| Tea plant?Camellia sinensis?L.?O.Kuntze?is a dicotyledonous tea family,mostly shrubs or small trees,mainly distributed in China,Japan and other countries.L-theanine is a unique amino acid in the tea tree.It is an important index to evaluate the quality of tea,and it has many functions such as strengthening immunity,lowering blood pressure and reducing fat.Caffeine is an important flavor of tea.Moderate consumption can refresh the brain and protect liver.Theanine physiological function is still not clear,however,is generally thought of nitrogen metabolism in vivo metabolism of theanine and tea tree are closely related,and different forms of nitrogen for the synthesis of theanine influence;Caffeine synthase?TCS1?plays a major role in the synthesis of caffeine,but its main role is not clear.In this experiment,the whole body culture system of Dahongpao was established.Dahongpao aseptic somaclone as material,studied the Dahongpao in exogenous salicylic acid treatment of theanine and caffeine content and the effect of using the fluorescent quantitative PCR analysis of theanine and caffeine synthase gene expression.The effects of foreign amino acids on theanine synthesis and precursor substances were also studied.The main results are as follows:1.The aseptic embryos of Camellia sinensis Dahongpao were used as explants,and the whole tissue culture system was established through the selection of embryo nduction,subculture and root induction culture.The results showed that the optimum induction medium was MS+sucrose 30 g·L-1 and the induction rate was 100%.The optimal edium for subculture was MS+TDZ 0.1 mg·L-1+sucrose 20 g·L-1,the average increment coefficient was 5.64.The effect of 2.5 mg·L-1 6-BA and 1.5 mg·L-1 IBA on seedling growth was obvious.The optimal rooting medium was 1/3MS+NAA 2.0 mg·L-1+IAA 10.0 mg·L-1+ascorbic acid 50 g·L-1+citric acid 50 g·L-1+sucrose 10g·L-1+agar 5.5 g·L-1,the rooting rate was more than 80%.2.The content of chlorophyll content,POD activity,proline and soluble sugar content in da red pao were not obvious in the treatment of exogenous salicylic acid,and the content of malondialdehyde and soluble protein was increased first and then decreased.The content of theanine was increased first and then decreased,the content of salicylic acid was 3.0 mmol·L-1,and the content reached the maximum,followed by a sudden drop,which was significantly lower than the control level.Theanine synthetase gene expression related to theanine synthetase?TS1?gene content change trend and the theanine content is consistent,and glutamate dehydrogenase?GDH?gene in decline in different degrees.When the salicylic acid concentration was 4.0mmol·L-1,the gene expression of theanine synthase and the content of theanine were inhibited.1.The treatment group of aspartic acid,glutamate and theanine significantly increased the content of theanine in the group,which increased by 19.8%,8.5%and36.8%respectively.There was no significant difference between the red robe and the control group.Not all exogenous amino acids can promote the synthesis of theanine in the tea tree.The aspartic acid treatment group decreased the expression of glutamate dehydrogenase?GDH?gene,and the glutamate was synthesized by the amino acid.Leucine treatment group increased the expression of glutamate dehydrogenase?GDH?gene,which is more decomposer of glutamate.Glutamic acid increases the precursor of theanine synthesis;Theanine can be absorbed directly by Da hong pao.2.By drying the leaves and using 75%ethanol for ultrasonic treatment,the extraction amount of caffeine can be improved effectively.The concentration of salicylic acid in da red coat reached the maximum at 1.0 mmol·L-1,then decreased,but the content of the caffeine content was lower than 1.0 mmol·L-1 and 3.0 mmol·L-1at 2.0 mmol·L-1.The change of the caffeine synthase?TCS1?gene was reduced first,and then reached the maximum at 2.0 mmol·L-1.Through the measurement of other substances in the coffee base synthesis pathway,the content of related substances in the caffeine synthesis pathway was lower than that of other treatment groups in the concentration of salicylic acid at 2.0 mmol·L-1.3.7-methyl xanthine,xanthine?1,7-dimethyl xanthine?precursor,theobromine and caffeine and theophylline five things have improved the purine metabolism in quasi xanthine,theobromine and caffeine and theophylline.The content of caffeine in the treatment group was the most significant,71%;Theophylline and the caffeine treatment group were second.In the treatment group of 7-methylxanthine,caffeine and theophylline,the increase in the content of pseudoxanthine was the most obvious,up to 38%,39%and 79%respectively.The increase in theobromine content was only10%,15%and 6%.The expression of theobromine synthase gene was the largest in theobromine treatment group,which was 15.7 times that of the treatment group.The expression of the caffeine synthase gene was more than 2.5 times that of the treatment group.Therefore,it can be speculated that the core pathway of caffeine synthesis is 7-methyl xanthine-pseudoxanthine-caffeine,and the main function of the caffeine synthase is to make the pseudoxanthine synthetic caffeine. |
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