Expression Characterization Of The MdAAT2 Gene And Its Regulation Mechanism Of Volatile Ester Biosynthesis In Apple(cv.Golden Delicious)

Apple (Malus domestica Borkh.) is the most important processing and table fruit all over the world. Aroma compounds are among the factors that determine fruit quality and influence on the final consumer acceptance of the commodity. Volatile esters are important aromatic components of apple fruit. In order to investigate the mechanism of ester biosynthesis and regulation, we isolated the gene (MdAAT2) of apple alcohol acyltransferase, which is the key enzyme that catalyzes the last step in ester formation by linkage of an acetyl moiety from acetyl CoA to appropriate alcohols. Results are as follows:1. Extraction and analysis of apple volatile estersThree types of SPME fibers were used and compared in esters extraction from apple fruit. The result showed that the PDMS/DVB 65μm Bonded SPME fiber is the best suitable fiber for esters extraction, and the optimal extraction is at 50℃for 30 minutes. SPME-GCMS analysis showed that the butyl acetate, hexyl acetate, hexyl 2-methylbutyrate, and hexyl butanoate are the most important characteristic ester compounds in ripened Golden Delicious apple fruit.2. Isolation and characterization of MdAAT2 geneAccording to the homologous sequences from other plants, degenerate primers were designed to amplify specific DNA fragment using cDNA prepared from apple fruit. By RT-PCR and RACE-PCR, we isolated two AAT genes from Jonagold and Golden Delicious apple fruit named MdAAT1 and MdAAT2, with the accession numbers AY512893 and AY517491, respectively. The full-length cDNA of MdAAT1 and MdAAT2 are 1,639bp and 1,628bp, respectively, and the deduced amino acid sequence of the two genes showed high identities, which is 94.26%. After analyzed by software, most of the deduced protein sequences of MdAAT2 are hydrophilic regions, which contain two speculated transmembrane(TM) segments at 148aa and 174aa, respectively. Sequence analysis shows that MdAAT2 exhibits the features of BAHD superfamily and other plant alcohol acyltransferases, including HXXXD and FGWG motifs. Further, phylogenetic analysis of various alcohol acyltransferases indicated that the apple AAT share one cluster with pear AAT, while have more distant with that of lemon, strawberry, and rose. Thus, it is most likely that we have isolated the apple alcohol acyltransferase gene.3. Expression of the MdAAT2 gene in E.coliAfter cloning the encoding region of MdAAT2 into prokaryotic expression vector pET-32a-c(+), we obtained the partial soluble fusion proteins in Origami B(DE3) strain.