Limonene Synthase Gene Cloning And Sequence Analysis Of Mentha Haplocalyx Briq. And Mentha Spricata L.

The plants of Mentha haplocalyx Briq.and Mentha spicata L.,which are perennial herbage,are of Mentha L..They,which are kinds of traditional Chinese medicine and the world's major perfume plants,are widely used in medicine,food, beverages,cosmetics,cigarettes and daily necessities etc.,and have a high ornamental and economic value.The essential oils are the main chemical components and effective components of Mentha haplocalyx Briq.and Mentha spicata L..According to the differences of the composition and content of essential oils,the can be divided into different chemo -types.There are large amount of monoterpenes in Mentha haplocalyx Briq.and Mentha spicata L.Monoterpenes,the C₁₀ members of the terpenoid family of natural products,are common constituents of plant-derived essential oils that serve a variety of different functions in basic and specialized metabolism.In addition,they are used in a wild range of industry such as foodstuff and medication.Monoterpenes are all synthesized from the common precursor geranyl diphosphate(GPP) via the plastid localized 1-deoxy-D-xylulose-5-phosphate pathway(DXP) pathway.Monoterpene synthases are to a large extent responsible for the diversity of monoterpene structures,but it was displayed that the gene-expression level often below or silencing.Limonene is the simplest one in all the cyclic terpenes and the intermediate of many cyclic terpenes.Therefore limonene sythase becomes one of the model for studying cyclic monoterpene synthase.Through limonene synthase gene cloning and sequence analysis of Mentha spricata L.and Mentha haplocalyx Briq,this paper aims at finding out that if there is obvious differences with the gene sequences of monoterpene synthases which are from two various species of Mentha L.and the same species but various chemotypes.The main results were as follow:1.The monoterpene synthase cDNAs were isolated by using RT-PCR from Mentha spricata L.(Carvone) and Mentha haplocalyx Briq.of two different chemotypes(Linalool and Menthone-Menthol) and marked as M3,M2 and M1.The length of M1 is 1632bp,M2 is 1680bp and M3 is 1587bp,including the open reading frame except the sequences coding leading peptide,which encoded 543,560 and 528 amino acid proteins,putative the protein molecular weights are 63.7kD,65.7kD and 62.1kD,isoelectric points are 4.73,4.74 and 4.87.The analysis result of nucleic acid and protein sequences speculated preliminarily that the gene cloned is limonene synthase.Bioinformatics analysis suggested that it is hydrophilic protein.2.In the experiment,the four characteristic sequences,RRx₈W,LQLYEASFLL whtich are sited on N-terminal and(N,D)D(L,I,V)X(S,T)XXXE,DDx₂D which are sited on C-terminal,are highly conserved in M1 and M2 which are two chemotypes of M. haplocalyx Briq..But the sequences of M3,which cloned from M.spicata L.,does not contain the conserved amino acid sequences(DDx₂D),the other three characteristic sequences are also conserved with M1 and M2.Through prediction and analysis of secondary structure,speculated influence of the sequence(DDx₂D) deletion on secondary structure and fuction of the gene cloned is little.3.Through clustering analysis of the amino acid sequences from the three genes cloned and the limonene synthase of other plants,found that Mentha,Agastache rugosa, Schizonepeta,Perilla and Lavender belonged to Labiatae,and their amino acid sequence similarity of limonone synthase are higher,so they could be closer,and vice versa.4.The relationships between the nucleotide sequences and amino acid sequences of three limonone sythases cloned and species and chemotypes were not significant.M2 and M3 of M.haplocalyx Briq.showed relatively high homologous with M1 of M. spicata L.on the nucleotide sequences and amino acid sequences,and the sequence similarity is higher than 91%.However,the sequence similarity of M1 and M3 is dslightly higher than that of M1 and M2 which are both of M.haplocalyx Briq..