Research On The Chloroplast Proteomics Of The AnJi BaiCha During Periodic Albinism

AnJi BaiCha is a natural albino mutation tea tree specie which is a rare resource and induced by low temperature. Its special performance are periodic albinism of the new shoot in AnJi BaiCha every spring and higher amino acid content at albinistic stage. Every spring the new shoot of AnJi BaiCha is tender green, then when the leaves unfold, the coulor of leaves turn to milky white quickly, two leaves and a bud is the most obvious, the coulor of leaves begin to turn to green gradually, until it recovers to the normal green fully. The amino acid content has a significant change between albinistic stage, the level of total amino acid content is 2-3 times higher than green leaves in the albinistic stage.The phenotypic traits change, the main biochemical characteristics, pigment content, chloroplast ultrastructure, gene and protein expression change of AnJi BaiCha are researched during periodic albinism. But the molecular mechanism of the stage albinism is unclear. To a certain extent, it limit the development of application and theory in AnJi BaiCha. Related findings showed that it was mainly due to chloroplast capsule membrane structure damaged so that chlorophyll biosynthesis was blocked, and the lack of chlorophyll would affect the plastid developing chloroplast, resulting to the phenomenon of periodic albinism, so the change of chloroplast was a key factor during periodic albinism. However, the information on the molecular mechanisms of periodic albinism was little in current stage. Therefore, in this study, the chloroplast proteomic research of AnJi BaiCha ware analysised and compared by using proteomics and bioinformatics. It aimed to obtain molecular mechanism to illustrate the stage albinism of AnJi BaiCha. The main results showed as follows:1. The study focused on the photosynthesis, pigment and biochemical components change systemly. The net photosynthetic characteristics of AnJi BaiCha ware measured by the LI-6400 portable photosynthesis meter during periodic albinism. The result showed that Pn, Gs and Tr ware rising during periodic albinism, and appeared down at albinistic stage slightly, while Ci had shown a trend of slow decline. The SPAD value was gradually decreasing from pre-albinistic stage to albinistic stage,and rised from albinistic stage to regreening stage. The total content of pigment was upward state from pre-albinistic stage to regreening stage, only declined at albinistic stage. The seven fat-soluble pigments of the thin layer chromatography separation ware Neo, Vio, Zea, Chlb, Xan, Chla and β-Car. The order of the total pigments was regreening stage, mid-greening stage, pre-greening stage, pre-albinistic stage, mid-albinistic stage, albinistic stage from big to small. The content of the total polyphenols was pre-albinistic stage>regreening stage>albinistic stage. The content of the total amino acid,18 kinds of the free amino acid and theanine ware showed a gradual decline from pre-albinistic stage to regreening stage,but appeared a rise at albinistic stage. The high performance liquid chromatography method was used to determine the total catechins, the result showed that the content was 8.79%-8.79%. The ratio of non-estered catechins and estered catechins was pre-albinistic stage<albinistic stage<regreening stage.2. The separation system of intact chloroplasts tea leaves was successful constructed. The pre-albinistic stage, albinistic stage, regreening stage of AnJi BaiCha chloroplast ware separated with the differential centrifugation and percoll density gradient centrifugation. The integrity of the chloroplast was 73% obtained by hill reaction and the result achieves the requirement of the following study. Identifing the purity of chloroplast based on the activity relative change rate of fumaric acid enzyme and hydrogen peroxide enzyme,the results showed that the significant difference was shown in optical density values of fumaric acid enzyme and hydrogen peroxide enzyme at the unit time in the crude chloroplasts, while not significant difference in the purified chloroplast. It explained that the crude chloroplasts had these enzymes pollution, but the purification of chloroplasts had no pollution of this two enzymes basically.3. The seperation of chloroplast proteins in AnJi BaiCha during periodic albinism. The pre-albinistic stage, albinistic stage, regreening stage of AnJi BaiCha chloroplast proteins ware extracted by TCA-acetone method, then segregated by two-dimensional gel electrophoresis, and the gel maps analysised through the PDQuest software.The results showed that each stage of the sample ware reproducibly detected about 700 protein spots. Quantitative analysis indicated that 59 chloroplast protein spots which showed a significant change in intensity by more than 1.5-fold well during the three periods of AnJi BaiCha and the different chloroplast protein spots could be divided into six expressed types. First,31 protein points showed downgrade from the pre-albinistic stage to the albinistic stage but upgrade from the albinistic stage to the regreening stage; Second,3 protein points showed upgrade from the pre-albinistic stage to the albinistic stage but downgrade from the albinistic stage to the regreening stage; Third,2 protein points showed upgrade during the three stages of the Anji Baicha; Fourth,3 protein points showed downgrade during the three stages of the Anji Baicha; Fifth,7 protein points showed expressed during pre-albinistic stage and regreening stage,but not expressed at albinistic stage; Sixth,13 protein points showed expressed only at albinistic stage, but not expressed during pre-albinistic stage and regreening stage.4. Identifieation of differentially expressed chloroplast proteins during periodic albinism. Twenty five representative chloroplast protein points ware exeised from different gel samples, identificated by MALDI TOF/TOF mass spectrometry, and 20 protein points ware successfully identified after retrieved database. According to their functions, these different identified proteins ware involved in five functional categories including metabolism of energy, photosynthesis, RNA proeessing, resistance and cell defense proteins.