Characterization Of Alternative Splicing And Functional Analysis Of Its Roles On Catechins Biosynthesis In Tea Plant(Camellia Sinensis)

Alternative splicing（AS）regulates m RNA at the post-transcriptional level to change genefunction in organisms.Catechins were main secondary metabolites in tea plant,determined the flavor of tea infusion.Many studies about mechanism of biosynthesis and regulation of catechins were reported,however,molecular mechanism of whether and how alternative splicing involved in its biosynthesis were still unclear.Hence,we used the transcriptome of different tissues,growing months and stages of tea leaves to analyze AS landscape and dynamics by WGCNA.To further uncover the regulation of catechins biosynthesis mediated by AS,we characterized the AS transcripts which related to catechins biosynthesis based on correlation analysis.Our study aimed to reveal the dynamics and mechanism of AS on catechins biosynthesis,and provide new insights into biosynthesis mechanism of catechins and development of new technologies on catechins biosynthesis regulation.The main results were listed as following:1.Characterization of alternative splicing of genes and its correlation analysis with catechins bionsynthesis in tea plant.We performed Illumina sequence of samples from different tissues（bud,young leaf,mature leaf,stem,root,flower,fruit）,growing months（April,June,August,September and October）and stages(bud,1^st leaf,2^nd leaf,3^th leaf and 4^th leaf in a round shoot)of tea leaves.Totally,18,295 genes were identified as AS genes and generated 105,845 AS transcripts.The most dominant（～20%）splicing pattern was intron retention.Tissue-specific AS genes were identified in eight tissues,while this specificity was not found in different months and stages of tea leaves.Furthermore,many AS events were characterized in catechins biosynthesis genes,for instance,4CL,DFR and ANR produced more than three different AS transcripts,and expression patterns of some of them positively correlated with accumulation of catechins.AS transcript of PAL was positively correlated with content of EGCG（r²=0.71）,while its full-length transcript exhibited opposite correlation（r²=-0.57）.2.The determination of JA involved in catechins biosynthesis and identification and dynamics of alternative splicing of critical genes（JAZ3 and LOXs）We constructed a co-expression network based on expression patterns of AS genes from different months of tea leaf,accumulation patterns of catechins and phytohormone by WGCNA.Results indicated that accumulation pattern of JA positively correlated（r²=0.85）with catechins.We further identified two hub genes from co-expression network Catechins,which exhibited correlation with catechins content.we found one full-length transcript（Cs JAZ3-1）and two alternative splicing variants（Cs JAZ3-2 and-3）were generated,all of them negatively correlated with accumulation pattern of catechins.Subcellular localization indicated that Cs JAZ3-1 localized in nucleus,while Cs JAZ3-2 and-3 localized in cytoplasm.Further,we identified eleven LOX members,and LOX3,9 and 11 positively correlated with C,GC,ECG and EGCG.Experiment of enzyme activity indicated that LOX3 and 9catalyzedα-linolenic acid into 13S-HPOT and 9S-HPOT,respectively,both of these products were generated under catalyst of LOX2 protein.Subcellular localization indicated that LOX2 localized in chloroplast,and LOX9 localized in cytoplasm.Additionally,six LOXs occurred AS events.3.Molecule mechanism of alternative splicing of Cs JAZ3 regulated catechins biosynthesis mediated by JAThe content of catechins were decreased after the expression of Cs JAZ3 was suppressed using as ODN experiment.Furthermore,Cs JAZ3-1 and-2 interacted with Cs MYC2 to suppress transcription of critical catechins biosynthesis genes（Cs DFR,Cs ANR and Cs LAR）by Y2H,Bi FC and SPR experiment,respectively.Meanwhile,Cs JAZ3-2 function as the alternative enhancer of Cs JAZ3-1,competed with Cs JAZ3-1 to interact with master regulator of JA signaling（Cs MYC2）,further repressed the transcription and expression of multiple catechins biosynthesis genes.While Cs JAZ3-3 exhibited JA-insensitivity and performed as repressor to stabilize Cs JAZ3-MYC2 complex by homologous dimerization with Cs JAZ3-1 and-2 in presence of JA,further repressed the transcription activity of Cs MYC2 on down-stream key genes in the catechins biosynthetic pathway.