Gene Isolation And Protein Expression Analysis Of LcIPS Families From Litsea Cubeba

Litsea cubeba is an important tree species of natural essential oil produced mainly from the fruits.L.cubeba essential oil is widely used in chemical industry,agriculture,pharmaceutical industry and so on.The biosynthesis pathway and primary gene of terpenoids,the major components of L.cubeba essential oil are unclear,so the exploration of primary gene in terpenoids biosynthesis can provide gene knowledge for genetic improvement of L.cubeba.Monoterpene and sesquiterpene are the main active substances in L.cubeba essential oil.Several key enzymes have been identified in terpenoids biosynthesis pathway so far,but there is no reports about isoprenyl diphosphate synthases(IPS)from L.cubeba,which play competitive regulation roles.We identified 14 LcIPSs based on transcriptome database of L.cubeba fruits from maturation and analyzed their expression patterns.In addition,we picked up 4 genes from L.cubeba geranyl diphosphate synthase(LcGPPS)and geranylgeranyl diphosphate synthase(LcGGPPS)families to conduct sequence and protein expression analysis,then predicted and verified the protein interaction from the two families,which provided a theoretical basis for clarifying the regulatory mechanism and improving yield and quality of L.cubeba essential oil,the main results are as follows:(1)The identification and expression pattern analysis of three LcIPS gene families were conducted.We found 14 LcIPS separately from farnesyl diphosphate synthase(FPPS),GPPS and GGPPS families based on transcriptome database,and analysed their expression patterns in different tissues and fruits in maturation.As result,14 LcIPSs exhibited unique expression patterns and there were at least one gene playing the main role.In LcFPPS family,LcFPPS2 revealed an obvious expression peak in fruits maturation.We found two types of genes separately encoded homodimer and heterdimer in LcGPPS family,and LcGPPS.SSU1 encoded the small subunit of heterdimer showed significant expression peak in fruits maturation.In LcGGPPS family,LcGGPPS1 expressed high level in most tissues,LcGGPPS2 expressed specifically,LcGGPPS3-7 play roles in flower and fruits maturation terpenoids biosynthesis,and LcGGPPS6 showed no obvious rhythm in fruits maturation,while the other six genes exhibited specific period of function.(2)Clone,sequences analysis and physical and chemical property prediction of LcGPPS1,LcGPPS.SSU1,LcGGPPS1 and LcGGPPS3 were investigated.Based on ExPASy prediction,the ORF of LcGPPS1 is 966 bp,encoding 321aa;the ORF of LcGPPS.SSU1 is 897 bp,encoding 298aa;the ORF of LcGGPPS1 and LcGGPPS3 are both 1152 bp,encoding 383 aa.Relative molecular weight of protein LcGPPS1,LcGPPS.SSU1,LcGGPPS1 and LcGGPPS3 are respectively 34.97,32.21,41.39 and 41.49 kDa;theoretical pI are 5.43,5.33,5.86 and 5.87;protein molecular formula are separately C1535H2499N421O477S15,C1413H2279N395O431S16,C1828H2960N504O551S18,C1820H2926N510O554S21.The prediction of subcellular localization showed that LcGPPS1 was probably located in nucleus,LcGPPS.SSU1 was probably located in chloroplast or cytoplasm,LcGGPPS1 and LcGGPPS3 is mostly located in mitochondrion.(3)Analyze the recombinant protein of LcGPPS1,LcGPPS.SSU1,LcGGPPS1 and LcGGPPS3 using eukaryotic and prokaryotic expression system.After constructing secretory expression vector,transforming into Pichia Pastoris strain X33 and methanol induction,we found that the four recombinant proteins were mainly expressed in cells in form of monomers and dimers,and little was secreted out of the cell.And we got LcGGPPS3 recombinant protein using Nickel column purification.Then we conducted pET28a-LcGGPPS1 and pET28a-LcGPPS.SSU1,and separately transformed into Escherichia coli BL21(DE3)and Rosetta strain,and got LcGGPPS1 recombinant protein after IPTG induction and chelating SFF(Ni)column purification.(4)Three dimensional structure prediction and yeast two hybrid were employed to verify the interaction of LcGPPS.SSU1 separately with LcGGPPS1 and LcGGPPS3.LcGGPPS1 and LcGGPPS3 were not classified into LSU clade,and LcGGPPS3 showed distance with most GGPPS in genetic.In the three dimensional structure of LcGPPS.SSU1,Lc GGPPS1 and LcGGPPS3,we found a common GPPS core,which constitute the most structure of LcGPPS.SSU1.Based on the prediction,both LcGGPPS1 and LcGGPPS3 can interact to LcGPPS.SSU1.While demonstrated by yeast two-hybrid test,only LcGGPPS3 could interact to LcGPPS.SSU1 weakly,LcGGPPS1 could not interact to LcGPPS.SSU1.