| Tea plant [Camellia sinensis (L.) O. Kuntze] is one of the most important economy crops. It is an evergreen bush and has over 3000 year history as a cultivation crop in China. Anji Baicha is a kind of specific variety with reversible albinism in spring shoots. During the albescent stage, the content of chlorophyll in albino leaves sharply reduces, but the amount of amino acids raise significantly. The amount of amino acids is about 2 times higher in albino leaves than that of normal leaves from ordinary varieties. L-Theanine is a characteristic ingredient of tea plant, and a non-protein amino acid, which accounts for 50?60% of total amount of amino acids in tea plant. L-Theanine is a kind of secondary metabolite with important physiology activities. Our previous research had isolated a bunch of differentially expressed genes related to the property of high amino acid level during stage albescent process of Anji Baicha by SSH and cDNA-AFLP. In this study, the full-length cDNA of two genes were cloned and their functions were further analyzed. The research results would support to regulate enhancing of the L-theanine level in tea plant from both theoretical and practical aspects. The main results were as follows:The components and contents of amino acids in albescent and green leaves were analyzed by AccQ.Tag pre-column derivative method according to Waters Company. The results showed that both albescent and green leaves contained 18 kinds of amino acids, but the total amount of amino acids and the content of each amino acid were different between two stages. The content of amino acids in albescent leaves was 5.93%, and it's 2.12 times higher than that of green leaves. The more tender the tea leaves was, the higher the amino acid level would be. There was a positive correlation between the content of L-Theanine and the total amount of amino acids in Anji Baicha. The content of L-Theanine in albescent leaves was 3.33%, and it's 2.31 times higher than that of green leaves. The results also indicated that AccQ.Tag pre-column derivative HPLC method established by this research could well separate the amino acid components of tea plant, so it can be uesd to analyze and compare the amount of amino acids in different tea samples.In order to separate the differentially expressed genes between albescent and green leaves of Anji Baicha, a subtracted cDNA library was constructed by the SSH method. According to the preliminary research from genetic level, Anji Baicha with high amino acid characteristic might trace to serine-glyoxylate aminotransferase gene and serine hydroxymethyl transferase gene. To reveal the miracle fact of amino acids enrichment in albino leaves, the further studies about these two genes are necessary and going on.Theanine synthase can convert L-glutamic acid and ethylamine into L-theanine while ATP providing energy. In this study, the full-length cDNA of theanine synthase gene was cloned. The cDNA sequence has the full-length of 1503 bp, and with an Open Reading Frame of 1071 bp. It can encode 356 amino acids. Bioinformatics analysis showed that theanine synthase has 356 amino acid residues; has a theoretical molecular weight of 39 250.3 Da and theoretical PI value of 5.79. Bioinformatics prediction showed that this protein is hydrophilic and located not within the transmembrane; there are 26 phosphorylation sites within the polypeptied chain. The signal peptide analysis showed that there is no signal peptide and no winded helix domain present within the sequence; the protein is non-secreted and it functions in the cytoplasm. This reported result about cloning full-length cDNA of theanine synthase gene would be beneficial for using biotechnology to improve tea plant variety resources and regulate enhanceing L-theanine level.The function of theanine synthase gene was preliminarily confirmed by prokaryotic expression and in vitro enzyme test. Via construction plasmid pMD 19-T-TS, the final recombinant plasmid pET-32a (+)-TS was constructed, then it was transfered to the expression vector Escherichia coli BL21. Afterward it was induced by IPTG to express recombinant protein. Result showed that a recombinant protein was produced with a molecular weight of about 59 kD, which was accordant with the predicted result. By in vitro enzyme test and HPLC analysis, the recombinant protein successfully catalyzed the synthesis of L-theanine.The full-length cDNA of CDC48 gene in Anji Baicha was cloned by SMART-RACE. The cDNA sequence has the full-length of 2859 bp, and with an Open Reading Frame of 2 424 bp. It can encode 807 amino acids. Bioinformatics analysis showed that CDC48 has 807 amino acid residues; has a theoretical molecular weight of 89 901.9 Da and theoretical PI value of 5.16. Bioinformatics prediction showed that this protein is hydrophilic and located not within the transmembrane; there are 38 phosphorylation sites distributing unevenly in the polypeptied chain. The signal peptide analysis showed that there is no signal peptide but there are three possible winded helix domains present within the amino acid sequence; the protein is non-secreted and it functions in the nucleus. Researches showed that CDC48 is a spindle decomposition related protein, and relates to protein biodegradation pathway relying on ubiquitin; and CDC48 gene is a vital element participating in cell cycle regulation. From this point of view, cloning full-length cDNA of CDC48 gene and analyzing the structure & function of its protein are meaningful for unfolding the molecular mechanism of amino acids enrichment in albino leaves of Anji Baicha.To analyze the differential expression of TS gene and CDC48 gene in albescent and green leaves, relative quantitation real-time PCR was applied. The results showed that the expression level of both TS and CDC48 genes were higher in albescent leaves than that of green leaves. TS catalyzes the synthesis of L-thenine and CDC48 is related to protein degradation into amino acids, so the over expression of these two genes in albino leaves could contribute to the property of high amino acid level during the albescent stage of Anji Baicha. |
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