Identification Of MYB Transcription Factors Related To L-theanine Biosynthesis And Functional Studies Of CsMYB73 And CsMYB38 Genes In Camellia Sinensis

Tea plant [Camellia sinensis(L.)O.Kuntze],a member of Theaceae family and Camellia genus,which originated in Southwest China and is widely planted in the world.Tea have become the second largest drink in the world apart from water.Tea has rich flavor and many benefits to human health.L-theanine is one of the important secondary metabolites in tea plants,and it mainly contributes to the umami taste of tea.L-theanine has the functions of relaxing,improving learning and immune system ability,influencing weight,and preventing cancer,among others.Although the key genes in L-theanine synthesis pathway have been reported,there are few studies on the transcription regulation mechanism of these key genes.In this study,we analyzed the accumulation patterns of free amino acids in leaves of “Baojing Huangjincha 1”(BJ)and “Baiye 1”(BY)in different growth stages.Based on RNA-sequencing technology,transcriptional database of “BJ” and “BY” leaves at different growing points was conducted.The CsMYB transcription factors related to L-theanine biosynthesis were further screened and identified.The full-length c DNA sequence of CsMYB73 and CsMYB38 were obtained by TA cloning.The transcription regulation of CsMYB73 and CsMYB38 protein on the key genes in L-theanine biosynthesis pathway was verified by molecular biological methods.The main results are as follows:1 The FAAs contents in the leaves of “BJ” and “BY” were determined by Ninhydrin colorimetry.The results showed that the FAAs contents in young leaves of “BJ” and“BY” were higher,which could reach to 67.85 and 78.89 mg/g,respectively.And that decreased with the growth and development of leaves.The levels of amino acid compositions(Glu,Ala and L-theanine)in the samples were determined by HPLC with pre-column derivatization.The results showed that the contents of L-theanine were the highest,with an average of 18.29 and 16.88 mg/g in “BJ” and “BY” leaves,while the contents of Glu in “BJ” and “BY” leaves were the second,with an average of 3.08 and 3.55 mg/g.The contents of Ala were the lowest,with an average of 0.18 and 0.29 mg/g in “BJ” and “BY” leaves,respectively.The accumulation of Glu,Ala and L-theanine was the highest in the leaves of “BJ” at the vigorous growth stage(4w),while the contents of Glu,Ala and L-theanine in the young leaves(1w)of “BY”were the highest.The content of total free amino acids and amino acid components in leaves at 7w growing stage were the lowest in the two varieties.2 The transcriptome libraries of “BJ” and “BY” leaves at different growth stages were constructed by RNA-sequencing technology.A total of 925,172,206 clean reads were obtained,and the average mapping ratio was 88.87%.In total,25 differentially expressed genes(DEGs)related to L-theanine metabolism were screened from transcriptome database,involving 14 L-theanine synthesis related KEGG pathways.The results of expression pattern analysis showed that most of the DEGs related to L-theanine metabolism were down regulated during leaf development,which was positively related to the content of free amino acids.The relative expression levels of11 selected DEGs related to L-theanine metabolism were verified by q RT-PCR.The results showed that the relative expression levels of these genes were generally down regulated during leaf development.Protein interaction prediction showed that the key DEGs in L-theanine synthesis pathway had interaction relationship.CsGS,CsGOGAT-Fd and CsGDH2 had close interaction with other proteins in the interaction network.It was speculated that these proteins might play core roles in L-theanine biosynthesis.3 Based on transcriptome data of tea leaves at different growth stages,20 CsMYBs transcription factors related to L-theanine synthesis were screened and identified.The candidate CsMYB transcription factors can be divided into 1R-MYB and R2R3-MYB subfamilies by conservative domain analysis and amino acid sequence alignment.With the growth and development of leaves,the expression of most CsMYB genes were down-regulated,which was positively correlated with the accumulation of free amino acids.The relative expression levels of six CsMYBs were verified by q RT-PCR.The results showed that the relative expression of CsMYB73 and CsMYB38 was significantly up-regulated in “BJ” and “BY” varieties with the increase of leaf maturity,while the relative expression levels of CsMYB12 and CsMYB4 were significantly down regulated during the development of leaves.The PPI network analysis showed that CsMYB5,CsMYB12 and CsMYB94 may form complexes with b HLH and WD40 proteins to regulate the biosynthesis of L-theanine.Many MYB transcription factors binding sites in the promoter region of structural genes.It is speculated that CsMYBs may participate in L-theanine metabolism by binding with the promoter region of structural genes.4 CsMYB73 and CsMYB38 both belong to the subgroup 22 of R2R3-MYB subfamily,and the full-length c DNA of these transcription factors were obtained by TA cloning.The recombinant plasmid CsMYB73-GFP and CsMYB38-GFP were constructed and expressed in tobacco leaves.The results showed that CsMYB73 and CsMYB38 are typical nuclear localization proteins.The recombinant vector p GBKT7-CsMYB73 and p GBKT7-CsMYB38 were constructed.The transcriptional activity of CsMYB73 and CsMYB38 were tested in yeast cells,respectively.The results showed that CsMYB73 did not possess any transcriptional activation,while CsMYB38 possesses transcriptional activation.The GST-CsMYB73 and GST-CsMYB38 fusion proteins were obtained by prokaryotic expression and protein purification technology.The obtained CsMYB73 and CsMYB38 fusion proteins were further used in EMSA experiment,and the results indicated that only CsMYB73 could bind to the promoter regions of CsGS1,CsGS2 and CsGDH2 in vitro.The results of dual-luciferase reporter gene system assay showed that the relative intensities of LUC/REN in tobacco cells of CsGS1 pro-LUC/p EAQ-CsMYB73,CsGS2 pro-LUC/p EAQ-CsMYB73 and CsGDH2 pro-LUC/p EAQ-CsMYB73 were obviously lower than that of the control groups,indicating that CsMYB73 could inhibit the transcription activities of CsGS1,CsGS2 and CsGDH2 promoters.The relative intensities of LUC/REN in tobacco cells co-transfected with CsGS1pro-LUC/p EAQ-CsMYB38 and CsGS2 pro-LUC/p EAQ-CsMYB38 were obviously lower than that in the control group,while the relative intensities of LUC/REN in tobacco cells co-transfected with CsGDH2 pro-LUC/p EAQ-CsMYB38 was significantly higher than that in the control group,indicating that CsMYB38 could inhibit the transcriptional activities of CsGS1 and CsGS2 promoters,and promote the activity of CsGDH2 promoter.These results suggest that CsMYB73 might negatively regulate L-theanine metabolism by inhibiting the transcriptional expression of CsGS1,CsGS2 and CsGDH2 genes.CsMYB38 inhibits the expression of CsGS1 and CsGS2,while it can also promote the expression of CsGDH2,indicating CsMYB38 has a complex regulatory role in the metabolism of L-theanine.