Prokaryotic expression, subcellular localization and enzymatic activity analysis of DXS gene from Platycodon grandiflorum / 药学学报

1-Deoxy-D-xylulose-5-phosphate synthase (DXS), the first key enzyme in 2-methyl-D-erythritol-4-phosphate (MEP) pathway, catalyzes the condensation of glyceraldehyde-3-phosphate with pyruvate to 1-deoxy-xylose-5-phosphate (DXP). In this study, PgDXS1, PgDXS2, and PgDXS3 genes were cloned from the root of Platycodon grandiflorum (P. grandiflorum). The open reading frame (ORF) of PgDXS1, PgDXS2, and PgDXS3 were 2 160, 2 208, and 2 151 bp in full length, encoding 719, 735, and 716 amino acids, respectively. Homologous alignment results showed a high identity of PgDXSs with DXS in Hevea brasiliensis, Datura stramonium and Stevia rebaudiana. The recombinant expression plasmids of pET-28a-PgDXSs were constructed and transformed into Escherichia coli (E. coli) BL21 (DE3) cells, and the induced proteins were successfully expressed. Subcellular localization results showed that PgDXS1 and PgDXS2 were mainly located in chloroplasts, and PgDXS3 was located in chloroplasts, nucleus and cytoplasm. The expression of three DXS genes in different tissues of two producing areas of P. grandiflorum were assayed via real-time fluorescence quantitative PCR, and the results showed that all of them were highly expressed in leaves of P. grandiflorum from Taihe. Under methyl jasmonate (MeJA) treatment, the expression levels of three PgDXS genes showed a trend of first decreasing and then increasing at different time points (3 - 48 h), and the activity of DXS showed a trend of first increasing and then decreasing in three tissues of P. grandiflorum. This study provides a reference for further elucidating the biological function of PgDXS in terpenoid synthesis pathway in P. grandiflorum.

Cloning and prokaryotic expression of 3-ketoacyl-CoA thiolase gene AIKAT from Atractylodes lancea / 中国中药杂志

In this study, the gene encoding the key enzyme 3-ketoacyl-CoA thiolase(KAT) in the fatty acid β-oxidation pathway of Atractylodes lancea was cloned. Meanwhile, bioinformatics analysis, prokaryotic expression and gene expression analysis were carried out, which laid a foundation for the study of fatty acid β-oxidation mechanism of A. lancea. The full-length sequence of the gene was cloned by RT-PCR with the specific primers designed according to the sequence information of KAT gene in the transcriptomic data of A. lancea and designated as AIKAT(GenBank accession number MW665111). The results showed that the open reading frame(ORF) of AIKAT was 1 323 bp, encoding 440 amino acid. The deduced protein had a theoretical molecular weight of 46 344.36 and an isoelectric point of 8.92. AIKAT was predicted to be a stable alkaline protein without transmembrane segment. The secondary structure of AIKAT was predicted to be mainly composed of α-helix. The tertiary structure of AIKAT protein was predicted by homology modeling method. Homologous alignment revealed that AIKAT shared high sequence identity with the KAT proteins(AaKAT2, CcKAT2, RgKAT and AtKAT, respectively) of Artemisia annua, Cynara cardunculus var. scolymus, Rehmannia glutinosa and Arabidopsis thaliana. The phylogenetic analysis showed that AIKAT clustered with CcKAT2, confirming the homology of 3-ketoacyl-CoA thiolase genes in Compositae. The prokaryotic expression vector pET-32 a-AIKAT was constructed and transformed into Escherichia coli BL21(DE3) for protein expression. The target protein was successfully expressed as a soluble protein of about 64 kDa. A real-time quantitative PCR analysis was performed to profile the AIKAT expression in different tissues of A. lancea. The results demonstrated that the expression level of AIKAT was the highest in rhizome, followed by that in leaves and stems. In this study, the full-length cDNA of AIKAT was cloned and expressed in E. coli BL21(DE3), and qRT-PCR showed the differential expression of this gene in different tissues, which laid a foundation for further research on the molecular mechanism of fatty acid β-oxidation in A. lancea.

Different development phase of transcriptome analysis from Crataegus pinnatifida Bge. and cloning, structure and function prediction of squalene epoxidase involved in triterpenic acid biosynthesis / 药学学报

italic>Crataegus pinnatifida is a traditional Chinese medicine, which contains organic acids, triterpenoid acids and other active components, has important medicinal and edible value. In order to study the difference of gene expression level in different developmental stages of hawthorn and explore the genes of active ingredient biosynthesis in Crataegus pinnatifida, high-throughput Illumina HiSeq 2000 technology were used to conduct transcriptome sequencing and bioinformatics analysis on Crataegus pinnatifida fruits from the same origin at different developmental stages. 78 496 Unigenes with an average length of 941 nt were obtained by Trinity software. Among them, 58 395 Unigenes can be annotated by NR, NT, Swiss prot, KEGG, COG, GO and other public databases. KEGG pathway analysis showed that 52 Unigenes encoding 15 key enzymes involved in the citric acid cycle. There are 62 Unigenes were involved in the triterpene biosynthesis pathway of Crataegus pinnatifida. Two key enzymes SQE of triterpenoid metabolism pathway in Crataegus pinnatifida were cloned and performed bioinformatic analysis. The results showed that ORF of CpSQE1 and CpSQE2 were 1 594 bp and 1 597 bp, respectively, encoding 530 and 531 amino acids. The molecular weight of proteins was 57.6 kDa and 57.5 kDa. Bioinformatics analysis showed that both CpSQE1 and CpSQE2 proteins have a PLN02985 superfamily conserved domain, belonging to the squalene monooxygenase superfamily. The phylogenetic tree shows that CpSQE1 and CpSQE2 are clustered together with SQE with squalene epoxidase function in other plants. This study provides a research basis for further exploring the key genes in the biosynthesis process of hawthorn active ingredients and analyzing the regulation pathway of its active ingredient biosynthesis.