| Menthae Haplocalycis Herba is a dry aboveground part of the Mentha haplocalyx Briq.in the labiate family.It has a pungent and cool taste,and has the effects of dispersing wind heat and clearing the head.It is clinically used for wind-heat colds,initial onset of wind heat,headaches,and redness of the eyes.M.haplocalyx contains chemical components such as essential oil,flavonoids,phenolic acids,and amino acids,and has pharmacological effects such as antiviral,antibacterial,antioxidant,and anti-tumor effects.M.haplocalyx is rich in resources and widely distributed,with widespread hybridization between humans and nature.The coexistence of sexual and natural reproduction results in many morphological and chemical variations in the population,leading to the formation of different chemotypes characterized by essential oil.Due to significant differences in the composition and content of essnetial oil in M.haplocalyx among different chemotypes,clinical efficacy also varies.The research group has been monitoring the dynamic changes in the content of the main characteristic components in essential oil of different chemotypes of M.haplocalyx for many years in the early stage.It was found that the L-menthol and carvone chemotypes of M.haplocalyx remained stable at different growth stages,while the main component of essnetial oil in pulegone chemotype of M.haplocalyx was L-menthone before early May.After mid to late May,the main component changed to be pulegone,and the chemotype remained stable during its harvesting period（July to October）.Therefore,through transcriptome sequencing of the pulegone chemotype of M.haplocalyx samples in May and August,the key enzyme genes related to the formation of the pulegone chemotype of M.haplocalyx were screened by comparative transcriptome methods,and the key enzyme genes were cloned,expressed and functional analysis,providing a basis for the molecular mechanism of the formation of the pulegone chemotype of M.haplocalyx.It also provides reference for the breeding and rational application development of M.haplocalyx varieties.The main research contents are as follows:1.Analysis of characteristic terpenoids in essential oils of chemotypes of M.haplocalyx at different growth stages.Gas chromatography was used to study the changes in the growth period of essential oils of three chemotypes of M.haplocalyx:L-menthol chemotype,carvone chemotype,and pulegone chemotype.The results showed that the main terpenoid component in the L-menthol chemotype of M.haplocalyx was L-menthol,which accounted for 21.53%to 50.86%of the total essential oil content at different growth stages.The main terpenoid component in the carvone chemotype of M.haplocalyx is carvone,which accounts for 49.44%～61.27%of the total essential oil content at different growth stages.Both chemotypes of M.haplocalyx maintain stable chemical types at different growth stages.Before early May,the highest content of terpenoids in the essential oil of M.haplocalyx was L-menthone.After mid to late May,the highest content of terpenoids in the essential oil of pulegone chemotype of M.haplocalyx transformed into menthone.In 2021,pulegone accounted for 9.44%to 54.36%of the total essential oil content,while in 2022,it accounted for 9.70%to 60.77%of the total essential oil content.The chemotype of peppermint in the traditional harvesting period（July to October）remained stable.2.Research on the differentially expressed genes in the monoterpene biosynthesis pathway in pulegone chemotype of M.haplocalyx.Transcriptome sequencing was carried out on the samples of the difference in the pulegone content in pulegone chemotype of M.haplocalyx by Illumina Hi SeqTM,and the differentially expressed genes were screened in combination with KEGG metabolic pathway（ko00902）.Seven differentially expressed genes were screened,encoding three enzymes,namely limonene synthase（LS）,limonene-3-hydroxylase（L3OH）and menthol dehydrogenase（MD）.Additionally,four genes encoding menthol reductase（PR）with certain differences in expression levels were screened.The relative expression levels of the 11genes were analyzed by q PCR,and it was found that the three genes encoding LS were DN74950＿c1＿g1,DN81505＿c1＿g1 and DN74282＿c2＿g1,among which the expressions of DN81505＿c1＿g1 and DN74282＿c2＿g1 were up-regulated.The expression level of DN74950＿c1＿g1 gene did not change significantly.The expression levels of DN71062＿c0＿g1 and DN77281＿c3＿g5 encoding L3OH were up-regulated.The expression of DN77156＿c2＿g2 gene encoding MD was down-regulated,and the expression of another DN89117＿c1＿g2 showed no significant change.The expression of four genes encoding PR was down-regulated,among which DN73496＿c1＿g2 had a more significant down-regulated trend,and DN81154＿c1＿g1 was second only to DN73496＿c1＿g2.Combined with the monoterpene biosynthetic pathway and the q PCR analysis results,three genes that may be related to the pulegone chemotype of M.haplocalyx were screened out.They are DN71062＿c0＿g1 gene encoding L3OH and DN81154＿c1＿g1（Mh PR1）and DN73496＿c1＿g2（Mh PR2）gene encoding PR respectively.3.Cloning and prokaryotic expression of the limonene-3-hydroxylase gene in pulegone chemotype of M.haplocalyx.The Mh L3OH gene was cloned from the leaves of the pulegone chemotype of M.haplocalyx.The ORF gene contains 255 bases and encodes 85 amino acids.The predicted secondary structure of the gene encoding protein shows that the helix contains 27 amino acids,accounting for 31.76%,and the extended chain contains 15 amino acids,accounting for17.65%,βfolding contains 10 amino acids,accounting for 11.76%,while irregular curling contains33 amino acids,accounting for 38.82%in the Mh L3OH protein.Mh L3OH protein is an extracellular protein without transmembrane domain and signal peptide,which belongs to non-secretory protein.The phylogenetic tree indicates that Mh L3OH has the closest genetic relationship with the L3OH of Mentha Canadensis.The recombinant plasmid p ET-32a（+）/Mh L3OH was constructed,and the recombinant protein was induced and expressed in E.coli.4.Cloning,expression,and in vitro functional study of the pulegone redutase gene in pulegone chemotype of M.haplocalyx.Mh PR1 and Mh PR2 genes were cloned from the leaves of the pulegone chemotype of M.haplocalyx.The ORF of the Mh PR1 gene contains 324 bases,encoding 108 amino acids.The ORF of the Mh PR2 gene contains 1032 bases,encoding 344 amino acids.The predicted secondary structure of the gene encoding protein shows that the the helix contains 53 amino acids,accounting for 49.07%,and the extended chain contains 21 amino acids,accounting for 19.44%,βfolding contains 10 amino acids,accounting for 9.26%,while irregular curling contains 24 amino acids,accounting for 22.22%in the Mh PR1 protein.The helix contains116 amino acids,accounting for 33.72%,and the extended chain contains 74 amino acids,accounting for 21.51%,βFolding contains 34 amino acids,accounting for 9.88%,while irregular curling contains 120 amino acids,accounting for 34.88%in the Mh PR2 protein.Both proteins are extracellular proteins without transmembrane domain and signal peptide,and belong to non-secretory proteins.The phylogenetic tree indicates that Mh PR1 has the closest PR relationship with Nepeta tenuifolia,while Mh PR2 has a closer PR relationship with Perilla frutescens var.hirtella.The recombinant plasmids p ET-32a（+）/Mh PR1 and p ET-32a（+）/Mh PR2 were constructed,and the proteins were induced and expressed in E.coli.In the subsequent protease catalyzed reaction,with（-）-menthone as the substrate,Mh PR1 did not show catalytic activity for the substrate,while Mh PR2 catalyzed（-）-menthone to produce（+）-pulegone,but further research and verification are needed. |
