Clone And Function Identification Of Carotenoid Cleavage Dioxygenase From Vitis Vinifera

In this study, the full length cDNA VvCCD1. VvCCD4a and VvCCD4b was cloned from grape berries (Cabernet Sauvignon). The sequence has been analysed and the expression of VvCCDs has been study in different tissue, years and varieties.Meanwhile, this research studied the relationship between expression of VvCCDs and single nucleotide polymorphism of VvCCD1s and the changes of norisoprenoids concentrations during development of grape berries.These three protein was successfully expressed in saccharomyces cerevisiae which can produce carotenoids by its own. And the protein activity of VvCCDs were studied by analysing the aroma of saccharomyces cerevisiae.The result showed as following:The full length cDNA sequences of VvCCD1, VvCCD4a and VvCCD4b in order is1641bp,1798bp and1767bp, and encode protein with predicted molecular mass of61.43kDa,65.70kDa and65.42kDa respectively. Neighbor-joining tree analysis shows that, VvCCD1belong to CCD1cluster, while both VvCCD4a and VvCCD4b are belong to CCD4cluster.This study confirmed15polymorphism site in nucleotide sequence of VvCCDls from five different grape varieties and7amino acids mutations in amino acids sequence. These mutations may caused difference content of norisoprenoids between grape varieties.The expression of VvCCDs in flower and berry is higher than other tissue. During the development of grape berry in two years, the expression of VvCCDs have few differences, but the trend of expression has little different because different phenological phase. The expression of VvCCDs increase during development of these five grape varieties berries, but it is different from norisoprenoids accumulates trend. This may result from grapes growth characteristics and metabolism regulation in carotenoids pathway.This study identify VvCCDs function in saccharomyces cerevisiae which can produce carotenoids by its own. Result show that:VvCCDs can cleave β-carotenoid and release β-ionone. Between cyclic and acyclic carotenoids VvCCDs tend to identify the former.