Study Of Molecular Mechanism Of L-Theanine Metabolism In Tea Plant(Camellia Sinensis)

Tea plant[Camellia sinensis(L.)O.Kuntze],belonging to theaceae family and camellia genus,is a perennial evergreen woody crop.The tea fresh leaves are the important raw materials for processing tea of non-.alcoholic beverages.Moreover,the tea leaves are rich of various secondary metabolites that are beneficial to human health,such as tea polyphenols(especially catechins),theanine and caffeine.Among these secondary metabolites,the theanine content is closely related with the quality and the healthy function of tea.The distribution of theanine content in tea plant has the specificity of cultivars and tissues,and has certain dynamic change tendency in processing of post-harvested tea leaves.Theanine metabolism is closely related with nitrogen metabolism.In the actual growth process,tea plant usually forced by nitrogen deficiency,which negatively affects the the accumulation of amino acids and the sensory quality of tea.However,the molecular mechanism of theanine metabolism in the above scientific phenomena and problems remains unclear.Therefore,in this study,bioinformatics analysis,molecular biology and omics research methods were applied to deeply reseach the correlation between theanine accumulation and related genes expression in different cultivars and tissues,the importance of the key enzymes of theanine metabolism in the dynamic changes of theanine at the protein level or gene level,and the molecular mechanism of differences between shoot and root under nitrogen deficiency or sufficiency.The specific research content and conclusions are as follows:1.The content of theanine in bud and 1st leaf,2nd leaf,3rd leaf,old leaf,stem and root of C.sinensis 'Huangjinya','Baiyeyihao' and 'Yingshuang' were detected by HPLC.The result suggested that the theanine content is gradually decreased with the increasing maturity of leaves.The theanine content in stem was the lowest,and in root is second to bud and 1st leaf.Among the cultivars,the theanine contents in 'Hlangjinya' leaves and root were the highest,followed by 'Baiyeyihao',and in 'Yingshuang' were the lowest.From the GenBank and tea transcriptome,17 genes involved in theanine metabolism were identified.The expression profiles of the 17 genes in different tissues of the three C.sinensis cultivars were compared and analyzed.Among the different cultivars,most of those genes showed higher expression levels in 'Huangjinya' than 'Baiyeyihao' and 'Yingshuang'.Moreover,the theanine contents were positively correlated with the genes expression levels in most cases.Among the tissues,the expression levels of CsGSl,CsALT,CsNADH-GOGAT,CsGDH2 in 'Huangjinya' and CsGGT1,CsGGT3 in 'Yingshuang' were positively related with theanine contents.The expression levels of most genes in 'Baiyeyihao' were positively related with theanine contents,except CsTS1,CsFd-GOGAT and CsGGT3.2.The post-harvest spreading leaves were treated with high temperature(38 0C),low temperature(4?)and shading.The differentially expressed proteins(DEPs)involved in theanine metabolism were detected from corresponding proteome data.The dynamic change of theanine content in different samples were analyzed by UPLC.The glutamate synthase of DEPs(CsFd-GOGAT and CsNADH-GOGAT)involved in theanine metabolism were identified.qRT-PCR was performed to analyze the expression profiles of the 17 genes,including CsFd-GOGAT and CsNADH-GOGAT.The interactions between CsFd-GOGAT and CsNADH-GOGAT,CsTS1,CsNiR were verified through yeast two-hybrid technology.The importance of glutamate synthetase in the theanine dynamic changes of post-harvested leaves was demonstrated through the correlation between theanine contents and related proteins and genes expression levels.3.The RNA-seq technique was used to analyze the transcripts of N6-R,N6+R,N6-S and N6+S(6:treatment for days;S:shoot(including 1st,2nd,3rd leaf;R:root;+:nitrogen sufficiency;-:nitrogen deficiency).The four transcriptome databases were mapped to genome,and the average mapping ratio was 62.99%.The total number of detected genes was 38,998,including 28,812 known genes and 10,186 novel genes.A total of 162 genes were screened and including 24 enzymes or transport proteins involved in the theanine metabolism.The correlation analyses showed that the theanine contents were correlated with these genes levels to a certain extent.The content of theanine was strong correlated with Glu,Ala,Arg,Val,Leu and Asp in N6+S,and strong related with Glu in all samples.There 31,14,90 and 107 differentially expressed genes(DEGs)involved in theanine metabolism were respectively identified from N6-R/N6+R,N6-S/N6+S,N6+R/N6+S and N6-R/N6-S groups.The number of up-regulated genes were signficantly more than that of down-regulated genes.qRT-PCR analysis suggested that regulation of the DEGs was consistent with transcriptome predictions.4.Through iTRAQ technique,a total of 4,912 proteins were quantified from the four samples,ie.N6-R N6+R N6-S and N6+S.There 37,24,246 and 273 differentially expressed proteins(DEPs)were identified from N6-R/N6+R,N6-S/N6+S,N6+R/N6+S and N6'R/N6-S groups.There 8,1,138,and 157 pairs of DEPs-DEGs were identified from the four groups after correlation analyses between DEPs and DEGs.The functional classes of correlated DEPs in N6-R/N6-S and N6+R/N6+S were further classified by GO and KEGG analyses.The proteins related with theanine metabolism were further screened from in each DEPs group and DEPs-DEGs pair.The activities of five key enzymes involved in theanine metabolism were determined,and the correlation between enzyme activity and theanine content was analyzed.5.Four samples,N6-R,N6+R,N6'S and N6+S,were used to construct small RNA library.The high-throughput sequencing technology was used to explore the information of theanine metabolism-related microRNAs.A total of 55,909,772 clean reads were obtained.After the clean reads of each sample were mapped to the genome,the kinds and total number of clean reads in shoot were higher than root,in control tissues were higher than nitrogen deficiency treated tissues.By alignment analysis and novel miRNA prediction,a total of 6028 known miRNAs and 2829 novel miRNAs were obtained.There 907,1432,2181 and 2416 differentially expressed miRNAs(DEMs)were identified from N6-R/N6+R,N6-S/N6+S,N6-R/N6-S and N6+R/N6+S groups.Correlation analysis of DEMs and DEGs revealed that there were 547,319,5877 and 5807 negative DEMs-DEGs pairs,and 1,2,5 and 6 pairs related with theanine metabolism.The DEGs of the negative DEMs-DEGs pairs were further used for GO and KEGG Pathway enrichment analyses.