| Objective:We performed gene cloning,expression analysis and in vitro catalytic function evaluation of IiCOMTs in this study.It is expected to lay a foundation for improving the content of lignans antiviral active components and cultivating high-quality strains of I.indigotica through molecular breeding and other methods.Meanwhile,taking oxygen methyltransferase(OMT)as an example,with the help of the model plant Arabidopsis thaliana(L.)Heynh.,a fluorescent probe-based method for detecting the activity of OMTs in plants in vivo was developed in this study.And it was used to characterize the OMT activity of I.indigotica in vivo.Methods:1.IiCOMTs genes were amplified by RT-PCR.Bioinformatics analysis of IiCOMTs was performed using online tools such as jcvi software,MEGA7.0.21 software,ExPASy,T-COFFEE,SignalP-5.0 and TMHMM Server v.2.0.The expression levels of IiCOMTs genes in roots,stems and leaves were detected by qRT-PCR.2.The recombinant protein of pET28a-IiCOMT was constructed in vitro,and its catalytic activity in vitro was verified by in vitro enzyme activity experiments.Using caffeoyl alcohol,caffeic acid,quercetin and luteolin as substrates,the enzyme kinetic parameters of IiCOMTs for four substrates were determined according to the Michaelis equation method.3.Taking the model plant Arabidopsis thaliana(L.)Heynh.as an example,the in vitro recombinant protein of A.thaliana OMT(including AtCOMT and AtCCoAOMT)was constructed.The substrate probe was screened by in vitro enzyme activity detection,and the enzyme kinetic parameters of A.thaliana OMT to the screened substrate probe were determined.The selected probe was used as a substrate to feed A.thaliana.After feeding for 12 h,the in vivo activity of OMTs in leaves of A.thaliana was detected by Confocal laser scanning microscope,and UHPLC-QTOF-MS was used to determine the metabolic components in A.thaliana after feeding with 3-BTD.The traditional method of detecting the subcellular localization of catalytic enzymes by recombinant fluorescent protein was compared with the new method,and the new method was used to detect the activity of OMTs in the leaves of I.indigotica.Results:1.Sequence and tissue expression characteristics of IiCOMTs:Four IiCOMTs genes were obtained by amplification.The number of bases was about 1000 bp,the encoded protein was about 360 amino acids,and the molecular weight was about 40 kDa.The homologous sequence alignment results showed that the IiCOMTs all had high homology with the COMT of A.thaliana(AtCOMT).The results of collinearity analysis showed that IiCOMT1-3 located on chromosome 5 of the genomes of I.indigotica has a collinear relationship with AtCOMT on chromosome 5 of the genomes of A.thaliana,but IiCOMT4 located on chromosome 7 of the genomes of I.indigotica had no collinear relationship with it.Their amino acid sequences of four IiCOMTs all contain five conserved domains of O-methyltransferase and three conserved domains related to the binding of methyl donor adenosylmethionine.The results of phylogenetic tree analysis showed that IiCOMT1 and IiCOMT3 were clustered into a group,and the homology with Saccharum officinarum L.and Miscanthus sinensis Anderss.was up to 87%;IiCOMT2 and IiCOMT4 were grouped together.IiCOMT2 shares 93%homology with Triticum aestivum L.and Lolium arundinaceum(Schreb.)Darbysh.,and IiCOMT4 shares 94%homology with Panicum virgatum L..The results of real-time fluorescence quantitative PCR showed that the expression of IiCOMT1 gene was the highest in roots,stems and leaves,while the expression of IiCOMT4 was the lowest;and the total expression of IiCOMTs genes was the highest in roots.2.Catalytic properties of IiCOMTs in vitro:Four IiCOMTs had the function of catalyzing the methylation of caffeoyl alcohol,caffeic acid,quercetin,luteolin and 5-hydroxyferulic acid.And four IiCOMTs had different catalytic efficiencies for caffeoyl alcohol,caffeic acid,quercetin and luteolin,and IiCOMT3 had the highest catalytic efficiency for the four substrates(caffeoyl alcohol:kcat/Km=6195.68 ± 147.25 mM-1 min-1;caffeic acid:kcat/Km=1363.26±91.44 mM-1 min-1;quercetin:kcat/Km=11091.14±161.45 mM-1 min-1;luteolin:kcat/Km=4171.57 ± 98.59 mM-1 min-1).In general,the order of affinity of IiCOMTs to different substrates is quercetin>luteolin>caffeoyl alcohol>caffeic acid;the order of catalytic specificity is quercetin>caffeoyl alcohol>luteolin>caffeic acid.3.Establishment of the detection method of plant OMTs activity in vivo:The substrate probe 3-BTD with the highest fluorescence intensity of the product was screened from 14 compounds.The results of its enzyme kinetic parameters showed that both AtCOMT and AtCCoAOMT had good affinity and catalytic specificity for 3-BTD probe(AtCOMT:Km=0.66 ± 0.03 μm,kcat/Km=54.43 ± 1.07 μM-1 min-1;AtCCoAOMT:Km=6.46 ± 0.41 μM,kcat/Km=1946.79 ± 105.01 μM-1 min-1).The observed results of Confocal laser scanning microscope showed that the wild-type A.thaliana fed 3-BTD showed fluorescent signals,while the wild-type A.thaliana without 3-BTD and the negative control group of COMT&CCoAOMT double mutant A.thaliana fed with 3-BTD showed no fluorescence.Meanwhile,the target methylated product 3-BTMD,which produces a fluorescent signal,was detected in wild-type A.thaliana fed 3-BTD.It is demonstrated that 3-BTD can be used as an ideal probe to accurately and truly characterize the enzymatic activity of OMTs in A.thaliana.In the leaves of A.thaliana,the active sites of AtCOMT and AtCCoAOMT detected by the 3-BTD probe were both located in the cytoplasm,while the traditional recombinant fluorescent protein method showed that AtCOMT was located in the cytoplasm,but AtCCoAOMT was located in the cytoplasm and nucleus.The established 3-BTD probe-based OMTs activity detection method in plants was used to detect the activity of OMTs in the leaves of I.indigotica,and it was mainly located in the cytoplasm.Conclusion:1.Four IiCOMTs genes were obtained from I.indigotica.The expression of IiCOMT1 was the highest in roots,stems and leaves,and the expression of IiCOMT4 was the lowest.And the expression level of each IiCOMTs in roots was significantly higher than other tissues,which was consistent with the pattern of higher accumulation level of lariciresinol in roots.2.Four IiCOMTs all have the functions of methylated caffeoyl alcohol,caffeic acid,quercetin,luteolin and 5-hydroxyferulic acid,but the catalytic efficiency is different.IiCOMT3 had the best catalytic activity among four IiCOMTs,and the selectivity of IiCOMTs to the substrates of alcohol and flavonoid was much greater than that of the substrates of acid.3.Taking the model plant A.thaliana as an example,a fluorescent probe-based method for detecting the activity of OMTs in vivo was established.This study improves people’s understanding of the localization and activity of OMTs enzymes,and detect the enzyme activities of OMTs in the leaves of I.indigotica mainly located in the cytoplasm. |
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