Study On Molecular Structure,biochemical Characterization And Expression Of Squalene Synthase From Siraitia Grosvenorii

Siraitia grosvenorii,scientific name as Siraitia grosvenorii（Swingle）C.Jeffery ex lu et Z.Y.Zhang,is a perennial vine of the Cucurbitaceae family endemic to the northern area of Guangxi province in China and an endemic plant for the country.Its fruit,commonly known as Luo Han Guo（LHG）,characterized by sweet and cool-natured,lung and large intestine meridian tropism,moistening lung for arresting cough,helping produce saliva and slake thirst,relaxing bowel,and thus applying to pyretic pulmonary disease or xeropulmonary cough,pertussis,the thirsty due to hotness and impairment of fluid,as well as coprostasis,is used as medicine.The main medicinal components of the LHG are mogrosides,of which the mogroside V is a low calorie,natural sweeting agent and also has the functions of anti oxidation,immune regulation,anti-cancer,lowering blood sugar,etc.The mogrosides at present can not be chemically synthesized.They are mainly obtained by extraction from LHG and their productions are restricted by the planting environment and conditions of LHG.Thus,the heterologous expression study of the key enzymes in the mogrosides synthesis pathway can laid the groundwork for improving the biosynthesis yield of mogrosides in plant,realizing industrial production of the mogrosides with plant genetic engineering and cell engineering.Chapter 1 Gene cloning and molecular structure analysis of squalene synthase from Siraitia grosvenoriiObjective:To clone squalene synthase（SS）gene from siraitia grosvenorii and obtain the complete sequence of SS gene open reading frame（ORF）,deduce its amino acid sequence,analyze with bioinformatics method the molecular structure and phosphorylation sites of the enzyme.Methods:The sequences of the SS genes from Terrestrial plants were downloaded from GenBank data base.The ORF of the SS genes were analyzed by comparison and found to be highly conserved in the sequences of their 5′end and middle,and relatively variable in the sequences of their 3′end.Based on the comparison result,PCR primers were designed using the conserved middle sequence of SS gene.An about 300 bp highly conserved DNA fragment of SS gene from siraitia grosvenorii was amplified.3′race primers were designed according to the DNA sequencing results to amplify 3′end sequence of the gene.The obtained3′end DNA fragment of SS siraitia grosvenorii gene was cloned and sequenced.Then,primers for amplifying the ORF of siraitia grosvenorii SS gene were designed according to the DNA sequencing results.The amplification product was cloned and sequenced and the whole siraitia grosvenorii ORF was obtained.The obtained DNA sequence of siraitia grosvenorii SS gene was analyzed for ORF on the ORF Finder of the National Center for Biotechnology Information（NCBI）.The ORF sequence was translated into amino acid sequence with DNASTAR software.ProtScal and Prot Param were used for hydrophobicity/hydrophily analysis of the siraitia grosvenorii SS protein.NetPhos2.0 Server was applied for phosphorylation site prediction of the enzyme protein.The secondary structure analysis of the enzyme was performed with the protean of DNASTART software and the online web site NPS@.Domain prediction analysis of the SS protein was conducted on the NCBI CD-Search website.Transmembrane domain of the SS was analyzed with TMHMM Server v.2.0.Tertiary structure prediction of the SS was performed with the online software SWISS-MODEL and followed the structure editing using RASTOP 2.0 software.Clustalx1.8 was used to analyze the SS gene sequences of gynostemma,cucumber,muskmelon,ginseng,Salvia miltiorrhiza,Arabidopsis,licorice,tomato,potato,tobacco,corn downloaded from GenBank as well as the deduced SS amino acid sequences of these plants.The phylogenetic tress was constructed with MEGA5.0 to perfume UPGMA and carry out 1000 times’Bootstrap test,using botryococcus braunii as outgroup.Results:The ORF of SS gene from