Effect of Strong Light Stress on Growth, Physiological and Biochemical and Gene Expression of Key Enzymes in Atractylodes lancea / 中国实验方剂学杂志

Objective::To explore the effect of strong light stress on the growth, physiological and biochemical and key enzyme gene expression of the Atractylodes lancea, in order to provide the scientific basis for the standardized cultivation of the A. lancea. Method::The two-year-old A. lancea seedlings were taken as experimental materials. Poplar forest (light transmittance between 18.26%-36.04%) was taken as control group(ck). Different density shading networks were used to simulate different degrees of high light stress (51.10%, 80.73%, 100%) in late July. The growth state of A. lancea was observed. On the 0th, 5th, 10th, 15th, 20th days, the physiological and biochemical indexes of malondialdehyde (MDA) content, cell membrane permeability, proline (Pro) content, antioxidant enzyme activity and chlorophyll content in the leaves of A. lancea were measured. The relative expression levels of 3-hydroxy-3-methylglutarate monoacyl coenzyme A reductase (3-hydroxy-3-methylglutaryl coenzyme A, HMGR) and farnesyl pyrophosphate synthase gene (farnesyl pyrophosphate synthase, FPPS) in leaves of A. lancea under intense light stress were determined by real-time fluorescence quantitative PCR(Real-time PCR). Result::After strong light stress, the color of the leaves of A. lancea changed from dark green to light green and yellowish green, and the burn of leaves became more and more serious. The contents of MDA, conductivity and Pro showed an upward trend with the increase of transmittance. Peroxidase (POD), superoxide dismutase (SOD) and catalase (CAT) tended to increase first and then decrease. The chlorophyll content decreased with the increase of light transmittance. The relative expression of HMGR in leaves of A. lancea decreased with the increase of light transmittance, while FPPS increased first and then decreased. Conclusion::The results showed that A. lanceaa could alleviate the inhibition of strong light stress by increasing the activity of antioxidant enzymes and regulating the content of osmotic pressure under certain strong light stress. Excessively strong intensity light stress leads to disequilibrium of metabolic mechanism of A. lancea, and seriously inhibits the plant growth.

Effect of endophytic fungi on expression of key enzyme genes in polysaccharide and alkaloid synthesis from Dendrobium officinale / 中草药

Objective: To analyze the effect of endophytic fungi GXRz2, GXRz3 and GXRz10 on content of poly saccharide and alkaloid, and the expression of key enzyme genes UGPase, HMGR, and FPS in Dendrobium officinale. Methods: The endophytic fungi liquid were added to D. officinale seedlings. Polysaccharide and alkaloid content were measured by spectrophotometry method. With 18S rRNA as internal control gene, the expression of key enzyme genes was detected by real-time quantitative PCR method. Results: It was found that the content of polysaccharide in D. officinale was higher, mainly concentrated in stem, but the lowest in root. And the content of alkaloid in D. officinale was lower, mainly accumulated in leaf, but the lowest in root. In addition, the three endophytic fungi strains could promote accumulation of polysaccharide and alkaloid in D. officinale to a certain extent. The expression of UGPase, HMGR and FPS genes in D. officinale induced by different strains was detected by real-time quantitative PCR. The results showed that endophytic fungi GXRz3 and GXRz10 could significantly increase the expression of UGPase, HMGR and FPS genes in D. officinale. In the polysaccharide synthesis pathway, the UGPase gene had the highest relative expression in the stem, followed by the leaf, and the least in the root. In the alkaloid synthesis pathway, HMGR gene had the highest relative expression in the stem, followed by the leaf, and the least in the root. However, FPS gene had the highest relative expression in the leaf, followed by the stem, and the least in the root. Conclusion: Endophytic fungi may affect the synthesis of polysaccharide by regulating the expression of UGPase. Considering the accumulation of polysaccharide, it is speculated that UGPase may be a key enzyme in the polysaccharide synthesis pathway of D. officinale. Endophytic fungi may affect the synthesis of alkaloid by regulating the expression of HMGR and FPS. Considering the accumulation of alkaloid, it is speculated that FPS may be a key enzyme in the alkaloid synthesis pathway of D. officinale.

Prokaryotic expression, function verification and immunoassay activity of farnesyl pyrophosphate synthase of Alisma orientale (Sam.) Juzep / 药学学报

Farnesyl pyrophosphate synthase of Alisma orientale (Sam.) Juzep. (AoFPPS) is considered as one of the important rate-limiting enzymes in the biosynthetic pathway of protostane triterpenes. In order to investigate the expression and function of AoFPPS, the gene (accession No. HQ724508) was cloned into a bacterial expression vector pCzn1, then the combined plasmid pCzn1-AoFPPS was transformed into Escherichia coli BL21, and a fusion protein was obtained after induction. The fusion protein was purified by Ni resin, and the function was verified through in vitro enzymatic reaction. High performance liquid chromatography (HPLC) analysis revealed that the products were able to catalyze the synthesis of farnesyl pyrophosphate (FPP). High purity recombinant protein was used to immunize New Zealand rabbits to generate a polyclonal antibody. The titer of the antibody was determined by enzyme linked immunosorbent assay (ELISA), and Western blot results demonstrated that the antibody could specifically recognize the AoFPPS protein in A. orientale (Sam.) Juzep. So,the method of rapid immunoassay to detect AoFPPS was established. This study lays the foundation for further study of the AoFPPS gene expression, regulation and mechanism of action in A. orientale (Sam.) Juzep., and it also provides a scientific basis on improving the quality of Alismatis Rhizoma using the plant genetic engineering.

