ABSTRACT
Isopentenyl diphosphate isomerase (IDI) is a key enzyme in the regulation of triterpenes biosynthesis and plays an important role in ginsenoside biosynthesis. In this study, two IDI genes, PvfIDI1 (GenBank No. MZ736417) and PvfIDI2 (GenBank No. MZ736418) were cloned from Panax vietnamensis var. fuscidiscus. The open reading frame of both PvfIDI1 and PvfIDI2 was 924 bp encoding 307 amino acids. The molecular weights of PvfIDI1 and PvfIDI2 were 34.84 kDa and 34.66 kDa, respectively, with theoretical pIs of 6.01 and 5.66. Bioinformatic analysis indicated that PvfIDI1 and PvfIDI2 contained two conserved sequences: TNTCCSHPL and WGEHELDY. Phylogenetic analysis showed that PvfIDI1 and PvfIDI2 were closely related to Panax notoginseng IDI. Expression analysis showed that both PvfIDI1 and PvfIDI2 genes are expressed in root, rhizome, stem and leaf of P. vietnamensis var. fuscidiscus. However, PvfIDI1 is highly expressed in the rhizome and PvfIDI2 is highly expressed in the stem. PvfIDI1 and PvfIDI2 recombinant proteins were expressed in E. coli; a functional coloration experiment showed that PvfIDI1 and PvfIDI2 could promote the accumulation of lycopene, indicating that both PvfIDI1 and PvfIDI2 encode functional IDI enzymes. The cloning and functional studies on PvfIDI1 and PvfIDI2 provide a foundation for the further study of IDI and the regulation of ginsenoside biosynthesis in P. vietnamensis var. fuscidiscus.
ABSTRACT
Objective: To clone the squalene epoxidase genes of Panax vietnamensis var. fuscidiscus(PvfSE),and perform bioinformatics analysis and prokaryotic expression. Method: Total RNA was extracted from root of P. vietnamensis var. fuscidiscus by trizol method, and reverse-transcribed into first stand of cDNA. Specific primers for PvfSE cloning were designed according to the transcriptome data of P. vietnamensis var. fuscidiscus,and the cDNA sequence of PvfSE gene was isolated. Bioinformatics of PvfSE was analyzed by relevant software. The prokaryotic expression vector pMal-c2X-PvfSE was built to express recombinant protein in Escherichia coli cells. Result: The PvfSE gene contained a 1 887 bp open reading frame,encoding a predicted protein of 628 amino acids. The calculated molecular weight was 68.8 kDa,the theoretical isoelectric point was 9.28,the aliphatic index was 95.18,the grand average of hydropathicity was -0.060, and the instability index was 40.36. The protein was unstable. Bioinformatics analysis showed that PvfSE had two transmembrane domains and no signal peptide. PvfSE was most likely to be located in chloroplast or cytoplasmic membrane. PvfSE was a mixed protein with FAD/NAD(P) binding domain and squalene epoxidase domain. Sequence alignment and phylogenetic analysis demonstrated that PvfSE had a relatively close relationship with CpSE1,CpSE3,OsSE1 and OsSE2,which was involved in the biosynthesis of triterpene saponins in Cucurbita pepo and Ononis spinosa. In addition,PvfSE protein was expressed in E. coli. Conclusion: In this study,PvfSE gene was cloned and expressed in BL21(DE3),which lays a foundation for the further study on the gene functions of PvfSE and the biosynthetic pathway of triterpenoid saponins in P. vietnamensis var. fuscidiscus.
ABSTRACT
Objective: The SSR loci information in the transcriptome of Panax vietnamensis var. fuscidiscus (PVF) was analyzed in this study to provide more powerful tools for molecular marker-assisted breeding in this plant. Methods: Simple sequence repeats (SSR) loci were searched in all 126 758 unigenes by using MISA. SSR loci information was analized and SSR primers were designed by Primer3. Furthermore, 30 pairs of primers were randomly selected for the polymorphic analysis on 13 PVF plants collected from different habitats. Results: A total of 21 320 SSRs were found in the transcriptome of PVF, distributed in 17 780 unigenes with the distribution frequency of 16.82%. Di-nucleotide repeat was the main type, accounted for as much as 52.52% of all SSRs, followed by tri-nucleotide repeat motif (28.08%). The dinucleotide repeat motifs of AG/CT and AT/AT were the predominant repeat types (46.25%). Using Primer3, a total of 39 336 pairs of SSR primers were designed. For validating the availability of those SSR primers, we randomly selected 30 pairs of primers for PCR amplification. Among them, 29 pairs of primers (96.67%) produced clear and reproductive bands, 15 pairs of primers (50.00%) showed polymorphism, and 13 PVF plants were divided into two groups by UPGMA. Conclusion: There are numerous SSRs in PVF transcriptome with high frequency and various types, this will provide the abundant candidate molecular markers for genetic diversity study and genetic map for this plant.