siraitia grosvenorii encodens 417amino acids and an isoelectric point of 7.56 with the protein.The ⁴⁷VSRSF⁵²、Y⁷⁰、R⁷⁴、⁷⁷DTVED⁸¹、Y¹⁶⁴、G²⁰²、L²⁰⁵、²¹²RDYLED²¹⁷、R²2525 in the enzyme amino acid sequence,which are related to SS activity,are highly conserved.NetPhos2.0 Server analysis suggested that there are phosphorylation sites in siraitia grosvenorii SS,in which there are 7 Ser phosphorylation sites(S⁴⁸,S¹⁹⁶,S²⁴⁹,S²⁸¹,S³⁴⁹,S³5858 and S⁴⁰⁷),6 Thr phosphorylation sites(T⁷⁸,T⁸³,T¹⁴⁴,T¹⁶¹,T³1818 and T³²⁷)and 4 Tyr phosphorylation sites(Y¹⁶⁵,Y²³,Y²4545 and Y²⁷³).All of the phosphorylation sites are located in the surface of the SS protein.The phosphorylation site S⁴88 locates in the activity center-related motif ⁴⁷VSRSF⁵²and S¹9696 is a positive selection site of terrestrial plant SS genes.The result of Prot Param analysis showed that siraitia grosvenorii SS is a hydrophilic protein.Secondary structure analysis displayed thatαhelix accounted for 64.99%,extended main chain accounted for 7.19%and random coil accounted for27.82%.Domain prediction study indicated that the SS is belonged to the isoprenoid biosynthase superfamily possessing the banding sites of farnesyl diphosphate and magnesian ion,and being in bundant of Asp motif.Transmembrane domain analysis suggested that there are two transmembrane regions in the SS,which are located in 282 to 304 and 386 to 408amino acid residues in the SS sequence.The phylogenetic tress constructed with the nucleotide sequences of siraitia grosvenorii and other plants SS genes showed that SS genes belonging to one subject of plants were combined with priority clustering and consistent with the natural evolution of plants.Prediction of the tertiary structure suggested that the SQS is a monomeric enzyme with a cave-like activity center formed byαhelixes.Conclusion:The siraitia grosvenorii SS is a monomeric enzyme.The main type of secondary structure of the enzyme isαhelix.The enzyme possesses the banding sites of farnesyl diphosphate and magnesian ion.There are two hydrophobic transmembrane helixes in the C terminal of the SS protein.And S⁴⁸and S¹9696 may be the critical sites for the regulation of the enzyme activity.Chapter 2 Construction of prokaryotic expression vector,purification and activity identification of squalene synthase from Siraitia grosvenoriiObjective:To construct the prokaryotic expression vector of the SS from Siraitia grosvenorii to realize the heterologous expression of the enzyme and thus laid the groundwork for further study of the molecular structure and function of Siraitia grosvenorii SS.Methods:The specific primers bearing BamHI and XhoI enzyme cutting sites were designed to amplify and clone the gene of Siraitia grosvenorii.The amplicon of the SS gene was inserted into the T-easy vector to construct pGEM-SS-ORF1.The p GEM-SS-ORF1,pET-21a（+）and pET-28a（+）were undergone double-enzymic digestionwith BamHI and XhoI.The DNA fragment of SS gene,p ET-21a（+）and pET-28a（+）were obtained respectively with DNA fragment gel recovery method and used to transfect the E coli JM109after legation.DNA sequencing of plasmids extracted were used to conform weather the pET-28a（+）-SS and p ET-21a（+）-SS transformed the BL21（DE3）and produced pET-28a（+）-SS and pET-21a（+）-SS expression strains or not.The pET-28a（+）-SS and pET-21a（+）-SS expression strains were induced to express Siraitia grosvenorii SS with IPTG.The Siraitia grosvenorii SS was purified with His nickel column and the enzyme activity was examined.Thermal stability and pH dependence of Sg SQS were investigated.Results:The pET-21a（+）-SS strain correctly expressed Siraitia grosvenorii SS while the p ET-28a（+）-SS strain did not do as good as pET-21a（+）-SS strain.The Siraitia grosvenorii SS purified by His nickel column showed enzyme activity.Siraitia grosvenorii SS activity was optimal at 37℃and p H 7.5.The Km value for FPP was 5.5μM.Conclusion:The prokaryotic expression system can be used to product Siraitia grosvenorii SS.Siraitia grosvenorii SS activity was optimal