Cloning And Functional Analysis Of Key Enzyme Genes CvSQS、CvSQE In Tea Saponin Synthesis Of Camellia Vietnamensis T.C.Huang Ex Hu

Camellia vietnamensis T.C.Huang ex Hu,which belonged to Camellia spp.,is a characteristic woody oil crop widely planted in China.Tea saponin is one of the important functional components of Camellia spp.,it has many functions such as lowering blood fat,disinfecting and inhibiting bacteria,and anti-tumor.Squalene synthase（SQS）and squalene epoxidase（SQE）are two key enzymes in the synthesis of tea saponin.Squalene synthase can promote 2 molecules of farnesyl pyrophosphate（FPP）to synthesize squalene,and squalene is further catalyzed by squalene epoxidase to 2,3-oxidizes squalene to form tea saponin after a series of biochemical reactions.In this study,transcriptome sequencing and bioinformatics analysis were performed on the samples of C.vietnamensis,and specific primers were designed on this basis.1683bp C.vietnamensis squalene synthase gene named Cv SQS was cloned the using rapid amplification of c DNA end（RACE）technology,and 1599 bp C.vietnamensis squalene epoxidase gene Cv SQE was cloned by homologous cloning,the two gene sequences above were analyzed by bioinformatics.Real-time Quantitative polymerase chain reaction（q PCR）was used to detect the expression changes of Cv SQS and Cv SQE in different tissues of C.vietnamensis.The Cv SQS gene was connected to the vector,transformed into Escherichia coli and expressed in prokaryotic cells.To identify catalytic activity of the obtained protein,enzyme activity was perform combined with gas chromatography-mass spectrometry technology.The main results of this study are as follows:（1）After sequencing the transcriptome of the C.vietnamensis sample,a total of73,274,082 read fragments were obtained.After assembly,All-Unigene was annotated to the NR,KOG,KEGG,and Swiss-Prot databases,and the number of Unigenes annotated in each database was counted.A total of 47,069 Unigenes had been annotated by the four major databases.46,521 Unigenes had been annotated by NR database.After comparison,it was found that unigenes of C.vietnamensis were similar to Vitis vinifera and Theobroma cacao;33549 Unigenes had been annotated by Swiss-prot database;28,630 Unigenes had been annotated by KOG database,and the Unigenes were divided into 25 categories according to their functions;17,567 had been annotated by KEGG database,of which ribosome have the highest gene abundance,followed by carbon metabolism.（2）Gene cloning:The full length of Cv SQS gene was 1683bp,of which the open reading frame（ORF）was 1245bp long and encodes 414 amino acids;The full length of Cv SQE gene was 1599 bp,of which the open reading frame size was 1569 bp,encoding522 amino acids.（3）Sequence analysis:Bioinformatics analysis showed that the molecular weight of Cv SQS protein is 47.3 k Da,with a molecular formula of C₃₈₁₁H₆₃₇₉N₁₂₄₅O₁₆₁₀S₂₃₂,the theoretical isoelectric point was 5.06.The Cv SQS protein had two transmembrane helical domains.And the protein was an unstable hydrophobin.As for secondary structure,theα-helix,random coil,β-turn and extended chains respectively accounts for 70.53%,20.77%,3.38%,and 15.71%;Cv SQS protein belonged to the isoprenoid synthase superfamily,and had typical squalene synthase conserved domains.Phylogenetic tree showed that Cv SQS and other plant SQSs were clustered together.Among them,The SQS of Camellia oleifera Abel.and Camellia sinensis had the highest homology with Cv SQS.The molecular weight of Cv SQE protein was 56.8 k D,with a molecular formula of C₄₇₇₄H₇₉₈₁N₁₅₆₉O₂₀₁₇S₃₅₇,the theoretical isoelectric point was 4.98.The Cv SQE protein had four transmembrane helical domains.And the protein was an unstable hydrophobin.As for the secondary structure,the proportions ofα-helix,random coils,β-turns,and the elongation chain were 38.51%,39.66%,6.13%and 15.71%.Cv SQE protein belonged to the cl39108 superfamily,and had typical squalene epoxidase conserved domains,including FAD binding domain and substrate binding domain.Phylogenetic tree showed that Cv SQE was clustered into one group with plant as Actinidia chinensis var.chinensis,Prunus persica,Glycine max,Glycyrrhiza glabra and Ononis spinose.Among them,the squalene epoxidase of Actinidia chinensis had the highest homology with Cv SQE.（4）Gene tissue-specific expression analysis:q RT-PCR showed that the expression of Cv SQS gene was the highest in leaves,followed by seeds,roots,and flowers.The expression of Cv SQE gene was highest in leaves,followed by seeds and roots,and less expression in flowers.（5）Prokaryotic expression and enzyme activity analysis:The last 165 amino acids at the C-terminal end of Cv SQS（both transmembrane helices were truncated）and the last 28amino acids at the C-terminal end（only the second transmembrane helix was truncated）were truncated to construct a vector for prokaryotic expression,After purification,Cv SQS1protein with a size of 32k Da and Cv SQS2 protein with a size of 47k Da were obtained by SDS-PAGE detection,and preliminary identification was carried out by Western-Blot,and then the enzyme activity reaction confirmed that Cv SQS2 had squalene synthase activity,while Cv SQS1 did not.