Cloning,Molecular Evolution And Genetic Transformation Of Limonene Synthase Gene From Mentha Haplocalyx Briq.(MhLS)

The genus Mentha are important medicinal plants and the second largest source of aromatic plants in the world.Due to the presence of volatile oils,Mentha play an unique roles in the pharmaceutical,cosmetic,flavor and food industries.Meantime,The genus Mentha is difficult to taxonomy because of altofrequent intra-and inter-specific hybridization and polyploidization.It is important to elucidate volatile oil biosynthesis pathway and phylogenetic development of Mentha.1.Cloning,bioinformatics and expression analysis of MhLS geneWith the homology-based cloning method,primers were devised according to limonene synthase in Mentha longifolia to amplify full-length cDNA sequence from the tender leaf of M.haplocalyx Briq.As a result,a length of 1900 bp fragment was obtained,which contains 1800bp ORF,encoding a polypeptide containing 599 amino acids.The deduced weight of the protein molecular is about 70 kDa,and theoretical pI is 5.36.Among the amino acid compositions in the protein,Leu shares a highest percentage,and Cys is the least.There is no signal peptide was found,and 45 phosphorylation reaction sites and no glycosylation site were detected in the gene.Furthermore,BLAST analysis indicates that it shares a high homological identity with LS from other Mentha species.Results of conserved domain prediction illustrated that this gene contains the plant-terpene-cyclase conseved domain,which belongs to the Isoprenoid Biosynthesis C1 super family.Based on these preliminary bioinformatics analysises,this gene fragment was named limonene synthase[Mentha haplocalyx Briq.(MhLS).2.Molecular evolution analysis of MhLS geneThe structure and molecular evolution status of MhLS gene was analysed among 13 mint samples and 2 outgroup samples.The results of estimation on Gene structure,sequence length,GC content of the different gene regions,Ts/Tv ratio showed that MhLS contains 7 exons and 6 introns,with a conservative structure between different samples.M.arvensis.M.haplocalyx 1?M.haplocalyx 2?M.haplocalyx 3?M.haplocalyx 4?M.rotundifolia 1?M.rotundifolia 2 and M.spicata var.crispa 2 share an equal length(1800bp)of ORF,others own a three-codon insertion than formers.The three-codon insertion happened in exonl.There was no more length variation in the section of exon among all mint linkages.Yet,the outgroup sequences showed several variations with Mentha samples,e.g.,exonl presented 2 codons insertion,exon 2 deduced 5 codons and exon 3 showed an insertion of 6 codons.Among exon 4 to exon 7,there was no length variation neither in Mentha group or in outgroup.For nucleotide constitution,A takes a percentage of 31.6,as C is 18.2,G is 20.3 and T is 29.9;the GC contents of different samples are varied from 35.1%to 39.4%in full-length DNA sequences,42.9%-46.5%in exons,and 27.3%-29.2%in introns.Codon bias was occured in majority sequences.Statistics for amino acid codon frequencies of MhLS illustrated its own favoritism.A total of 305 evolutionary information sites providing sufficiant variation information was detected,of which,134 of which are transition sites and 171 of which are transversion sites.In particular,the events of A replace to G were mostly happened in 42 times.The minimum was the transition of G to C,which had happened 10 times.Substitutions happened most frequently in the third codon,and least in the second codon.In general,sequences of MhLS show a conserved gene structure among the genus and/or outgroups.Meanwhile,there are plenty of substitutions in sequences,which could provide sufficient species evolution information for illustrating genetic relationship between species in Mentha.3.Phylogenetic analysis of Mentha L.based on MhLSMolecular evolution analysis of MhLS gene indicated that this gene posses sufficient genetic information to illustrate the evolution story of the genus Mentha.Consensus phylogenetic tree was obtained using maximum likelihood,neighbor-joining,and maximum parsimony,respectively,based on the full-length genomic DNA sequences,complete ORF coding sequences and predicted amino acid sequences of MhLS.According to the tree Mentha forms a well-supported monophyletic lineage,made up of four different clades.Clade ? consists of M.haplocalyx and two accessions of M.spicata var.crispa(M.spicata var.crispa 1 and M.spicata var.crispa 2).Clade ?,sister to Clade I,includes M.cardiaca and the other two collections of M.spicata var.crispa(M.spicata var.crispa 3 and M.spicata var.crispa 4).This indicates that M.spicata var.crispa seems to be a polyphyly.Inspite of a poorly supported bootstrap value(53),the next clade(Clade ?)includes only M.rotundifolia.Finally,M.arvensis comprised the last clade(Clade ?),which appears to be a sister taxon to the group formed by Clade ?+Clade ?+Clade ?.Results of phylogenetic analysis clearly indicated that M.haplocalyx verified from other species,and M.spicata var.crispa are showing an obvious interspecific differentiation.M.rotundifolia shared close relationship with M.arvensis.4.Transformation of MhLS in Mentha haplocalyx Briq.To clearly identify the function of MhLS during volatile oil biosynthesis,an over expression vector and an inhibitor expression vector were constructed.The plant-expressed vectors promoted by CaMV 35S had been brought into mint genome by the Agrobacterium-mediated transformation method.The adventitious buds were obtained from the transformed mint stem on the MS medium containing 3.0 mg·L-1 TDZ,0.2 mg·L-1 IAA,25%(v/v)coconut juice,50 mg·L-1 kanamycin and 500 mg·L-1 cephalosporin after about 40 days cultivation.After bud initiated,seedlings are sub-cultivating on the antibiotic resistance medium for positive selection.In this study,the transformation system of mint was established.