at 37℃and pH 7.5.The Km values for FPP were 5.5μM.Chapter 3 Effects of amino acid residues S⁴⁸and S¹⁹⁶of squalene synthase from siraitia grosvenorii on enzymatic activeObjective:To research the effects of amino acid residues S⁴88 and S¹9696 of squalene synthase from siraitia grosvenorii on enzyme active.Methods:The primers including Kozak sequence,restrict enzyme sites of BamHI and XhoI was designed to amplify the squalene synthase gene of siraitia grosvenorii,then the results was cloned into pcDNA3.1 vector to construct recombinant pcDNA3.1-SS.The recombinant plasmid pcDNA3.1-SS was transfected into HepG2 cells,HepG2 cell was cultured with medium containing Geneticin to creation of Stable Cell Line,then lysised and squalene synthase was extracted.The reaction system containing farnesyl pyrophosphate（FPP）and NAPDH was prepared.The conversion of FPP into squalene under catalysis of the purified SgSQS was analyzed by using GC–MS,catalytic activity of SS was determined with the conversion of FPP to squalene.The primers including mutation site was designed respectively.The PCR was carried with high-fidelity thermostable DNA polymerase and pcDNA3.1-SS as template.The results of PCR was digested by DpnI,and then transferred into XL10 competent cells.The positive clone was picked with PCR,and was identified finally by sequencing.Three plasmids which were pcDNA3.1-SS-S⁴⁸,pcDNA3.1-SS-S¹⁹⁶,pcDNA3.1-SS-S⁴⁸-S¹9696 were constructed and transfected into HepG2 cell.The stable Stable Cell Lines HepG2.SS.48,Hep G2.SS.196,Hep G2.SS.48.196 was created by using medium containing Geneticin and lysised to extracted SS.Catalytic activity of SS was determined with the conversion of FPP to squalene.Results:The pcDNA3.1-SS was confirmed correctly by restriction endonuclease digestion and sequenced;the HepG2.SS was got after culturing 30days with medium containing Geneticin.The results of catalytic activity verified that the SS which expressed by pcDNA3.1-SS vector had enzymatic activity.Monoclonal colony PCR and the results of sequencing confirmed that the mutation of S48A,S196A was successful,and the plasmids of mutation were got,after culturing with medium containing Geneticin,the cells of HepG2.SS.48,HepG2.SS.196,and Hep G2.SS.48.196 were created.The enzymatic activity of SS-S48A、SS-S196A、SS-S48A-S196A was detected,the enzymatic activity of SS-S48A is 1.21 fold to wild-type SS（P（27）0.01）,SS-S196A is 1.01 fold to wild-type SS,and SS-S48A-S196A is 1.16 fold to wild-type SS（P（27）0.01）.Conclusion:The amino acid residues S⁴88 is the key site of enzymatic activity regulation.Chapter 4 Analysis of expression profiling of squalene synthase from Siraitia grosvenorii in plant tissues and organsObjective:To investigate the expression profiling of SS from Siraitia grosvenorii in plant tissues and organs and thus laid the groundwork for increasing the accumulation of mogrosides in fruit.Methods:Total RNA was extracted from the roots,stems,leaves and peel of Siraitia grosvenorii 30 days after pollination.Reverse transcription was performed with random primers.The gene of Siraitia grosvenorii 18S rRNA was cloned and sequenced,and the primers for amplification of Siraitia grosvenorii18S rRNA gene with fluorescence quantitative PCR were designed based on the result of the 18S rRNA gene DNA sequencing.The expression levels of Siraitia grosvenorii gene were examined by using a relative quantitative method of Sybgreen fluorescence PCR with 18S rRNA as reference gene.Data were processed using 2-^{（35）（35）C T}method.Results:Higher expression level of siraitia grosvenorii SS gene in stems and roots was found while lower one was observed in leaves and peel.The expression level in peel was 1.25 times that in leaves,the one in stems was 2.60times that in leaves and the one in roots was 2.51 times that in leaves.Conclusion:The secondary metabolite of siraitia grosvenorii triterpenes may be biosynthesized mainly in roots and stems of the plant and then transported to the fruit for storage via the stems.