| Tea plant(Camellia sinensis)is a vital economic plant in China,and tea is one of the three major non-alcoholic drinks in the world,which has important economic value.Tea aroma is one of the essential indexes of tea quality.As the main volatiles of tea aroma,monoterpene alcohols and aromatic alcohols etc frequently occur as glycosides.In tea leaves,primeverosides and glucosides are the main aroma precursors.It is generally held that,tea aroma is released when the the glycosides hydrolyzed by the endogenous glycosidases,after the fresh tea leaves physical damaging,and the subcellular compartments destroying.The endogenous goycosidases mainly include β-Primeverosidase and β-Glucosidase.The tea volatile fragrance components,which released from aroma precursors,are not only the basis of tea aroma,but also play key roles in tea plant defense of diseases and insects.Though there are numerous researches on endogenous glycosidases and progresses on the cloning and expression were gained,they have not been immobilized yet.What’s more,studies on the antibody preparations are few,no commercial antibody sold,yet.It makes difficult for us to study the endogenous glycosidases in protein level.In this paper,we study on the cloning and expression of the genes encoding endogenous glycosidases,prepare the polyclonal antibody of the endogenous glycosidases,analysis differential expression of β-Primeverosidase and the β-Glucosidase in 13 tea cultivars and study on the variation of the water content and the β-Glucosidase activity in tea leaves during the withering process,so as to get these goals: we can get further insights on the tea aroma biosynthesis.Hence,we can improve the tea aroma quality;we try to make it possible to comprehend the endogenous glycosidases metabolism in protein level;we can obtain gene markers in tea plant breeding identification field;we can provide theoretical direction for tea processing.Main results are as follows:1.According to the ORF sequence of β-Glucosidases(Glu),whose purification had been reported before,and β-Primeverosidase(Pri)from tea plants(Camellia sinensis),the primers were designed and synthesized and the gene segments of Pri and Glu were gained then.Inserting the gene segments into pET-32a(+)and pMAL-c5 X,we obtained the recombinant expression vectors pET-32a(+)-Pri/Glu,pMAL-c5X-Pri/Glu and pMAL-c5X-Pri/Glu without signal peptide.pET-32a(+)-Pri and pET-32a(+)-Glu were expressed in e-coli using the Reastta(DE3)pLysS vector,pMAL-c5X-Pri without signal peptide and pMAL-c5X-Glu without signal peptide were expressed in e-coli using the BL21(DE3)vector.Induced by IPTG,we analysised the fusion protein by SDS-PAGE,the fusion proteins of pET-32a(+)-Pri and pET-32a(+)-Glu were highly expressed in the form of inclusion body,while the pMAL-c5X-Pri/Glu without signal peptide fusion proteins were expressed as soluble protein.However,pMAL-c5X-Pri and pMAL-c5X-Glu were not expressed.They all had no bioactivity.2.The pET-32a(+)-Pri and pET-32a(+)-Glu fusion proteins were purified by the Ni chelating resin affinity chromatography,and then we prepare the rabbit polyclonal antibodies against Pri and Glu.ELISA detection of the antibody recommended dilution ratio of 1:100000.We verified the specificity of the antibodies by Western-blot.By on the specific binding of Pri antibody and the total proteins of tea seeds,we speculated that Pri maybe exist in tea seeds,too.3.Based on the ORF of the Glu,two pairs of specific primers were designed,and the gene segments of Glu obtained by RT-PCR.The recombinant expression vector By inserting the Glu into pESC-HIS vector,pESC-HIS-Glu was constructed.Induced by galactose,we analysised the fusion protein by SDS-PAGE and Western-blot,Glu can be expressed by the recombinant expression vector.The activity of Glu,assayed by using the pNPG substrate,is 0.24±0.03 U.4.Results of real time quantitative PCR of Pri and Glu in 13 tea cultivars showed that,the gene expression levels had such rules: Both genes were highly expressed in Zaofengchun and Anhui 7,Duokangxiang and Yueminxiang,which are famous for the outstanding tea aroma,showed higher expression values,compared with the medium-sprouting cultivars,most early sprouting cultivars showed higher gene expression values.5.We studied on the variation the water contents and β-Glucosidase activities of the tea leaves from five cultivars,which were withered after 0,2,4,6,8,10 h indoor.Results indicated: There is no significant difference between time and β-Glucosidase activities(P>0.05).While,the effect of cultivar and β-Glucosidase on enzyme activity reached very significant level(P<0.01).The tea cultivars play crucial roles in tea aroma.During the withering process,the water content decreased linearly.In the former 6 h,there was a sharp enzyme activity variation and a dramatic decline in 4 h of the most tea cultivar leaves.The β-Glucosidase enzyme activity kept a higher level in 6-8 h,while after 10 h the level approached to level of 0 h. |
PDF Full Text Request