Cloning And Analysis Of 1-aminocyclopropane-1 Carboxylate Synthase Gene And Promoter In Watermelon

The fruit of watermelon is crisp, sweet and juicy. It has a high nutritional value because it contains a wealth of mineral salts and vitamins. The area under cultivation and total production in the fruit out of the world’s top ten.But with the fruit ripening and the extension of storage time, the internal of fruit tissues can produce a large number of ethylene. That affects the color, smelling, tasting and quality of watermelon, and it can even decay. Therefore huge economic losses have been brought to farmers. In 1979, the confirmed of ethylene biosynthetic pathway make people recognizing that the ACC sythase is the rate-limiting enzyme of the ethylene biosynthetic pathway. A large number of scholars showed that fruit ripening is the result of expression of differentiation and regulationed by gene. The objectives of the present study are to cloned gene ang promoter sequence of the three white watermelon by RT-PCR, RACE and genome walking approach. The results are as follows:1. The cDNA fragments of ClACS1 gene were obtained by RT-PCR. Using RACE technique and genome walking approach the whole ClACS1 gene was cloned. The nucleotide sequence of ClACS1 cDNA was 1,881 bp, which contained an open reading frame (ORF) of 1,434 bp encoding a putative polypeptide of 447 amino acids. The molecular mass of the predicted protein was 53.9147 kDa and the isoelectric point was calculated to be 8.90. Its accession in GenBank is JQ710336.2. The bionformatic analysis showed that ClACS1 protein without transmembrane domain, but with signal peptide, which localized in the nucleus and belonged to hydrophobic protein. The putative functions were predicted, e.g. it may participate in transport and binding, energy metabolism, fatty acid metabolism, central intermediary metabolism, cell envelope, biosynthesis of cofactors. The amino acid sequence of ClACS1 protein contained a conserved domain AAT_I.3. Homology comparison and cluster results showed that the ClACS1 gene and Cucumis sativus, Cucumis melo were grouped together.4. Using genome walking approach, the promoter region of ClACS1 has been isolated. The conserved regulatory elements functional on transcriptional regulation, such as TATA box and CAAT box and some important cisacting element were identified before translate start site (CTCATCA). This study has been cloned the sequence of ACC synthase gene and its promoter, and analysised its bioinformatic capabilities. Be to provide a theoretical basis for delaying maturity and senescence of plant by genetic engineering techniques, extending shelf life and storage period, and modifying the storage stability of watermelon.