Oleanolic And Ursolic Acid Content Analysis And Molecular Biology Study Of Their Biosynthesis In Persimmon Fruit

Triterpenoids, which include many varities, are widespread in plants. The most common compouds are oleanane and ursane-type pentacyclic triterpenoids. The representative compounds were oleanolic acid (OA) and ursolic acid (UA) and their derivatives. At present, the research of OA and UA in persimmon (Diospyros kaki L.) is mainly focused on leaves and pedicles, while OA and UA content in the peel and flesh of persimmon fruit has not been examined.In this study, persimmon fruits of 32 cultivars (23 astringent and 9 non-astringent) were used as materials, and OA and UA content in these fruits were determined. OA and UA content in typically selected cultivars'Niuxinshi'and'Nishimurawase'at different developmental stages were also analysised. The full-length or fragment of SQS and OSC genes were cloned, and invesgated the expression levels of these genes. Research results are found as follows:1. OA and UA contents in the flesh of 32 different cultivars were determined. Results showed that the contents of OA and UA in the fleshs of different persimmon cultivars were significantly different. The contents of OA and UA were 0~88.57μg.g-1 FW and 0 ~27.64μg.g-1 FW for astringent cultivars, respectively. While the contents of OA and UA were 1.24~26.43μg.g-1 FW and 0 ~12.23μg.g-1 FW for non-astringent cultivars, respectively.2. OA and UA contents in different parts of persimmon fruits and at different developmental stages of two representative cultivars'Niuxinshi'and'Nishimurawase'were analyzed. The data showed that the contents of OA and UA varied according to different parts of persimmon fruits measured, pedicle ranking first, next peel and then flesh. Moreover, the contents of OA and UA differed significantly with their growing periods. The contents of OA and UA in flesh and peel were comparatively high in the initial growing period, while that of pedicle in mature period were higher than other periods. If comparisons were made between astringent and non-astringent persimmons, the latter's contents of OA and UA were higher than that of the former regardless of the three different parts and various growing periods.3. On the basis of conserved sequence of squalene synthase gene in other plants, a pair of PCR primers were designed to clone the fragment of squalene synthase gene in persimmon fruit with RNA as template by the support of RT-PCR techonology. Then, primers were designed for 3' and 5'-RACE, and finally the 1467 bp long full-length cDNA was spliced. The gene encoded 415 amino acids by sequence analysis. The result of homology research in GenBank showed that amino acids encoded by this sequence and other squalene synthase gene -encoded ones were much homologous, and the registration number was FJ687954.4. On the basis of 2,3-oxidosqualene cyclase gene sequenc in other plants, primers were designed to amplied 3' partial sequence of 2,3-oxidosqualene cyclases gene cDNA in persimmon fruit with RNA as template through 3'-RACE method. This fragment was 522bp long and encoded with 98 amino acids, with an ORF 298 bp long, UTR 224 bp long and a complete PolyA tail.5. The comparative expression researches of SQS and OSC genes in the flesh of persimmon fruit at different developmental stages indicated that SQS and OSC genes expressionswere not significantly correlated with the biosynthesis of OA and UA in persimmons flesh.