Tea ¦Â-glucosidase Gene Cdna Cloning And Sequence Analysis

Tea [Camellia sinensis(L.) O.kutze] is one of the most important economic plants in China, Aroma is one of the keys index of weighing the quality of tea. p-Glucosidase(β-Glc) take a very important function to tea aroma and stress. So we tried to get the cDNA of β-Glc from tea plant. This contributes to make clearly position and function of β-Glc in forming course of tea aroma, substantiate the theory that the aroma of tea forms and also establish the foundation that utilize genetic engineering techniques to improve the cultivar of tea plant, quality of tea and insect-resistance and disease-resistance afterwards. In the paper, the complete cDNA squence of β-Glc of tea was cloned in the tea leaf at the first time. The results are reported as follows:Total RNA from young leaves of tea was extracted by chomczynsk's single step method of RNA isolation, which was partially modified on the basis of the features of biochemistry composition in fresh tea leaves.Two degenerate primers were designed and synthesized according to the highly conservative sequences among the known β-Glc genes. A cDNA fragment of 208bp was amplified by RT-PCR, which was subsequently cloned into PMD18-T Vector for sequencing analysis. The result of sequencing analysis showed that the nucleotide and deduced amino acids sequences had high identity with the corresponding parts of β-Glc cDNAs of other published plants. The results indicate that the special PCR product is a cDNA fragment of β-Glc of tea plant.Two special primers of RACE were designed based on the above sequence of the RT-PCR product. Two fragments of 5'cDNA(895bp) and 3'cDNA (581 bp) were amplified by RACE reaction. Two complete squence primers were designed based on the result of RACE. A 1475bp sequence was amplified by PCR. The analysis of the product shows that the nucleotide and deduced amino acid sequences share 40%-60% homologous to the corresponding parts of β-Glc gene family of Pinus contorta, Cucurbita pepo, Arabidopsis thaliana by the Blast_W program comparison.The nucleotide sequence and the primary and secondary structure of the protein were determined and predicted by using molecular biology software. The β-Glc squence of tea plant consists of 1475 bp, including 189bp 3'UTR,233bp 5'UTR and 1050bp open reading frame. The deduced amino acids(AA) sequence is a 350AA β-Glc precursor which consists of an 82AA transit peptide and a 257AA mature protein.Predicted by the method of Gamier, the secondary structure of β-Glc in tea plant which may contain helical conformation 14.33%, coil conformation 25.43%, extended conformation 33.81%. The molecular weight of the precursor protein is 40kDa and isoelectric point is 5.2. β-Glc should be water-soluble protein.