| Longan (Dimocarpus longan Lour.) is a kind of famous subtropics fruit originedfrom south of China. It is also an important fruit in Fujian province. According toliteratures, longan has abundant flavonoids in fruits and leaves, and it is considered asa medical resource. Studies on flavonoid in longan mainly focused on componentsand contents in fruits. The mechanism of flavonoid biosynthesis in longan was still inpuzzled. To confirmed key genes in flavonoid biosysthesis pathway in longan leaves,rutin and quercetin contents in4cultivar varieties were determined, some relatedgenes such as Dlpal, Dlf3h and Dlfls cDNA were cloned, the promoter sequences ofDlpal, Dlf3h, Dlfls, CHI and CHS were also obtained, and these genes' expressionlevels in longan leaves were studied in this article. The mainly results as follows:1) Rutin and quercetin contents were measured in4cultivar varieties'Songfengben','Shixia','Lidongben' and 'Wulongling' by HPLC. The resultsshowed that there was significant difference in rutin contents among4varieties. Therutin content in 'Wulongling' is highest in4varieties with439.8μg/g. The quercetincontent in 'Songfengben' is lowest in4varieties with69.6μg/g. There was nosignificant difference between 'Shixia' and 'Lidongben' with the content of131.8μg/gand129.1μg/g respectively. There was no quercetin detected in 'Wulongling'.2) Full length cDNA of Dlpal and Dlf3h were cloned by RACE technology, andpartial cDNA of FLS was also cloned using cDNA of longan leaves. Results ofbioinformatical analysis showed that the three sequences were highly homologouswith PAL, F3H and FLS respectively. The prediction results of Dlpal and Dlf3hcoding sequences showed that there were the conserved domains of PAL and F3H. Itwas inferred that Dlpal, Dlf3h and Dlfls were structural genes in longan leaves, andinvolved the processing of flavonoids biosynthsis.The molecular weight of Dlpal coded by Dlpal cDNA is306.4KD, the protein ishydrophilici and belonged to aromatic amino acid ammonia lyase containing PAL(PLN02457) and PAL-HAL conserved domain, and was similar with PAL and HAL.Transmembrane structure of Dlpal is outside-in. The Dlpal is synthesized onEndoplasmic reticulum and catalyzed phenylpropanoids. The molecular weight of Dlf3h coded by Dlf3h cDNA is45.3KD. The protein ishydrophilici and located on endoplasmic reticulum. Transmembrane structure ofDlf3h is outside-in and inside-out.3) The upstream sequences of CHI,CHS,PAL were cloned by TAIL-PCR kit,and the upstream sequences of F3H was cloned by I-PCR. The sequences CHI-P,CHS-P, PAL-P and F3H-P contained the promoter sequences of CHI, CHS, PAL andF3H each other, and were analyzed by PlantCare. There are light responsed elementsin all5sequences, such as SP1, Box4, L-box and so on. The results showed that thereare complex light responsed expression models in those genes involved in flavonoidssynthesis, and the expression of PAL, CHI, CHS and F3H are light dependent.Except for the light responsed elements, there are some element related to planthormone. The results showed that there are abscisic acid responsed element inpromoter sequences of F3H, CHS and CHI, according to that, it was concluded thatF3H, CHS and CHI are coordinated expression and regulated by endogenoushormones. Besides. In addition, CHI-P contains gibberellin responsed elementsTCCACCT-motif, CHS-P and PAL-P contain MeJA responsed elementsCGTCA-motif, CHS-P contained salicylic acid responsed elements TCA-element.4) There were different expression model of Dlpal,CHI,CHS,Dlf3h,Dlfls in4cultivar varieties. Considering the results in chapter2, it was concluded that thosegenes'transcript level were positively related to rutin and quercetin contents.Therefore, the genes Dlpal, CHI, CHS, Dlf3h and Dlfls played key roles in flavonoidssysthesis.
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