Research On WlTPSs Of Wurfbainia Longiligularis And Its Comparison With WvTPSs Of Wurfbainia Villosa

1 ObjectiveThe Zingiberaceae plants,Wurfbainia longiligularis and Wurfbainia villosa are both the Fructus Amomi.The medicinal parts of these plants are their mature and dried fruits,which are rich in monoterpenoids such as bornyl acetate,borneol,camphor,camphene,and pinene.Bornyl acetate is considered the quality index in the China Pharmacopoeia.We have identified 66 members of the TPS gene family from the W.villosa genome,and have identified the catalytic function of 18 TPSs,including bornyl diphosphate synthase（BPPS）,the key enzyme responsible for synthesizing bornyl diphosphate precursor,the main effective component of W.villosa.We have also identified TPS（WlTPS24）in W.longiligularis,which can catalyze the production of BPP,but its activity is weak.It is speculated that there may be other TPSs in W.longiligularis that can catalyze the production of BPP,and the TPSs of W.longiligularis have not been screened and studied from the genome level.Therefore,we aim to identify the TPS gene family of W.longiligularis and compare it with that of W.villosa to increase our understanding of the terpene synthase gene family of both plants,analyze the molecular basis of the difference of terpene species and content in different medicinal Fructus Amomi,and provide a theoretical basis for improving the germplasm of Fructus Amomi and synthesizing terpenoids and other medicinal effective components.2 Method2.1 Identification of TPS gene family of W.longiligularis and its evolutionary analysis with that of W.villosaTo screen the W.longiligularis TPS gene family,we retrieved hidden Markov models（HMM）corresponding to TPSs in the Pfam protein database,screened the annotation information of the W.longiligularis genome database,and performed local blast on the obtained genes in the transcriptome database.We named the screened TPS gene family with conservative motif and complete open reading frame as WlTPS.We will construct a phylogenetic tree of WlTPS and WvTPS using the MUSCLE tool of TBtools and beautify it with the online tool ITOL（https://itol.embl.de/）.2.2 Chromosome localization,domain visualization and expression thermography analysis of WlTPSsChromosome localization and expression thermogram analysis of WlTPS gene family were carried out by using chromosome localization function module and thermogram analysis module of TBtool software respectively,and the domain of WlTPS gene family was analyzed by using MEME online website（http://MEME-suite.org/tools/meme）,and the meme file obtained by analysis was visualized and beautified by using TBtool domain analysis module.2.3 Screening,cloning and functional identification of candidate WlTPS genesWe screened the genes that had a close clustering relationship with WvBPPS and high expression level in seed,as well as genes that had a close clustering relationship with identified WvTPS and high expression level or organ specificity,as candidate genes.We used the c DNA of the organ site with a high expression amount of the candidate gene as a template for cloning,and used a prokaryotic expression system to induce the fusion protein.We then used the purified protein to perform in vitro enzymatic reaction using geranyl pyrophosphate（GPP）as a substrate.For the purified protein that may catalyze the generation of BPP,GPP was used as a substrate and alkaline phosphatase was added for dephosphorylation.GC-MS was used to detect the catalytic product.2.4 Real-time fluorescent quantitative PCR of BPPS-related genesA specific primer was designed for q RT-PCR based on the gene sequence using c DNA from various tissue parts of W.longiligularis（60 days after anthesis）and W.villosa seed as templates.The gene expression level was normalized using the 2^-△△Ct method and the data were analyzed using Graph Pad Prism software.2.5 Enzymatic kinetic analysis of WlBPPs and WvBPPSThe optimum reaction time and protein concentration of the enzyme were determined,and the in vitro enzyme activities of WlBPPS and WvBPPS were measured under a range of substrate concentrations.The content of borneol generated by their catalysis was detected by GC-MS and enzyme kinetic parameters were calculated using Graph Pad Prism8.0 software.2.6 Construction and functional identification of WlTPS24 and WvBPPS mutantsBased on the results of the previous active pocket analysis of WvBPPS binding to substrates by the research group,site-directed mutagenesis was performed at the sites that were not subjected to mutation validation.The in vitro enzyme activity detection method was shown in item 2.3.2.7 Comparative analysis of pinene synthase between W.longiligularis