Cloning And Functional Verification Of The Key Gene Limonene-3-hydroxylase In Monoterpene Biosynthesis Of Schizonepeta Tenuifolia Briq.

The Schizonepetae Herba is the dry aerial part of Schizonepeta Tenuifolia Briq.It is mostly cultivated and mainly produced in Jiangsu,Hebei and other places.Schizonepetae Herba is pungent and mild.It has the effects of relieving the surface,dispelling wind,clearing eruptions,and reducing sores.Clinically,it is often used to treat colds、measles、rubella and pruritus.Monoterpenoids are the main medicinal components in the volatile oil of Schizonepetae Herba.Among them,menthane type monoterpenes(+)-limonene,(-)-pulegone and(+)-menthone are the main components,which have strong anti-inflammatory,anti-influenza virus and other pharmacological effects.At present,the volatile oil of Schizonepetae Herba for clinical use is extracted from S.tenuifolia,but the content is relatively low.Secondly,due to the influence of soil,climate and cultivation conditions in different growing areas,the quality of the volatile oil of S.tenuifolia is uneven.Therefore,in order to ensure the quality of Schizonepetae Herba,it is necessary to clarify the biosynthetic pathway of the main medicinal components of S.tenuifolia from the perspective of molecular biology.In this paper,based on the S.tenuifolia transcriptome database established in the early stage of the research group,the first cytochrome P450 key enzyme gene limonene-3hydroxylase gene(StL3OH)in the biosynthetic pathway of S.tenuifolia monoterpenes was screened out.The cDNA sequence of the StL3OH gene was cloned and the yeast eukaryotic expression system was constructed,and the function of the StL3OH protein was verified.It is expected to lay the foundation for elucidating the biosynthetic pathway of S.tenuifolia menthane monoterpenes,and also to increase the yield of S.tenuifolia volatile oil.This study provides theoretical support for the application and sustainable development of S.tenuifolia.The specific research contents and results of this paper are as follows:1 Cloning and bioinformatics analysis of StL3OHThe cDNA sequence of StL3OH gene was cloned from the young leaves of S.tenuifolia by RT-PCR.The StL3OH gene cDNA sequence length was 1 598 bp,containing a 1 497 bp complete open reading frame which encoded 498 amino acids.Bioinformatics analysis showed that StL3 OH protein had no signal peptide and had a transmembrane domain,which might be located in endoplasmic reticulum.According to the conserved domain prediction,this protein belongs to the cytochrome P450 superfamily.Multiple sequence alignment showed that the amino acid sequence of MsL3OH(Mentha spicata)protein had a high similar with StL3OH protein,both of which contained cytochrome P450 specific conservative sequences(FGAGRRICPG、AGTETS).Cluster analysis showed that StL3OH protein and mentha L3OH protein converged in one branch,indicating the highest genetic relationship.2 Correlation between the spatiotemporal expression characteristics of StL3OH gene and the accumulation of monoterpenoidsQuantitative real-time fluorescence PCR(qRT-PCR)was used to analyze the expression characteristics of StL3OH gene in different growth stages and tissues of S.tenuifolia.Meanwhile,the main monoterpenoids of S.tenuifolia were detected by gas phase mass spectrometry(GC-MS).The results showed that the expression of StL3OH gene was spatiotemporal.The expression level was high in the early growth stage of S.tenuifolia and tended to zero with the growth of the plant.In the same period,StL3OH gene was expressed in root,stem,leaf and spike,with the highest expression level in spike and the lowest in root.Correlation analysis showed that the expression of StL3OH gene was correlated with the accumulation of(+)-limonene and(-)-pulegone content,which laid a foundation for the function and expression regulation research of StL3OH gene in the biosynthesis of S.tenuifolia monoterpenes.3 Eukaryotic expression and functional verification of StL3OHThe recombinant plasmid pYeDP60-StL3OH of the StL3OH gene and the eukaryotic expression vector pYeDP60 was constructed using seamless cloning technology.The recombinant plasmid PYEDP60-STL3OH was transformed into Saccharomyces cerevisiae strain WAT11 for inducing expression.The enzyme activity reaction was carried out by feeding substrate to Saccharomycetes liquid,and the enzyme activity product was detected by GC-MS.The results showed that the StL3OH protein could catalyze substrate(+)-limonene to produce trans-isopiperitenol.4 Comparison of L3OH protein function between S.tenuifolia and Mentha×piperitaBased on the constructed yeast eukaryotic expression system,by cloning the MpL3OH gene,expressing the MpL3OH protein,and using different chirality(±)limonene as substrates to compare the L3OH proteins function from two plants of S.tenuifolia and Mentha × piperita.The results showed that the StL30H and MpL3OH had the same catalytic activity for(±)-limonene,but had no selectivity for the chirality of the substrate,and had no deterministic effect on the cis-trans isomerization of the product.