and high salt stress. The CaMV 35S promoter of pBI121 was replaced by the upstream sequence of GAPDH to build a fusion plasmid in purpose to verify the promoter activity of the sequence. The results of Agrobacterium-mediated transient expression showed that the promoter exhibited obvious activity to drive GUS gene expression under ABA, low temperature, and especially under PEG6000. In conclusion, the promoter region of GAPDH gene was cloned by genome walking and the analysis showed that it was a strong inducible promoter.
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Received: 26 February 2014
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