摘要
安吉白茶是在浙江省安吉县山区发现的温度敏感型珍稀茶树资源。在早春季节,新长出的芽叶呈淡绿色,随后叶片逐渐转白,并在完全白化后持续一段时间,然后叶片开始复绿,直至完全恢复正常颜色,这个过程被称为阶段性返白过程。在安吉白茶阶段性返白过程中,随着叶片颜色的变化,氨基酸含量也发生了显著改变。在叶片最白期,氨基酸含量为普通绿茶品种的2-3倍,非常适合于名优绿茶的生产。目前关于安吉白茶的研究大多集中在生理生化和叶绿体结构等方面,关于安吉白茶阶段性返白的分子机理尚缺乏深入研究,在一定程度上限制了安吉白茶的应用发展。另外,由于茶叶中的茶氨酸具有重要的生理功能,茶氨酸的高效制备技术一直被广泛关注。随着生物技术的发展,利用微生物酶定向合成茶氨酸已成为高效制备茶氨酸的发展趋势。本研究首先从建立适合于茶树蛋白质组学研究的双向电泳技术体系着手,成功构建了适用于茶树蛋白质组学研究的双向电泳技术体系,并对安吉白茶新梢生育期间蛋白质表达谱的变化进行了研究；同时,系统分析了安吉白茶阶段性返白过程中叶绿体超微结构的变化。本研究第二部分利用基因工程技术成功构建了催化茶氨酸体外合成的基因工程菌,并对其产酶条件进行了研究。
1.安吉白茶新梢生育期间叶绿体超微结构的分析
系统分析了安吉白茶从萌发期到完全复绿期6个不同时期叶片中叶绿体超微结构的变化。研究发现,安吉白茶返白前与复绿后叶片中叶绿体超微结构与正常叶片无明显区别,都具有典型的叶绿体结构。但是,在白化期间叶片中叶绿体数目明显减少且叶绿体超微结构存在严重缺陷,已发育的叶绿体内无基粒结构,嗜锇颗粒较多,有些甚至已经空化,但叶绿体双层膜结构完好；同时,大多数叶绿体发育停滞于前质体阶段,且未发育出明显的片层膜结构。结果表明,安吉白茶白化期叶绿素缺乏是出于低温破坏了已发育的叶绿体中的片层膜结构,使得叶绿素生物合成受阻；而叶绿素的缺乏又影响到质体向叶绿体的发育过程,从而导致白化现象的产生。
2.茶树蛋白质组学双向电泳技术体系的建立
通过对双向电泳技术体系中样品制备、IPG预制胶条选择、裂解液配方等关键步骤的优化,构建了一套适合于茶树蛋白质组学研究的双向电泳技术体系。利用此技术体系获得了高质量的蛋白质点1117±9.89个,大部分蛋白质的等电点集中在p15-7之间,分子量集中在15.00KDa-95.00KDa之间,大大提高了双向电泳所获得的茶树蛋白质组信息量。
3.安吉白茶新梢生育期间差异蛋白质的分离
首次采用双向电泳技术对安吉白茶新梢生育期间三个典型时期的叶片蛋白质组进行分离。每个时期样品在双向电泳凝胶上能检测到约1000个蛋白质点,且不同时期蛋白质的表达发生了明显改变。在三个时期之间,表达丰度变化均在1.5倍以上、重复性好的差异蛋白质点共计60个。根据差异蛋白质点的表达情况,可将差异蛋白质点分为四个表达模式：模式一,在返白前期和白化期之间上调表达,而在白化期和完全复绿期之间下调表达的蛋白质点,共计30个；模式二,在返白前期和白化期间下调表达,而在白化期和完全复绿期间上调表达的蛋白质点,共计21个；模式三,在返白前期、白化期和完全复绿期间均上调表达的蛋白质点,共计7个。模式四,在返白前期、白化期和完全复绿期间均下调表达的蛋白质点,共计2个。
4.安吉白茶新梢生育期间差异蛋白质的质谱鉴定及功能分析
通过MALDI TOF/TOF MS对安吉白茶新梢生育期间30个重复性好,且在不同生育期间表达丰度发生1.5倍以上变化的蛋白质点进行质谱鉴定,其中有26个蛋白质点被成功鉴定。根据其功能,利用生物信息学数据库可将它们分为7类,分别为：与碳、氮、硫代谢相关；与能量代谢相关；与光合作用相关；与蛋白质加工、翻译相关；与RNA加工相关；与植物抗性相关及一些未知功能的蛋白质。其中与代谢及蛋白质加工相关的蛋白质最多,约占所鉴定蛋白质数的二分之一。
5.安吉白茶新梢生育期间差异蛋白质的实时荧光定量PCR分析
应用实时荧光定量PCR技术对6个可能与安吉白茶阶段性返白现象及高氨基酸含量相关的差异蛋白质的mRNA表达情况进行分析。研究发现,在不同生育期间仅有2个差异蛋白质的mRNA表达与蛋白质表达的变化相一致。这一研究结果表明,在安吉白茶返白过程中mRNA的表达与蛋白质表达有一定的相关性,但也不完全相符合,说明蛋白质组和转录组的表达之间有一定的差异性及互补性。
6.催化茶氨酸体外生物合成基因工程菌的构建及产酶条件的优化
通过基因工程技术成功构建了能高效表达γ-谷氨酰转肽酶的基因工程菌,并对其重组酶的表达条件进行了优化。结果表明,以0.1mmol/L IPTG,于20℃诱导表达6 h时,粗酶液的酶活达(4.4131±0.1658)U/mL,大约是出发菌株E-coliBL21 (DE3)的18.7倍。工程菌粗酶液以200 mmol/L谷氨酰胺、1.5 mol/L乙胺盐酸盐为底物,于pHl0、20℃下反应6 h,茶氨酸的合成量达到12.6 mg/mL,L-谷氨酰胺转化率为41.05%。
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 tea
Study 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 cultivars
The 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 tea
It 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 analysis
Thirty 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 tea
To 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 enzyme
A 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%.
引文
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