Fagopyrum Tataricum Flavonol Synthase Gene's Cloning And Its Expression Analysis During Florescence

Flavonoids, including flavonols, flavones, flavanones, anthocyanins, chalcones, etc, exist widely in plants. Long being famous for potential pharmacological activities in anti-carcinoma, anti-aging, anti-virus, cardiovascular protection, high blood-lipids prevention, etc, these compounds have become the hot-spots of the current research in plants and medicine. As an excellent resource of both food and medicine, tartary buckwheat (Fagopyrum tataricum) is not only rich in nutrition, but also rich in flavonols, (rutin in specific). As one of the key enzymes involved in the synthesis of flavonols, flavonol synthase (FLS) catalyzes the C3-hydroxylation of flavonoids. This study mainly focuses on the cloning of Fls gene in F. tataricum and its tissue-specific expression during florescence, therefore to further reveal relationship between flavonol synthase and accumulation of flavonoids in F. tataricum. Main results of this study are as follows:1. A pair of degenerate primers was designed based on the conserved regions of reported FLS amino acid sequences to amplify conserved fragment of Fls gene from F. tataricum through RT-PCR. Sequence analysis verified the 532bp fragment to be part of the Fls gene. RACE was then performed to obtain cDNA sequence of Fls gene in full length. Sequence analysis indicated that, ORF of Fls gene is 1008bp in length encoding 335 amino acid residues (Accession Number in Genbank:JF274262). The amino acid identity among FLSs from different species was high (ranging from 68% to 77%). Identity of the C terminal region (202-335 amino acid residues) is higher,86% in specific, while identity of the N terminal (1-113 amino acids) is only 62%.2. Result of homologous modeling using SWISS-MODEL indicated that, FLS protein of F. tataricum contained eight amino acid residues, Gly44, His81, Pro 195, His232, Asp234, Gly248, His288 and Arg289, which were all strictly conserved in dioxygenases, participating in protein folding and catalytic sites'formation. These amino acid residues were distributed in three highly conserved regions:GLFQVVNHG, NYYPPCPRPDLALGVVAHTDMS, and GDQLEIMSNGKYKAVLHRTTVNKEKVRMS WPVFLEPP.3. Using the housekeeping gene Histone 3 (H3) from F. tataricum as reference gene, semi-quantitative RT-PCR was performed to detect the expression of Fls gene in stems, leaves and flowers during florescence. Result showed that Fls gene expressed in all these organs, but with different values. The highest expression was detected in leaves, while the least in stems. Fls gene in leaves showed the highest expression value during the early stage of florescence, and then gradually reduced. The highest expression of Fls gene in flowers was detected druing flowering stage. In stems, no significant change was found during the whole florescence process.