Polymorphism And Functional Analysis Of Monoterpene Synthase Gene From Lilium

Lily has a variety of floral fragrance,the floral fragrance is different among hybrids.Most Asiatic hybrids lily has no floral fragrance,but Oriental hybrids lily has a strong floral fragrance,which can not obtain the love of consumers.The aim of Lily breeders is to increase the fragrance of Asiatic hybrids lily and to reduce the fragrance of Oriental hybrids lily.The key ingredient in floral fragrance substance of Oriental hybrids lily is monoterpene,so the study about lily floral fragrance gene is mainly concentrated on the monoterpene syntheases gene.The composition and content of monoterpene vary with varieties.In order to study the polymorphism of monoterpene syntheases gene in different Lily species,the monoterpene syntheases gene of Lily was cloned in 66 lily cultivars.A total of 32 monoterpene syntheases genes were obtained.These genes could be divided into three categories,They were named LTPS-a,LTPS-b,LTPS-c respectively.Among the 32 genes,23 belong to LTPS-a,2 belong to LTPS-b,and 7 belong to LTPS-c.LITPS-a is found in 71.01%cultivars,it contain an open reading frame(ORF)of 1761 bp that encodes a protein with 586 amino acid.We obtain Minimum number of LTPS-b from 66 lily cultivars,with insertion of 12 nucleotides,it contain a 1773 bp ORF that encode a protein with 590 amino acid.LTPS-c have a deletion of 114 bases.It contain a 1647 bp ORF that encodes a protein with 548 amino acid.The three kinds of the LTPS have highly conserved regions,the active site lid residues RRX8W motif that is appear in all monoterpene synthases gene and substrate-Mg2+ binding site DDXXD motif that is appear in all terpene synthases gene.LTPS-a and LTPS-b contains substrate binding pocket RX8H,but LTPS-c have no the pocket.In the correlation analysis of mutation site and the content of a-pinen,the correlation coefficient of 230,872 is higher and greater than 0.5.In the correlation analysis of mutation site and the content of ocimene,the correlation coefficient of 99,101,407,1108,1150,1225 is higher and greater than 0.5.The three gene were obtained from O11 which has a strong floral fragrance,them were named Lso TPS-a,Lso TPS-b,LsoTPS-c,two gene were obtained from AT6,them were named LrlTPS-a,LrlTPS-c.Five gene were subcloned into expression vector,expressed in Escherichia coli,After induction,genes were expressed in Escherichia coli,but the expression level of LrlTPS-c is low.To analysis enzyme activity,protein is extraction by sonication device,adding a substance of monoterpene syntheases:geranyl pyrophosphate(GPP)into protein extract,5 genes can transfom GPP into ocimene.