The Effects And Mechanism Studies Of Theanine Biosynthesis Of Tea Plant(Camellia Sinensis L.) Roots Under Different Cultivation Mediums

Theanine is the most abundant free amino acid in tea plant,accounting for 1～2% of the dry weight of tea plant.Theanine was positively correlated with the quality of fresh leaves.In addition,theanine has many pharmacological functions including antidepressant,antitumor,Neuroprotection,memory improvement and so on.Thus,it’s widely used in food and drugs area.In order to facilitate the study of theanine biosynthesis,a GC detection method was established and found that theanine content of tea plant root which were grown under two different culture mediums(soil and sand)were 3.88 % and 1.99 % respectively.In order to reveal the molecular mechanism of cultivation mediums on theanine biosynthesis,the genes and metabolites of tea seedling roots under different mediums were analyzed by transcriptome and metabolome,three candidate genes possibly involved in theanine biosynthesis were selected,the overexpression vector was constructed and transferred into tea somatic embryos for homologous transient expression to verify the function of the genes.This paper provides a new detection method for the biosynthesis research of theanine,and lays a foundation for revealing the mechanism of theanine biosynthesis and molecular regulation mechanism.The experimental results are as follows:1.A gas chromatography-flame ion detection method of theanine was established.The high boiling theanine generated to a low boiling theanine derivative and thus can separated on the GC column.The entire analysis process,including theanine extraction,derivation,and chromatographic separation,can be completed in 40 minutes,with a recovery rate of between 92.59 and 104.77 %.Intra-day precision was 0.57 to 2.28 %,and inter-day precision was 1.57% to 13.48%.The intra-day accuracy was-6.84 ～ 5.26 %,and the inter-day accuracy was-1.08 ～ 3.12%.The limit of detection was 10.28 μg/m L and the limit of quantification was 13.47 μg/m L.The results show that this method is convenient,fast,accurate and economical,and provides a rapid detection method for the biosynthesis of theanine.2.Significant difference of theanine in the roots of soil culture(T)and sand culture(S)was detected.Metabolome and transcriptomic analysis of S and T were performed.Five KEGG pathways involved in amino acid metabolism in the metabolome found that amino acids such as alanine,glutathione,cystine,homocysteine,and proline were all upregulated in Tvs S,among which alanine played an important role in theanine biosynthesis.A total of 18 related genes involved in amino acid metabolism were selected from the top ten significantly enriched KEGG pathways,including eight upregulated genes and 10 downregulated genes,and of which four genes were associated with theanine biosynthesis.A total of 17 coding genes of the five enzymes involved in the theanine biosynthesis process were selected from the transcriptome.Among them,11 genes were upregulated and 6 genes were downregulated.The Cs SDC48 gene in the upregulated genes was annotated as serine decarboxylase(SDC),which has the highest expression in the transcriptome and has been confirmed to be alanine decarboxylase(Ala DC).It indicates that alanine and its related enzymes such as Ala DC may be the main reason account for the difference in theanine content under different cultivation mediums.Therefore,three genes,Cs SDC48,Cs SDC37 and Cs SDC29,were finally identified as candidates,and all three candidate genes were annotated as serine decarboxylase(SDC).3.Bioinformatic analysis results showed that the CDS lengths of the 3 genes were1437,1434,and 1455 bp,and were extremely similar in both physicochemical information and protein structure.Based on this analysis,functional validation analysis was continued,two pairs of specific primers were designed according to the CDS sequence,and gene fragments was obtained by RT-PCR,then inserted into p CXSN vector to construct the overexpression vector;recombinant plasmid containing target gene was transferred into Agrobacterium GV3101 and then infected somatic embryo.The results showed that the components in somatic embryo which was infected by agrobacterium containing Cs SDC48 and Cs SDC29 genes changed little compared with the normal somatic embryo,and in the somatic embryo infected by agrobacterium which containing Cs SDC37 gene,the content of alanine was decreased and the content of theanine was increased.It proved that the Cs SDC37 has the effect of alanine decarboxylase and catalyzed alanine to ethylamine in tea plant.