Cloning And Characterization Of Alcohol Acyltransferase Encoding Genes From Muskmelon And Transformating Antisense Vector To Tomato

Muskmelon (Cucumis melo L.) is one of the major horticultural crops. Aroma is an important measurement of its quality and one of the important factors to attract consumers and enhance the market competitiveness.Melon aroma is attribute to some volatile substances, such as esters, alcohols, aldehydes, terpenes and volatile phenols. Esters as the characteristic aroma component, a variety of enzymes involved in the regulation of its metabolism but alcohol acyltransferase (AAT) catalyzes the final step in ester biosynthesis. Therefore, it has great significance to change alcohol acyltransferase activity on muskmelon breeding by modern molecular biology methods .In this study, we cloned the AAT full-length cDNAs from muskmelon fruit by RT-PCR and 3', 5' RACE and analyzed its time-specific expressions. We constructed the sense and antisense expression vector of AAT gene and transformed antisense gene to tomato by agrobacterium tumefaciens strain LBA4404. Three transgenic plants were obtained by PCR detection. The main results are as follows:1. The full-length cDNAs of AAT gene from muskmelon fruit were cloned by RT-PCR and 3', 5' RACE. The sequence analysis showed that AAT gene contained 1386 bp and coded 461 amino acid residues. This gene has been registered in GenBank (Accession EU431334 )2. Quantitative real-time RT-PCR analysis reveal that in different developmental stages of fruit,the expression level of AAT gene gradually decreased and then gradually increased before 15 day after pollination(DAP), and the expression level will be highest in mature fruit .3. Full length gene (A 700 bp fragment) of the AAT was removed from pMD18-T simple vector by XbaI and Smaâ… (SalI and BamHI).And the fragment was ligated into the plant expression vector pROK3 (pBI121). We constructed the sense and antisense expression vector of AAT successfully by PCR and digested with restricted enzymes.4. Anti-sense gene was transformed to tomato by agrobacterium tumefaciens strain LBA4404 and three transgenic plants were obtained by PCR . The transformation rate was approximately 27.3 %.