and W.villosaThe amino acid sequence of Pinene synthase（PS）was downloaded from Uniprot and sequence alignment was conducted with WlPS and WvPS,as well as the prediction of PS key sites.Correlation analysis was conducted on the expression of WlPS and WvPS and pinene content in each tissue part based on transcriptome data of W.longiligularis and W.villosa,as well as terpene content data obtained by the research group.3 Results3.1 Identification and biological information analysis of TPS gene family in W.longiligularisA total of 75 candidate sequences with full-length CDS of WlTPS were obtained through screening.These were classified into 5 subfamilies of TPS,namely TPS-a,TPS-b,TPS-c,TPS-e/f,and tps-g.The 75 WlTPS were distributed across 13 chromosomes,with a total of 14 tandem repeat gene clusters.Fragment replication events were observed in 15genes.3.2 Screening,cloning and functional identification of candidate genes involved in the synthesis of major monoterpenoid components from W.longiligularisBased on the clustering relationship and expression spectrum information of the transcriptome,15 WlTPS candidate genes were screened in the TPS-b subfamily.Of these,11 were successfully cloned and identified.pinene synthases,namely WlTPS5 and WlTPS8.WlTPS21 and WlTPS33 were identified as ocimene synthases,while WlTPS26,WlTPS28,and WlTPS30 were found to generate BPP.WlTPS32 generated pinene and limonene,WlTPS35 was linalool synthase,and WlTPS36 and WlTPS51 were bifunctional synthases that catalyzed the production of linalool and nerolidol from GPP and FPP,respectively.3.3 Expression patterns of BPPS-related genesWlTPS26 and WlTPS28 were specifically expressed in flowers,while WlTPS24 was mainly expressed in stems,and WlBPPS was mainly expressed in seeds and flowers.The expression level of WvBPPS in seeds was much higher than that of WlBPPS,while WlTPS24,WlTPS26 and WlTPS28 were almost not expressed in seeds.3.4 Enzymatic kinetic analysis of WlBPPS and WvBPPSEnzyme kinetic analysis of WlBPPS and WvBPPS showed that the Km value of WvBPPS was small,indicating high affinity for substrates.However,the Vmax and Kat/Km values of WlBPPS were higher than those of WvBPPS,indicating that WlBPPS had better catalytic activity for GPP than WvBPPS.3.5 Construction and functional identification of WlTPS24 and WvBPPS mutantsBased on previous analysis of key sites of WvBPPS by our research group,a site V455 close to the conservative motif"DTE"was identified,and a mutant V455R of WvBPPS was constructed.Additionally,the mutant V450I was constructed based on the mutant V450A of early WvBPPS.At the same time,the"I"at this site of WlTPS24 was mutated into"V,"and the mutant I450V was constructed.The catalytic results showed that the activity of V450I was significantly decreased,while that of V455R lost its catalytic activity.However,the catalytic activity of mutant I450V of WlTPS24 was not significantly different from that of the wild-type mutant.3.6 Comparative analysis of PS between W.longiligularis and W.villosaThe amino acid sequence identity ofα-pinene synthase（APS）WlAPS,β-pinene synthase（BPS）WlBPS,APS WvAPS,and BPS WvBPS was found to be 98.33%.The key site of APS WlAPS and APS WvAPS,which are the main products ofα-pinene,is consistent with that of Paeonia lactiflora APS（Pl APS^）,which exhibits high selectivity forα-pinene product,and both of them have Phe（F）at this site,while BPS WlBPS,BPS WvBPS,and other reported PS have Leu（L）at this site.Based on the products of WlAPS/WlBPS/WvAPS and WvBPS and their expression patterns in different tissue sites of W.longiligularis and W.villosa,it was speculated that WlAPS and WvAPS might be the key enzymes responsible for the synthesis ofα-pinene in the pericarp,seed mass,and flower of W.longiligularis and W.villosa,and WlBPS and WvBPS might be the key enzymes responsible for the synthesis ofβ-pinene in the pericarp and flower.4 ConclusionIn this study,the TPS gene family of W.longiligularis was identified for the first time,and 11 WlTPS belonging to the TPS-b subfamily were identified in vitro,including WlBPPS,the key enzyme responsible for BPP synthesis in W.longiligularis.WlTPS26 and WlTPS28 have the function of catalyzing GPP to generate BPP.The TPS gene families of W.longiligularis and W.villosa were clustered for the first time,and the BPPS and PS of W.longiligularis and W.villosa were compared and analyzed from amino acid sequence differences,enzyme kinetics,catalytic products,and expression patterns.This provides a theoretical basis for comprehensively understanding the metabolic pathway of monoterpenoids in W.longiligularis and W.villosa and improving the germplasm of Fructus Amomi and synthetic terpenoids.