| Terpenoid metabolic profile in plant includes primary and secondary metabolites,and is directly involved in plant growth and resistance.IPPI(Isopentenyl pyrophosphate isomerase),FPPS(Farnesyl pyrophosphate synthase)and GGPPS(Geranylgeranyl pyrophosphate synthase)which catalyze the synthesis of terpenoid precursors,exist in plant as a small subfamily.The cooperation of family member determines the synthesis and distribution of terpenoid precursors,and directly impacts the constitution of terpenoid metabolic profile.Target genes were cloned by RACE and RT-RCR from ‘Longjing 43'(Camellia sinensis cv.Longjing43).Combined with the results of gene expression pattern analysis,bioinformatics analysis and function complementary experimental analysis,we made a prediction of the catalytic function of target genes.The main results of this dissertation are as follow:1.Nine genes belonging to Isopentenyl pyrophosphate isomerase and Isoprenyl pyrophosphate synthase gene subfamily were cloned,including two IPPI,two FPPS and five GGPPS.Besides,CsGGPPS9 had three allelic genes(CsGGPPS9-1,CsGGPPS9-2 and CsGGPPS9-3).2.The quantitative Real-time PCR(qRT-PCR)analysis showed that CsIPPI had a low abundance in tea plant,whereas higher in tender root and mature leaf,and CsFPPS got a highest expression in tea leaves,while each number of GGPPS gene family in tea plant had its specific pattern of gene expression.CsGGPPS1 had a low expression level in most tea plant tissues,only higher in two and a bud;CsGGPPS2 was highly expressed in different tissues especially in flowers;The expression level of CsGGPPS3 was higher in leaves and tender root than in flowers,and was stable during the development of flower;The expression level of CsGGPPS4 was similar in different tissues,it shared a same pattern with CsGGPPS2 through flower blooming;CsGGPPS9 was highly expressed in flower than that in tender root and leaves.3.Bioinformatics analysis showed that CsIPPI had a Nudix domain structure,and a catalytic pocket formed by ? folds surrounded by several ? helices;The three-dimensional structure of CsFPPS and CsGGPPS contained a complete terpenoids synthase fold,and two aspartate rich motif used to combining the allyl substrate.Chain-length elongation pocket in the fold,constituted by the ? helices D,E and F,possesses some amino acid residues in three floor loci that determine the chain length of products.The floor 1 loci of CsFPPS1 and CsFPPS2 existed two amino acid residues with an aromatic side chain,thus the catalytic product of FPPS is FPP;CsGGPPS1 and CsGGPPS2,which contained amino acid residues with a middle size side chain in floor 1 loci and thus produced GGPP as their main product,were bona fide GGPPS;Although CsGGPPS9 shared a similar structure with others,the main product of it could be an isopentenyl pyrophosphate which had a chain length more than 30,considering the residuses in all floor loci had a small side chain;CsGGPPS3 was the type?small subunit of heterodimer GPPS,without any aspartate rich motif,and containing two CxxxC motif,and had a terpenoid synthase fold in three-dimensional structure.Although CsGGPPS4 shared the same pattern in FARM with bona fide GGPPS,the last aspartate residue in SARM transformed to glutamate make it a type?GPPS.SSU.A function complementary experiment revealed that the proteins that CsGGPPS1,CsGGPPS1,CsGGPPS9-1,CsGGPPS9-2 and CsGGPPS9-3 encoded had catalytic activity.4.Based on the results of gene expression pattern analysis,proteins structure analysis and function complementary experimental analysis,we made a prediction of the catalytic functions of GGPPS in tea plant.CsGGPPS3 and CsGGPPS4 could be the GPPS.SSU,playing an important role in controlling the direction of terpenoid metabolic flux.CsGGPPS9 might provides precursor to synthase the isoprene chain of plastoquinone and ubiquinone as it functioned as solanesyl pyrophosphate synthase.CsGGPPS1 and CsGGPPS2 were bona fide GGPPS,supplying the precursor for GGPP-drived terpenoid in tea plant. |
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