ABSTRACT
Screening suitable reference genes is the premise of quantitative Real-time PCR(qRT-PCR)for gene expression analysis. To provide stable reference genes for expression analysis of genes in Aconitum vilmorinianum, this study selected 19 candidate re-ference genes(ACT1, ACT2, ACT3, aTUB1, aTUB2, bTUB, 18S rRNA, UBQ, eIF2, eIF3, eIF4, eIF5, CYP, GAPDH1, GAPDH2, PP2A1, PP2A2, ACP, and EF1α) based on the transcriptome data of A. vilmorinianum. qRT-PCR was conducted to profile the expression of these genes in the root, stem, leaf, and flower of A. vilmorinianum. The Ct values showed that 18S rRNA with high expression level and GAPDH2 with large expression difference among organs were not suitable as the reference genes. NormFinder and geNorm showed similar results of the expression stability of the other candidate reference genes and demonstrated PP2A1, EF1α, and CYP as the highly stable ones. However, BestKeeper suggested EF1α, ACT3, and PP2A1 as the top stable genes. In view of the different results from different softwares, the geometric mean method was employed to analyze the expression stability of the candidate re-ference genes, the results of which indicated that PP2A1, EF1α, and ACT3 were the most stable. Based on the comprehensive analysis results of geNorm, NormFinder, BestKeeper, and geometric mean method, PP2A1 and EF1α presented the most stable expression in different organs of A. vilmorinianum. PP2A1 and EF1α were the superior reference genes for gene expression profiling in different organs of A. vilmorinianum.
Subject(s)
Aconitum , Gene Expression Profiling , Genes, Plant/genetics , Real-Time Polymerase Chain Reaction , Reference Standards , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
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
Triterpenoids are active constituents of many medicinal plants. The biosynthetic pathways of triterpenes have been fully studied and significant progress has been made. CYP450s are mainly involved in the post-modification process of triterpenoids biosynthesis, which plays a key role in the diversity of triterpenoids. Currently, a lot of CYP450s, which related to the biosynthesis of triterpenoids, have been cloned from many plants. In this paper, CYP450s involved in the biosynthesis of triterpenoids were reviewed, which provided a reference for the analysis of the biosynthetic pathway of triterpenoids and the functional study of CYP450s.
ABSTRACT
<p><b>OBJECTIVE</b>To study the feasibility of the application of ultra high-pressure processing (UHPP) as an anticorrosion and anti-mould method by comparing the total numbers of bacteria and mould colonies and the content of ginsenosides before and after UHPP.</p><p><b>METHOD</b>The total numbers of bacteria and moulds colony were determined by microbiological test method. The contents of 12 ginsenosides were determined by HPLC.</p><p><b>RESULT</b>Under the three selected conditions, the total number of bacterial colony decreased significantly, while the mould was not detected in UHPP samples; and the contents of 12 ginsenosides were increased significantly in methanol extracts and water extracts.</p><p><b>CONCLUSION</b>UHPP not only shows anticorrosion and anti-mould effects, but also enhances the leaching rate of ginsenosides. It is a highly effective, safe and environmental friendly anticorrosion and anti-mould technique for Ranax ginseng worth in-depth study.</p>
Subject(s)
Bacteria , Chromatography, High Pressure Liquid , Corrosion , Feasibility Studies , Ginsenosides , Panax , Chemistry , Microbiology , PressureABSTRACT
<p><b>OBJECTIVE</b>To study the genetic diversity of C. morifolium on the chemical constituents.</p><p><b>METHOD</b>Chemical constituents of four cultivars cultivated with the same conditions were compared in three types of index: chlorogenic acid, flavonoid and volatile oil.</p><p><b>RESULT AND CONCLUSION</b>With different cultivars and processing methods, the contents of chlorogenic acid, flavonoid and volatile oil extracted from C. morifolium vary great extent.</p>