Jasmonate signal pathway involved in phytoalexin biosynthesis in Sorbus aucuparia cell elicited by Hpa1 / 中国中药杂志

The Harpin protein Hpa1 can induce defense responses in plant. This study aimed at investigating the role of jasmonate (JA) signal pathway in the process of biosynthesis of secondary metabolite in Sorbus aucuparia cell eliciting by Hpa1 crude extract (Hpa1 CE). The results showed that Hpa1 crude extract (Hpa1 CE) could induce phytoalexin synthesis in S. aucuparia cell， most of which was noraucuparin and its glycosides. Meanwhile Hpa1 CE treatment resulted in methyl jasmonate (MeJA) production increased and noraucuparin was de novo synthesized in large quantities. Combination of Hpa1 CE and salicylhydroxamic acid (SHAM, JA signaling inhibitor) caused the decreased MeJA and noraucuparin in the S. aucuparia cell compared with that in Hpa1 CE group. Real-time PCR results indicated that Hpa1 CE treatment caused down-regulation of JAZ and up-regulation of mcy2 in transcription level. Therefore Hpa1 CE elicited defense mechanism and JA signaling pathway involved in phytoalexin biosynthesis in S. aucuparia cell. It presented information to elucidate the role of JA signal pathway in stress response in the perspective of secondary metabolism of plant.

Research of genes expression related to ginsenosides biosynthesis by methyl jasmonic acid in ginseng hair roots / 中国中药杂志

In order to obtain the expression of ginsenoside biosynthetic pathway related enzyme gene in ginseng hairy root under the control of elicitors, methyl jasmonate (MeJA) was added exogenously as elicitors. Ginseng hairy root clones induced by 4-year-old ginseng root was used as material, total saponin content in ginseng hairy root before and after MeJA treatment was determined by vanillin-sulfuric acid colorimetry, Meanwhile, relative expression of squalene synthase genes, squalene epoxidase genes, oxidized squalene cyclase genes, dammarenediol synthase genes, β-amyrin synthase genes, cycloartenol synthase genes before and after MeJA treatment were determined by Real-time PCR. The optimum conditions of MeJA which added to ginseng hairy root were obtained, the optimum additional concentration was 6×10⁻⁴ μmol•L⁻¹, the optimum additional time was 22 d, and the optimum action time was 5 d. The addition of MeJA could improve the enzymatic activity of peroxidase (PPD), catalase (CAT) and peroxidase (PPD) in ginseng hairy root. The expression of SQS，SQE，OSC，DS and β-AS genes of ginsenoside biosynthetic pathway increased significantly after MeJA treatment, while the change of CAS gene expression were not significant. The expression of key enzyme SQS，SQE，OSC，DS and β-AS genes in ginsenoside biosynthetic pathway was consistent with the changes of PPD，CAT，PPO enzymatic activity.

Effects of Elicitors on Fucoxanthin Production and ZDS Gene Expression in Phaeodactylum tricornutum / 中国药学杂志

OBJECTIVE: To explore the effects of methyl jasmonic acid(MeJA), arachidonic acid(AA) and acetylsalicylic acid(ASA) on fucoxanthin production and PtZDS gene expression in Phaeodactylum tricornutum. METHODS: The full length cDNA of PtZDS gene in P. tricornutum was obtained by HiSeq™ Illumina 2000 and bioinformatics analysis. HPLC was used to determine the production of fucoxanthin in Phaeodactylum tricornutum, and RT-PCR was used to determine the expression level of PtZDS. RESULTS: The total length of PtZDS gene was 1 905 bp, containing an open reading frame of 1 776 bp encoding 591 amino acids. The deduced amino acid sequence analysis showed that PtZDS protein contained a typical amino oxidase domain, NAD(P)-binding Rossmann-like domain and a chloroplastic transit peptide sequence. The phylogenetic analysis demonstrated that PtZDS was homologous with Thalassiosira pseudonana CCMP1335(76%). Under the treatment of 0.1 mg·L-1 AA, 25 mg·L-1 ASA and 100 μmol·L-1MeJA, the highest yield of unit cell fucoxanthin content was achieved. Upon the observation of PtZDS regulation expression, the expression level of PtZDS reached a peak value under the treatment of 0.1 mg·L-1 AA, 50 μmol·L-1MeJA and 10 mg·L-1 ASA. CONCLUSION: The expression of PtZDS gene has certain relationship with the fucoxanthin synthesis of P. tricornutum. Under the treatment of 100 μmol·L-1 MeJA, the ability of synthetic fucoxanthinis the strongest in P. tricornutum.

Gene cloning, sequence, and expression analysis of GpSS1 and its regulation by MeJA in Gynostemma pentaphyllum / 中草药