Cloning and bioinformatic analysis of FPPS gene from Paeonia lactiflora / 中草药

Objective: To clone the full-length cDNA sequence which encodes one of the key enzymes of terpene biosynthesis, farnesyl pyrophosphate synthase (FPPS) and analyze the bioinformation. Methods: Based on the transcriptome data of Paeonia lactiflora (Pl), a pair of specific PCR primers were designed. The objective PCR band of PlFPPS was successfully amplified using RT-PCR technique and then it was cloned to pMD18-T vector. After clone and plasmid PCR characterization, the recombinants were sequenced and then a series of bioinformatic analysis was carried out including sequence homology search, construction of molecular phylogenetic tree, domain search, and 3D structure prediction, etc. Results: Sequencing results showed that the full-length PlFPPS was 1 315 bp, which contained 60 bp 5'-UTR, 1 050 bp CDS, and 205 bp 3'-UTR. It encoded 349 amino acids and the accession number of GenBank was KP708571.Blastn and Blastp analysis revealed that PlFPPS (KP708571) and its encoding protein (AKJ26301) had high homology with FPPS genes and proteins from several plants at the nucleotide and amino acid levels. Phylogenetic tree analysis indicated that the genetic relationship of PlFPPS with FPPS from other plants was relatively far. It predicted that the molecular weight and isoelectric point of PlFPPS were 40200 and 5.33, which was a hydrophilic and stable protein, located in cytoplasm without transmembrane domain and signal peptide. It contained seven conservative domains like substrate binding pocket, substrate-Mg2+ binding site, catalytic site, aspartate-rich region 1, and aspartate-rich region 2.The proportion of α-helix in its 3D structure was high and the α-helix was linked by β-loop. The central cavity was composed of 10 core α-helixes. Conclusion: The full-length cDNA sequence of FPPS is cloned firstly from P. lactiflora and the bioinformatic analysis is then carried out.

Analysis on codon bias of farnesyl pyrophosphate synthase gene in Astragalus membranaceus / 中草药

Objective: Farnesyl pyrophosphate synthase (FPS) is the key enzyme in biosynthesis pathway of astragaloside IV. The purpose of the experiment was to provide the theory basis for selecting appropriate expression systems and regulating the content of astragaloside IV. Method: FPS gene coding sequence was cloned based on Astragalus membranaceus from Changbai Mountain. Synonymous codons usage of FPS gene was analyzed by EMBOSS and Codon W programs and compared with the genome of other seven plants, such as Zea mays and Artemisia apiacea, and E. coli. Results: FPS gene of A. membranaceus was bias toward the codon with A and T at the third codon position and there are 22 codons showing the significant differences between FPS gene of A. membranaceus and E. coli genome. Conclution: The codons need to be optimized to improve the expression level of FPS gene in E. coli.

Cloning and sequence analysis of Farnesyl pyrophosphate synthase gene in a mangrove tree Sonneratia alba / 中草药

Objective: To clone the full length cDNA encoding Farnesyl pyrophosphate synthase (FPS) which played an important role in terpenoid biosynthesis pathway in Sonneratia alba and to provide the basis for the further studies on biosynthesis and gene regulation of terpenoid. Methods: According to the annotation of root transcriptome in S. alba, primers were designed and cDNA of S. alba Farnesyl pyrophosphate synthase (SaFPS) gene was cloned from S. alba by PCR. Results: The complete coding sequence of SaFPS gene was 1029 bp and encoded a protein of 342 amino acids. The deduced SaFPS amino acid sequence exhibited 86% identity to the FPS of Glycyrrhiza uralensis. The predicted SaFPS shared two conserved functional domains. Phylogenetic analysis on the amino acid sequence of SaFPS with those of other plants showed that SaFPS was closely related to G. uralensis, Medicago truncatula, and Lupinus albus. It showed that SaFPS was highly expressed in flowers while lowly expressed in fruits, stems, and leaves by real-time PCR analysis. Conclusion: The SaFPS gene is cloned from a mangrove tree S. alba for the first time and the expression of SaFPS is tissue specific. This research lays a foundation for studying the gene expression pattern and regulating the functions of SaFPS in terpenoid biosynthesis.