Mining Of Genes Involved In Terpenoid Biosynthesis Based On Transcriptome Analysis And Cloning Of Several Terpene Synthase Genes From Amomum Villosum Lour

1 ObjectiveTo dig out the related volatile terpene synthase genes from transcriptomes and expression profiles data of Amomum villosum Lour, and to study the expression characteristics and functions of the selected candidate genes, it would lay the foundation for a comprehensive understanding of volatile terpenoid biosyntheis pathways and molecular regulation mode.2 Methods2.1 Transcriptome analysis of volatile terpene synthase genes in A. villosum Lour.Based on the analysis of two transcriptome obtained preliminarily of A. villosum, we screened and counted up the number of unigenes which were annotated in volatile; terpenoid biosynthesis pathway in KEGG, then we re-annotated the unigenes by homology-based BLAST with downloaded terpene synthases (TPS) sequences from NCBI which were relat-ed to A. villosum volatile terpenes, such as monoterpene synthases (mono-TPS) like (+)-bornyl diphosphate synthase, pinene synthase,1,8-cineole synthase, and sesquiterpene synthases (sesqui-TPS) like bicyclogermacrene synthase. Finally, by phylogenetically ana-lyzing well-characterized TPS and BLAST between 2 different transcriptomes, the candi-date terpene synthase genes(AvrPSs) for further research were determined.2.2 Cloning of full-length coding sequence and bioinformatics analysis of encoding protein of AvTPSsAfter forecasting the open reading frame (ORF) of AvTPS candidate genes, the AvTPSs with full-length ORF were cloned by PCR method and constructed of recom-binant vector. Then their protein coding sequences were bioinformatics analyzed for functional characterization, including protein molecular weight, isoelectric point, hydrophilicity and other physical and chemical properties, subcellular localization and protein structure prediction, homology-based BLAST and phylogenetic analysis with well-characterized TPSs.2.3 Gene expression and correlation analysis of AvTPSsThe absolute quantification real-time PCR (qRT-qPCR) was used to gene expression analysis of cloned-AvTPSs among different tissues and different development process. Then correlation analysis was taken between RPKM from A. villosum expression profiles data of AvTPSs, with differentiation change among the content of volatile terpenoids in MeJA induced experiment.3 Results3.1 Seletion of candidate volatile terpene synthase genes in A. villosum Lour.27 candidate unigenes involved in volatile terpenoid biosynthesis pathway were found out from 2 different transcriptomes of A. villosum, including 9 annotated unigenes and 18 re-annotated unigenes. And comprehensively considering the results of phylogenetic analy-sis and BLAST,11 candidate genes, which were re-named as AvTPS1～11 were finally determined.3.2 Cloning of full-length coding sequence (CDS) and bioinformatic analysis of encoding protein of AvTPSsThe full-length CDS of 4 AvTPSs was obtained. The cloned AvTPS1 contains a 1803bp ORF, encoding a protein of 600 amino acids, while AvTPS3 contains a 1791bp ORF, encoding a 597 aa protein, and AvTPS8 contains a 888bp ORF, encoding a 295 aa protein, and AvTPS8 contains a 1650bp ORF, encoding a 549 aa protein. Except for AvTPS5, other 3 AvTPSs shared a common molecular mass of 60-70kDa and isoelectric point near 5.0, which were similar to other known TPS, then they also contained the highly conserved motifs such as DDXXD and closely resembled with other well-characterized TPSs.Thus we could classify AvTPSl and AvTPS3 in monoterpene synthase, and classify AvTPS8 in sesquiterpene synthase. AvTPS5 was more like a dehydrogenase or reductase.3.3 Gene expression and correlation analysis of AvTPSsThe qRT-qPCR result of 4 AvTPSs showed that AvTPS1, AvTPS3 and AvTPS8 were highly expressed in specialized tissue, and AvTPS1, AvTPS3 and AvTPS5 showed in a high level in certain development stages. The expressing specificity of AvTPSs was as common as other TPSs.And the correlation analysis proved that most of candidate AvTPSs had a strong correlation with total content of volatile terpenes in A. villosum. Therein, AvTPS2, AvTPSS, AvTPS7 and AvTPS9 exhibited more consis-tent correlation pattern, while AvTPS6 and AvTPS 11 showed another related patterns. And in the correlation analysis with the specific terpenoids content, the pericarp-higher-expressed genes, AvTPS1 and AvTPS2, and seed-higher-expressed genes, AvTPS3 and AvTPS6, showed a positive correlation with the main component in spe-cialized tissue respectively, which may play an important role in the biosynthesis of these volatile terpenods in A. villosum Lour.4 ConclusionBinding the lucubrating of transcriptome and expression profiles data and the correlation with the content of volatile terpenoids, we effectually find out 11 terpene synthases candidate genes from A. villosum which are worthy for further research. The cloning and bioinformatic analysis of some AvTPSs not only lays a foundation for further study on gene functional characterization, but also provides a reference for further study of volatile terpenoid metabolic engineering research of A. villosum Lour.