Cloning,Expression And Protein Function Analysis Of TPS Genes In Lagerstroemia

Both Lagerstroemia caudata and L.indica ’Baiyunyingxia’ are white flowers,and monoterpenoids are the main different volatile components of the flowers during their blooming period.However,the biosynthesis-related genes and functions of Lagerstroemia are not yet clear.In order to analyze the molecular mechanism of the biosynthesis of monoterpenoids in Lagerstroemia,full-length transcriptome sequencing was performed to screen and obtain differentially expressed TPS genes.Use L.caudata and L.indica ‘Baiyunyingxia’ as materials to isolate TPS genes,Analyze the expression pattern of genes and conduct preliminary verification of their functions.The main results are as follows:1.Full-length transcriptome sequencing was performed on mixed RNA samples of the flowers of L.caudata at three stages,and Unigene sequences with read length better than the second-generation sequencing transcriptome was obtained,database annotation function,SSR locus and TFs family were searched.By searching the conserved domains of terpene synthase protein and combining the differential expression data of second-generation transcriptome genes,two genes,Lc TPS1 and Lc TPS14,which are consistent with the aroma release law of L.caudata were screened out.2.Lc TPS1 and Lc TPS14 have been isolated in L.caudata and the homologous sequences Li TPS1 and Li TPS14 have been isolated in L.indica ‘Baiyunyingxia’.Lc TPS1 CDS is 1779 bp long and encodes592 amino acids.Li TPS1 CDS is 1776 bp long and encodes 591 amino acids.Both contain terpene synthase conserved domains RRX8 W,DDXXD and DTE/NSE.The phylogenetic tree assigns them to TPS-b.Subfamily.The Lc TPS14 and Li TPS14 genes are both 1575 bp in length and encode 525 amino acids.They contain DDXXD and DTE/NSE domains,and RRX8 W is deleted.They are typical members of the TPS-g subfamily.3.The results of q RT-PCR indicated that the expression of Lc TPS1 was the highest during the blooming period of L.caudata,while the expression of Li TPS1 reached the peak during the immature flower bud period of L.indica ‘Baiyunyingxia’.In all parts of the flower during the blooming period,Lc TPS1 expressed higher in the pistil,while Li TPS1 expressed higher in the stamens of L.indica‘Baiyunyingxia’.Both Lc TPS14 and Li TPS14 had the highest expression levels during the blooming period.Lc TPS14 was specifically expressed in pistil,Li TPS14 was highly expressed in petals and pistils,and the expression was lower in other parts.4.Combined with signal peptide,transit peptide,and subcellular location prediction,Lc TPS1 and Li TPS1 are very likely to be expressed in the plastid,while Lc TPS14 and Li TPS14 are predicted to be expressed in the cytoplasm.The plant expression vector is constructed and verified by the transient expression technology of tobacco leaves,Lc TPS1 and Li TPS1 are located in the chloroplast,which means they are expressed on the plastid;Lc TPS14 and Li TPS14 are expressed in the cytoplasm.5.Use the prokaryotic expression system to construct different prokaryotic vectors,select p ET30 a to express the fusion protein,obtain recombinant proteins Lc TPS14 and Li TPS14 in the supernatant,and obtain Lc TPS1 and Li TPS1 inclusion bodies in the precipitate.In vitro enzymatic reaction combined with GC-MS,the recombinant proteins Lc TPS14 and Li TPS14 catalyze GPP to produce linalool and FPP to produce E-nerolithol,indicating that Lc TPS14 and Li TPS14 recombinant proteins are terpene synthase with dual catalytic functions.The inclusion bodies Lc TPS1 and Li TPS1 have no biological activity.In this paper,we screened out terpene synthase genes that are consistent with the aroma release rules based on full-length transcriptome data,and successfully isolated the genes in L.caudata and L.indica‘Baiyunyingxia’,and carried out bioinformatics analysis on them,and subcellular location And prokaryotic expression function verification,to further understand the molecular mechanism of Lagerstroemia floral biosynthesis,to provide a basis for the breeding of Lagerstroemia floral.