Cloning Of Terpene Synthase Gene(TPS) In Chrysanthemum Indicum Var.Aromaticum And Genetic Transformation Of Chrysanthemum Indicum

Chrysanthemum indicum var.aromaticum is a new variant of Chrysanthemum indicum.The main chemical components of the whole plant are a-tuphibines,β thujone,borneol and other anthraquinones.Chrysanthemum indicum var.aromaticum is a kind of rare aromatic flower in the Chrysanthemum and its flowers,leaves,and roots are full of aromas.The terpene synthase(TPS)is a key enzyme in the terpenoid metabolic pathway.It plays an important regulatory role in the aromatic substances in plants.The purpose of this study was to investigate the role of the TPS gene of Chysanthemum indicum var.aromaticum on the regulation of monoterpenoids in plants.The regulatory potential of CiTPS has been studied in Nicotiana tabacum by developing transgenic plants constitutively expressing CiTPS,and the genetic transformation of chrysanthemum was performed in order to better dig the fragrant value of Chrysanthemum indicum var.aromaticum,improve the aroma quality of Chrysanthemum indicum,and lay the foundation for the cultivation of color-flavored chrysanthemum varieties.In this study,Chrysanthemum indicum var.aromaticum was used as the experimental material,and the monoterpene synthase gene was cloned from the basis of the transcriptome data on Chrysanthemum indicum var.aromaticum in the previous period of this laboratory,and the gene was analyzed for bioinformatics.The over-expression plant vector was constructed and transformed into Nicotiana tabacum,and the function of the CiTPS gene was preliminary explore.Then the genetic transformation system of Chrysanthemum indicum was optimized,and the CiTPS gene was transferred into Chrysanthemum indicum finally.The results of the study are as follows:1.The open reading frame of CiTPS is 1818bp,which encoded a protein sequence of 605 amino acids;the gene has a monoterpene synthase conserved domain and highly conserved sequence:DDXXD(N,D)D(L,I,V)X(S,T)XXXE and RRX8W,which belongs to the monoterpene synthase family,has the function of synthetise monoterpene synthase.The gene encoding a protein,which molecular weight is 70.11kDa,theoretical isoelectric point(pI)is 5.80,it belonged to unstable protein;and its hydrophilicity was strong;multiple sequence alignment and phylogenetic analysis showed that the CiTPS gene similarity with trans linalool QH1 gene similarity in Artemisia annua reached 91%,the CiTPS gene belonged to TPSb family,and CiTPS is more homologous to Artemisia annua and thistle.2.On the basis of the CiTPS gene in Chrys’anthemum indicum var.aromaticum,weconstructed the pBI121-TPS-GFP overexpression vector with the method of double enzymedigestion.The pBI121-TPS-GFP recombinant plasmid and pBI121-GFP empty vector plasmid were introduced into Agrobacterium by electroporation.CiTPS gene and pBI121-GFP vector were transferred into tobacco plants by Agrobacterium-mediated method.The results showed that the several positive lines of CiTPS gene and pBI121-GFP empty vector had been successfully obtained by resistance rooting detection,PCR detection of resistant plant DNA and RNA.and qRT,PCR Three transgenic lines expressing CiTPS and one transgenic pBI121-GFP line were selected to extract leaf secretions contrasted with wild-type tobacco during the vigorous growth stage of the T1 generation.It was found that the CiTPS gene could increase the monoterpene content in plants.3.The leaves of Chrysanthemum indicum was induced best in MS medium additional 1.0 mg/L 6-BA and 0.8 mg/L NAA.The kanamycin optimal critical tolerance concentration is 8 mg/L,which is most suitable the Chysanthemum indicum leaves differentiation.And the optimum rooting critical tolerance concentration is 10 mg/L.The inhibitory concentration of cefotaxime was selected to be 200 mg/L in the screening culture stages.The genetic transformation rate was increased with the procedures of the 2 d pre-culture,10 min infection,3 d co-cultivation.Agrobacterium-mediated method was used to transfer CiTPS gene and pBI121-GFP empty vector into wild-type Chrysanthemum indicum.Through the selection of resistance rooting and PCR detection of resistant plants DNA and RNA,it was proved that several positive lines of CiTPS gene and pBI121-GFP empty vector had been successfully obtained to wild-type Chrysanthemum indicum.