Cloning And Functional Analysis Of Jasmonic Acid Carboxyl Methyltransferase (JMT) From Camellia Sinensis

Methyl jasmonate(Me JA)is an important component of perfume.It is an organic ester that can release volatile organic compounds(VOCs)like flowers.Me JA is widely distributed in angiosperms,gymnosperms,ferns and algae,and plays an important role in plant life activities such as stress,growth and development.Tea is one of the most popular non-alcoholic drinks in the world.Different cultivar,processing technologies,and geographical locations of tea plants will lead to different aroma of tea.Although the content of these VOCs in tea is trace,it plays a vital role in the aroma and quality of tea.Me JA is one of the important compounds that give tea high quality aroma and taste.Among the six types of tea(black tea,green tea,yellow tea,white tea,oolong tea and dark tea)processed by the same tea cultivar(Longjing43),Me JA content was the highest in the black tea sample.In this study,the changes of methyl jasmonate and jasmonic acid during the processing of black tea were used as the reference point to study and identify a new JMT(Jasmonate carboxymethyl transferase)—— CsJMT in tea plant,which can catalyze the synthesis of Me JA.CsJMT was successfully cloned from c DNA of tea leaves,and the activity of CsJMT was verified by using Escherichia coli(E.coli).Meanwhile,the function and expression characteristics of CsJMT were studied by bioinformatics analysis,subcellular localization,prokaryotic expression,enzyme activity detection,three-dimensional structure prediction,site-specific mutation,real-time fluorescence quantitative analysis,etc.The results of this study give a new understanding of the formation,optimization and regulation of tea aroma,and have a positive significance for improving the aroma quality of black tea.The main research results are as follows:(1)The content of Me JA in the processing of black tea was detected by SPE and GC-MS,and it was found that the content of Me JA was in an increasing state during the process of fresh leaves,withering,rolling and fermentation.At the same time,the content of JA in tea leaves during the processing of black tea was extracted and detected by LC-MS.It was found that the content of JA also showed a trend of regular change.(2)Two complete segments of ORF were cloned,with the length of 1077 bp(ORF-1)and 1161 bp(ORF-1),respectively.Enzymatic reaction of recombinant p GEX-4T-1-ORF-1 showed that no Me JA was detected when JA was used as substrate.The enzymatic reaction of recombinant protein p GEX-4T-1-ORF-2revealed that Me JA was detected when JA was used as substrate.ORF-2 was named CsJMT(login number NW407182).CsJMT has the highest homology with JMT in strawberry(Fragaria vesca subsp.vesca).The three-dimensional structure of CsJMT was predicted,and its SAM/SAH binding site,substrate binding site,JA binding site and key catalytic amino acid residues were determined.(3)Recombinant vector p K7GWF2-CsJMT was introduced into Agrobacterium tumefaciens.The transient expression of CsJMT was confirmed by injection into tobacco leaves and Arabidopsis protoplasts,and the localization of CsJMT was confirmed in the cytoplasm.(4)qRT-PCR was performed on root,stem,bud,first leaves,second leaves,old leaves,flower and fruit of tea plant.It was found that the expression of CsJMT was high in bud and leaves,the highest in the second leaves.In black tea processing(fresh,withering,rolling,fermentation),qRT-PCR showed that the expression level of fermentation samples was the highest.