Molecular Cloning Bioinformatic And Real-Time Qaulitative PCR Analysis Of Flavone Synthase Ⅱ Gene Of Tea Plant

As rich in flavonoids, tea is recognized as a non-alcoholic healthy beverage. Flavones, a class of flavonoids, have a unique pharmacological efficacy which is superior to other flavonoids on anticancer and antitumor. Flavone synthaseâ…¡gene (FSâ…¡), much envolutionary conservative, is the key enzyme direct catalyzing the biosynthesis of of flavones. The molecular cloing and the expression analysis of FSâ…¡gene in tea plant will provide theoretical supports to genetic improvement, and to study the evolution and classification of tea plant. The main results are as follow:1. The degenerate primers were designed, based on flavone synthaseâ…¡amino acid consensus region and codon preference published in the NCBI. Using RT-PCR technology, a 671bp internal fragment was cloned in tea plant. On the basis of the internal sequence, the FSâ…¡gene full-length cDNA was cloned using RACE techniques. It was 1824bp in length with 1605bp coding region, encoding 534 amino acids with the putative molecular weight of 60.4 kD. The GenBank Accession No. was FJ169499. Alignment of amino acid sequences of tea plant FSâ…¡with other plant CYP93 and CYP75 families showed that tea plant FSâ…¡belongs to cytochrome P450 superfamily. It has three conservative domains in envolution, heme-binding region (LPPSPXXXXP), proline-rich region (AGTDT(T/S)), and catalytic property(PFGXGRRXCPG). FSâ…¡has much closer relationship with CYP93B2 subfamily in the phylogenetic analysis.2. The expression levels of flavone synthaseâ…¡were analyzed in tea germplasms using Real-Time PCR technology. The FSâ…¡gene expressed in mature leaves, spring'two and a bud', seeds, petals, stamen and pistils of Longjing 43. The relative expression levels in mature leaves, spring'two and a bud', and seeds were significantly higher than petals, stamen and pistils. In petals, stamen and pistils, the expression is not significant different.The analysis of the expression levels of FSâ…¡gene in different color young shoots of tea cutivars indicated that the expression of yellow-green shoots in Baiye 1, Baijiguan, Huangjinju and purple-green Luoding Hongya were significantly higher than CK. Lvya Foshou is lower than CK. Therefore, FSâ…¡gene expression level is related with the color of the shoots, Using Longjing 43 as control.The relative expression levels of the FSâ…¡gene in different types and provinces tea germplasm showed the similar trends with the evolution and the spread route. In the different type germplasms, the expression level of FSâ…¡gene in landraces and improved cultivars is higher than the wild. The expression level of FSâ…¡gene in 21 tea germplasms of seven provinces showed an increasing trend from north to south or along the coast line, similar to the spread route of tea plant.