Objective: To clone Gynostemma pentaphyllum squalene synthase gene (GpSS1) and analyze its sequence and expression pattern as well as its regulation in response to MeJA. Methods: Primers were designed based on the sequence of GpSS (GenBank accession numbers: FJ906799) and GpSS1 was cloned by using RT-PCR method. Physicochemical properties and transmembrane regions of the deduced GpSS1 protein were predicted via Protparam and Tmpred programs, respectively. Conserved domains involved in catalytic activity of SS enzyme were identified using MotifScan and multiple sequence alignment was achieved using the BioEdit software. Expression pattern of GpSS1 and its regulation by MeJA were analyzed by quantitative real-time RT-PCR. Results: GpSS1 contains an open reading frame of 1 254 bp encoding a putative protein of 417 amino acids. The protein has an aspartate-rich motif and an endoplasmic reticulum membrane anchoring region in addition to three conserved domains involved in catalytic activity of SS enzyme. The expression level of GpSS1 in young leaves was the highest, followed by that in old leaves, and the lowest was in rhizomes. The expression of GpSS1 was significantly upregulated in G. pentaphyllum leaves sprayed with different concentration of MeJA. The greatest upregulation of GpSS1 occurred in G. pentaphyllum leaves treated with 50 μmol/L MeJA. In both young and old leaves, the transcription of GpSS1 gradually increased and then decreased to varying degrees at 6-96 h after MeJA treatment. However, the expression level of GpSS1 was always higher in young leaves than that in old leaves. Conclusion: The cloning of GpSS1 and analysis on the expression regulated by MeJA will be helpful for further elucidating the function of GpSS1 and its mechanism regulated by MeJA, as well as for improving the quality and content of gypenosides.

Establishment of cell suspension culture system of Carthamus tinctorius and analysis of compounds by UPLC-Q-TOF/MS / 中草药

Objective: To study the establishment of cell suspension culture system and build a platform for exploring the functional genes in the biosynthesis pathway for Carthamus tinctorius. Methods: Using the cotyledon of C. tinctorius as explants for callus induction, callus with good quality was selected for suspension cultivation. The factors influencing the establishment of C. tinctorius cell suspension culture system were investigated, such as hormone, inoculum amount, pH, and cell activity. The effect of MeJA at different concentration on suspension cell growth rate was studied. Chemical compounds in suspension cell system were preliminary identified by UPLC-Q-TOF/MS. Results: The optimal medium for the cell suspension culture was MS + 1.0 mg/LTDZ + 0.1 mg/L NAA, the pH was 5.5-6.0, and inoculum amount was 0.02 g/mL. MeJA at different concentration had effects on suspension cell growth rate: 50 μmol/L MeJA increased cell growth rate, 500 μmol/L MeJA depressed cell growth rate greatly. Thirteen potential chemical compounds in suspension cell system were preliminary identified. Conclusion: The cell suspension culture system of C. tinctorius is established, and MeJA with optimal concentration has effects on suspension cell growth rate.

Effects of exogenous MeJA, SA and two kinds of endophytic fungi on physiology and total phenols content of seedlings of Bletilla striata / 中国中药杂志

Tissue culture seedlings of Bletilla striata were treated with MeJA, SA and two kinds of endophytic fungi in order to study the effects of those treatments on the physiology and total phenols content. The method of tissue culture was used to culture seeds into seedlings, and then different treatments were applied on them to observe and measure the changes of physiology and total phenols content. We find that the growth of seedlings treated with SA was poor, which treated with 40 μmol•L⁻¹ MeJA, 50 mL•L⁻¹ Hypocrea koningii and 10 mL•L⁻¹ Trichoderma koningiopsis showed better. The activity of SOD, POD and CAT was at a high level under SA treatment of each concentration. The activity of SOD and POD increased as the rise of MeJA concentration, while CAT was highest at 80 μmol•L⁻¹. The activity of SOD and POD increased with the increasing of the concentration of H. koningii treatment, while CAT reached the highest at 1 mL•L⁻¹. The activity of SOD, POD and CAT increased first and then declined with the concentration of T. koningiopsis increasing, and the highest activity was at 10 mL•L⁻¹. The contents of MDA, soluble protein and proline were increased more or less under the four treatments. The content of polysaccharide was at a high level under 60 μmol•L⁻¹ of MeJA. The total phenols content was at a high level under 40 μmol•L⁻¹ of MeJA, 60 μmol•L⁻¹ of SA, 1 mL•L⁻¹ of H. koningii and 10 mL•L⁻¹ of T. koningiopsis. The results indicated that the addition of exogenous MeJA, SA and endophytic fungi under certain concentrations could improve the resistance of B. striata and increase the content of total phenols at some degree and the trearment of MeJA, H. koningii and T. koningiopsis could promote the growth of seedlings under certain concentrations.

Full-length cDNA cloning of AsNINJA1 gene in Aquilaria sinensis and MeJA-induced expression in callus / 中草药

Objective: To clone the full-length cDNA of interacting protein of JAZ (NINJA) gene in Aquilaria sinensis, and to provide the basic information for further study on gene function in sesquiterpenes biosynthesis pathway. Methods: With the total RNA as template, the full-length cDNA of NINJA in A. sinensis was cloned through RACE technique and RT-PCR method. The bioinformatics of cloing NINJA gene was analyzed as well. The expression mode of this gene was with MeJA treatment in A. sinensis callus detected by qRT-PCR method. Results: The full-length cDNA (1 982 bp) of NINJA gene in A. sinensis, named as AsNINJA1 was obtained with an open reading frame of 1 221 bp and encoding 406 amino acids. The relative molecular mass of AsNINJA1 protein calculated was 43 697, and the isoelectric point was 6.02. The qRT-PCR results indicated that MeJA treatment could stimulate the increase of mRNA expression of AsNINJA1; There was a sharp rise at 4 h with nearly 100 times higher than the control (without MeJA treatment), then dropped significantly. Conclusion: The full-length cDNA sequence of AsNINJA1 gene is obtained; AsNINJA1 is extremely sensitive to MeJA treatment, and responded to the early damage.