Proteomic Analysis Of Shoot Tissue During The Developmental Stages In Anji White Tea [Camellia Sinensis (L.)O.Kuntze] And The Biosynthesis Of Theanine In Vitro

Anji white tea is a typical albino tea cultivars found in Anji County, Zhejiang province. The color of its shoot is sensitive to temperature.The new shoot is light green in the early spring, then it gradually change from light green to completely white. After about two weeks at the albinistic stage, the leaves gradually turn as green as those of common tea cultivars. This process is called stage albinism. A significant change of the level of amino acid was occurred during the stage albinism. The level of total amino acid was 2 or 3 times higher than the common green tea cultivars in the albinistic stage. Most previous studies focused on physiology, biochemistry and chloroplast structure of Anji white tea, but little information about molecular mechanism of the stage albinism. To a certain extent, it limit the development of application and theory in Anji white tea. In addition, since a series important physiological functions of theanine were found, the efficient preparation technology of theanine has receive much attention. With a number of advantages of enzymatic reaction, using microbial enzymes synthetize theanine become the mainly methods for the preparation of theanine. In this study, the technical system of 2-DE for the tea leaves was estabilished and was firstly used for the proteomic analysis of Anji white tea during the developmental satges. The ultrastructure of chloroplast in Anji white tea was also studied. In the second part of this paper, a recombinat strain was successful constructed, which could catalyze the synthetic reaction of theanine in vitro and the optimal conditions of the synthetic reaction were studied.1. The analysis of chloroplast ultrastructure during the developmetal stages in Anji white teaStudy on the change of chloroplast ultrastructure during six developmental stages. It is found that there is no significant difference between the pre-albinistic stage and regreening stage compare to the normal tea cultivars. They all have a typical chloroplast structure at the pre-albinistic stage and regreening stage of Anji white tea. However, the number of chloroplast was reduced markly and the ultrastracture of chloroplast was severely damaged at the albinistic stage of Anji white tea. The grana disappeared, more osmiophilic granules appeared and some chloroplasts even cavitated, but the chloroplast membrane structure was still intact. Meanwhile, most of the plasmid development was suppession. The results showed that the lack of chlorophyll may be a consequance of destroying the chloroplast membrane structure at the albinistic stage in Anji white tea. The lack of chlorophyll will retarded the development process of the pre-plastid to chloroplast and resulting albinism.2. The technical system of 2-DE for proteomic analysis of tea cultivarsThe system of 2-DE for proteomic analysis of tea cultivars was successful constructed by optimizing the sample preparation method, IPG strips and lysis buffer. Using this system can obtain 1117±9.89 high quality protein spots and their isoelectric points were found to lie between 5-7. The relative molecular weight of proteins was between 15.00 KDa-95.00 KDa. This technical system could greatly increase the proteome information of tea plant obtained by 2-DE.3. The seperation of proteins during the developmental stages in Anji white teaIt is the first time that proteins from Anji white tea during the developmental stages were separated by 2-DE. Among all the tested samples, more than 1000 protein spots were reproducibly detected with PDQuest 8.0.1 software on each CCB G-250-stained gels. Quantitative analysis revealed that 60 protein spots showed a significant (p<0.05) change in intensity by more than 1.5-fold from pre-albinistic stage to albinistic stage and from albinistic stage to regreening stage as well. In total, 60 differentially expressed protein spots could be classified into four expressed patterns:patternⅠ,30 protein spots showed up-regulation from the pre-albinistic stage to the albinistic stage but down-regulation from the albinistic stage to the regreening stage; patternⅡ,21 protein spots showed down-regulation from the pre-albinistic stage to the albinistic stage but up-regulation from the albinistic stage to the regreening stage; patternⅢ, seven protein spots showed up-regulation from the pre-albinistic stage to the albinistic stage also from the albinistic stage to the regreening stage; and pattern IV, two protein spots showed down-regulation from the pre-albinistic stage to the albinistic stage and also from the albinistic stage to the regreening stage.4. Identification of differentially expressed preorins during the developmental stages in Anji white tea and their functional analysisThirty differentially expressed protein spots were excised from gels, digested in-gel by trypsin and identified by MALDI-TOF/TOF MS. In total,26 protein spots were successfully identified. According to their fuctions, these identified proteins were involved in seven functional categories including metabolism of carbon, nitrogen and sulfur, metabolism of energy, photosynthesis, protein processing, stress defence, RNA processing and unknow proteins.5. Gene expression analysis by qPCR during the deveopmental stages in Anji white teaTo investigate the changes in gene expression at the mRNA level, qPCR analysis of six identified proteins, which may be related to the albinism phenomena and high level of amino acid, were performed. The result indicated that just two proteins, whose mRNA expression and protein expression were consistent. The mRNA expressed level were not well related to protein expressed level. It suggests the complementary nature of the two methods.6. The construction of the recombinat strain for the synthsis of theanine and optimize the expressing conditions of recombinat enzymeA recombinant strain, which could highly produce y-glutamyltranspeptidase, was successful constructed and the expressing conditions of recombinat enzyme were studied. The result showed that the recombinant strain incubated with 0.1mmol/L IPTG for 6h at 20℃, the activity of crude extract from recombinant strain was (4.4131±0.1658) U/mL, which was about 18.7 times than the activity of E·coliBL21. When the synthetic reaction condition were 200mmol/L L-glutamine,1.5mol/L ethylamine, pH10, and incubation at 20℃for 6h, the yield of theanine was 12.6mg/mL, and the rate of conversion from L-glutamine to theanine was 41.05%.