Clone, Expression And Localization Of β-glucosidase Gene In The Tea [Camellia Sinensis (L.)O.kutze]

Various matter metabolism,energy transmission,message transferation and growth of tea plant are all enzymatic processes.Enzymes are extremely important to the tea quality.studies of enzyme discipline are known to be the major tea theory. P -glucosidase has important effects on the alcoholic aroma precursors, tea aroma are the important factors in determining the tea quality.P -glucosidase belongs to hydrolytic enzyme.It can hydrolyze p-nitrophenyl β-D-galactopyranoside specially.lt can also hydrolyze cellubiose and salicin.P -glucosidase is an important enzyme which is responsible for the liberation of aroma constituents from their glycosidically bound precursors. Monoterpene alcohols (linalool, geranial etc) and aromatic alcohol (benzyl alcohol, 2-phenylethanol etc) are known to be the major flower tea aroma constituents. Flower aroma is mainly produced during the processing of black tea,Oolong tea and high-grade green tea by the action of endogenous glucosidase. Takeo T (1981) studied the phenomina for the first time. Recent studies on P -glucosidase have deepened to its relationship with tea cultivars, zoological environment and processing techniques. The molecular clone and expression of P -glucosidase gene in the oat, sorghum, nasturtium, Madagascar periwinkle and Dalbergia cochinchinensis have been reported, but there are no reports in tea plant.This paper studied on cDNA clone, prokaryotic expression, mRNA expression and localization of P -glucosidase gene in the tea leaves. The results are as follows:cDNA of P -glucosidase in the tea[Camellia sinensis(L.)O.kutze] was cloned. Total RNA from young leaves of tea was extracted by JIANG Chang-jun's method of RNAisolation, which was partially modified on the basis of the features of biochemical composition in fresh tea leaves, we used the RACE(rapid amplification of complementary DNA ends) technique to degenerate gene-specific primers based on the amino-terminal sequence of P -glucosidase. The 5' untranslated sequence and 3' untranslated sequence of the cDNA fragment were isolated by 573' RACE . The isolated cDNA, termed P -Glc (GenBank accession no.AF537127), consists of 1475 base pairs and encodes a protein of 450 amino acids which includes a signal peptide of 28 amino acids. The P -Glc sequence includes 92bp 3'UTR, 33bp 5'UTR and 1350bp open reading frame. The secondary structure mainly contains 14.33% a -helical conformation, 25.43% P -sheet conformation. According to the putative amino acid sequence, there are 4 function domains of N-glycosylation sites(NSTK202^ NFTK20(\ NRTQ238 NRSS307), 5 protein kinase C phosphorylation sites (SNRI85 STK202TSK24\ TLR27(\ STR371), 2 casein kinase II phosphorylation sites (SIVE1 TLNE350) and 5 N-myristoylation (GGHASK67, GQASGS28Is GIRCCR288, GGLCAA317, GQKGSG331 ) . The nucleotide sequence shares 40-60% similarity to corresponding parts of P -glucosidase gene of other plants through the Blast_W program comparison.P -glucosidase had been expressed in pET-32a expressional system. The P -glucosidase gene was cloned into pET-32a vector, and the recombinant plasmid was transformed and expressed high-efficiently in E.coli BL21(DE3), and the molecular weight of target protein was 37KDa. The results of in vitro enzymatic reaction showed that the expressional products had normal bioactivity. pET-32a/ P -Glc induced for Oh was 0%. pET-32a/ P -Glc induced for 0.5h was a little weaker. pET-32a/ P -Glc induced for llu 2h was beneficial to the increasing of fusion protein.pET-32a/ P -Glc induced for 3h was the highest expressional products(32.2%).pET-32a/P-Glc induced for 4h7h like 3h was the highest expressional products.Expressional products by different temperature after IPTG induction were analyzed by SDS-PAGE,IPTG induced for 3h at 26 was one tenth of 37PTG induced for 3h at 45 was the same as 37.Expressional products by different concentration of IPTG induction were analyzed by SDS-PAGE,IPTG(3h) induced at K 0.5 > 0.1mmol.L'1are basically similar.Expressional products by different parts of cell after IPTG induct...