Mechanism Of Improving The Accumulation Of GABA In Fresh Tea Leaves By Anaerobic Treatment Based On Metabolomics

GABA,a class of four-carbon non-protein amino acids,is widely found in mammals and has the function of inhibiting neurotransmitters,so it has extensive health effects on human.Furthermore,it shows that metabolic of GABA be related to response to biotic and abiotic stresses in plant.It plays an important role in maintaining the balance of carbon and nitrogen in plants."Gabaron Tea"is rich in GABA,for rapidly increasing the content of GABA in tea fresh leaves,in production,the processor always apply anaerobic treatment to process"Gabaron Tea".Research shows that the treatment methods of vacuum filling N₂and vacuum filling CO₂ has obvious effect on the GABA enhancement.In this experiment,picking leaves of“Longjing 43”then treated leaves under different anaerobic treatment for1 hours and 6 hours.Stalling in room temperature was as a control.In this paper,the mechanism of GABA accumulation in tea plant which treated by different anaerobic treatment was analyzed from two aspects by UPLC-QTOF-MS and gene real-time expression detection,in order to comprehensively and systematically analyze the stress response and adaptation strategy of tea plant under different anaerobic conditions.The results are as follows:1.Non-target analysis of samples based on UPLC-TOF-MSUsed UPLC-TOF-MS,the separation and identification of metabolites in fresh tea leaves samples were carried out by electrospray ionization platform,and 1756 and 1523compounds were identified using the data mapping and matching degree identification with compound database in positive ion mode and anion model.Principal component Analysis（PCA）showed that the different process groups could be well distinguished.Orthogonal partial least square method-discriminant analysis（OPLC-DA）results showed that the experimental group distinguished from each other,and the model is of good quality and strong robustness.Using univariate variables statistical analysis and multivariate statistical analysis screened and identified differential compounds.The number of differential metabolites in different groups was in the range from 90 to 408.2.Screening of differential compounds and analysis of metabolic pathwaysThe difference metabolites of different groups mapped to the authoritative metabolite databases such as KEGG and PubChem,and then analyzed the metabolic pathway.Through comprehensive analysis and screening of the pathway of different metabolites（including enrichment analysis and topological analysis）,the key pathways with the highest correlation between metabolites were obtained.The results showed that compared the N₂ treated group the control the metabolic pathways mapped by main differential compounds were:flavonoids and flavonoids synthesis,galactose metabolism,glyoxylate and dicarboxylate metabolism and TCA cycle.Compared the CO₂ group and the control the metabolic pathways mapped by main differential compounds were:Carbon fixation,phenylalanine metabolism,pyruvate metabolism,galactose metabolism,TCA cycle,starch and sucrose metabolism and alkaloid metabolism.3.Selection of key enzymes in differential pathways and the obtaining of gene sequencesAccording to the comparison results,having picked out five metabolic pathways,such as TCA cycle,glycolysis/gluconeogenesis,pentose phosphate pathway,arginine biosynthesis,alanine,and aspartate and glutamate metabolism.And having determined 41key enzymes through KEGG.Using NCBI data to screen the required gene sequence in the whole genome database of the tea tree,we obtained at least 2 homologous gene sequences,and 116 gene sequences were obtained,98 gene sequences could expresses in tea leaves.4.Detection and dynamic analysis of gene expressionApplying the technique Real-time quantitative PCR（qRT-PCR）to detect the relative expression of 98 genes.The results showed that vacuum-filled N₂ and vacuum-filled CO₂were inconsistent in regulating the key genes.With the treatment vacuum filling CO_2,the geneexpressionofphosphoenolpyruvatecarboxykinase,Enolase,glycerol-aldehyde-phosphate dehydrogenase were higher than treatment vacuum filling N₂.Vacuum-filled N₂ treatment facilitates the expression of argininosuccinate lyase,ornithine carbamoyl transferase and arginase.The above results shows that under anaerobic treatment,tea plant initiated the response modes;however,there are differences between the results of two processions.Anaerobic treatment made tea plant subject rapidly to hypoxia damage,later the cells enter the degradation state soon.Under filling CO₂ treatment,the glycolysis facilitated,through it little energy produced for cell metabolism and help tea plant resist anaerobic stress.Long time anaerobic will cause the leaves into catabolism,dipeptide and free amine increased.Procession with filling CO₂ of"Gabaron tea"will be more conducive to the formation of aldehydes,small organic acids and other flavor substances.