Cloning and characterization of two rhamnose synthases from Sorbus aucuparia / 药学学报

Rhamnose synthase (RHM) is a key enzyme in the biosynthesis of uridine diphosphate rhamnose (UDP-Rha), reversibly converting uridine diphosphate-glucose (UDP-Glc) into UDP-Rha in the presence of NADH or NADPH. In this research, yeast extract (YE) was used to stimulate Sorbus aucuparia suspension cells. Based on a previous study of the transcriptome database of S. aucuparia suspension cells, two RHMs were cloned from S. aucuparia and named SaRHM1 (GenBank No.: MK213340) and SaRHM2 (GenBank No.: MK213341). The SaRHM1 gene contained a 2 007 bp open reading frame (ORF) encoding a polypeptide of 668 amino acids with a molecular weight of 75.25 kD, and a theoretical isoelectric point (pI) of 7.24. The SaRHM2 gene contained a 2 040 bp ORF encoding a polypeptide of 679 amino acids with a molecular weight of 76.26 kD and pI of 6.41. Bioinformatic analysis indicated that SaRHM1 and SaRHM2 contained two special sequences of GxxGxxG/A and YxxxK. Multiple sequence alignments and phylogenetic trees show that SaRHM1 and SaRHM2 have high sequence similarity with other plant species of RHMs. The results of enzyme activity assays in vitro revealed that both recombinant SaRHM1 and SaRHM2 are able to convert UDP-Glc into UDP-Rha. SaRHMs displayed maximum activity at 40 ℃ and a pH of 8 and 9, respectively. The Km values of SaRHM1 and SaRHM2 for UDP-Glc were 212.4 ± 56.70 and 361.0 ± 63.74 μmol·L-1, respectively, with Vmax values of 235.5 ± 18.98 and 516.5 ± 22.30 nmol·min-1·μg-1, respectively. This study reports the cloning and sequencing of RHMs from S. aucuparia and verifies their function, which likely provide rhamnose donors for the subsequent biosynthesis of rhamnosides.

Preliminary study on memory function of Sorbus aucuparia suspension cell to biotic stress / 中国中药杂志

Plants have a memory function for the environmental stress they have suffered. When they are subjected to repeated environmental stress, they can quickly and better activate the response and adaptation mechanism to environmental stress, thus realizing long-term stable reproduction. However, most of the relevant studies are applied to crops and Arabidopsis thaliana rather than medicinal plants about the improvement of plant growth status and the effect on phytoalexin biosynthesis. In this study, yeast extract(YE) was used as an elicitor to simulate biotic stress, and the changes in biomass and the content of some secondary metabolites were measured by giving repeated stresses to Sorbus aucuparia suspension cell(SASC). The results showed that the accumulation levels of biomass and some secondary metabolites in SASC subjected to repeated stress are significantly increased at some time points compared with single stress. A phenomenon that SASC can memorize biotic stress is confirmed in this study and influences phytoalexin accumulation in SASC. Furthermore, the work laid the groundwork for research into the transgenerational stress memory mechanism of medicinal plant.

Full-length Cloning and Protein Expression Analysis of Glycosyltransferases Gene SaUGT1/SaUGT2 in Sorbus aucuparia / 中国实验方剂学杂志

Objective: To obtain the glycosyltransferase gene involved in modification reaction of phytoalexin from Sorbus pohuashanensis suspension cell,and conduct sequence analysis and prokaryotic expression analysis. Method: Based on the transcriptome data,specific primers were designed to obtain 2 cDNA sequences of SaUGTs genes,construct prokaryotic expression vector HIS-MBP-pET28a-SaUGTs and induce the expression of recombinant SaUGTs protein. Result: SaUGT1 and SaUGT2 sequences were cloned and obtained from glycosyltransferases,then bioinformatic analysis of the sequence and prokaryotic expression analysis were conducted. SaUGT1 gene contained 1 458 bp open reading frame (ORF),encoding a polypeptide of 485 amino acids,with a relative molecular weight of 54.27 kDa and theoretical isoelectric point (pI) of 5.50.SaUGT2 gene contained 1 431 bp ORF,encoding a polypeptide of 476 amino acids,with a relative molecular weight of 53.49 kDa and theoretical pI of 5.63. Bioinformatics analysis indicated that SaUGT1 and SaUGT2 protein had no signal peptide,and the conserved domains of glycosyltransferase family were detected. Phylogenetic results showed that SaUGT1 and SaUGT2 proteins had the closest relationship with the UGT85 family of A. thaliana. Differential expression analysis revealed that the relative expression levels of SaUGT1 and SaUGT2 were increased significantly after being induced by yeast extract (YE), with the highest expression level found at 24 h and 12 h. The recombinant SaUGT1 and SaUGT2 proteins were successfully expressed in Escherichia coli DE3 cells and finally,the recombinant SaUGT1 and SaUGT2 proteins were purified through Ni2+ affinity chromatography. Conclusion: The glycosyltransferase gene was cloned from the S. aucuparia for the first time,and the prokaryotic expression vector was successfully constructed,laying foundation for further study of the function of this gene.

Cloning and Sequence Analysis of Pathogenesis Related Protein 1-like Gene from Sorbus aucuparia / 中国实验方剂学杂志

Objective: To explore the functions of pathogenesis related protein 1-like (PR1-like) in suspension cell of Sorbus aucuparia (SaPR1-like) under biotic stress. Method: The full sequence of SaPR1-like gene was cloned and analyzed by multiple bioinformatic tools. The expression of SaPR1-like in the suspension cell of S. aucuparia in response to harpin protein stress was analyzed by real-time fluorescence quantitative polymerase chain reaction (PCR). Result: A SaPR1-like gene was identified and cloned, which contained a complete open reading frame and encoded 161 amino acids. The coding protein was hydrophilic and stable, had transmembrane structure and signal peptide, and belonged to secretory protein. Amino acid sequence alignment data indicated that the C-terminal of SaPR1-like contained a highly conserved domain[cysteine-rich secretory protein, antigen 5, and pathogenesis-related 1 peptides (CAPE)], which could induce defense genes to produce immune responses against biotic stresses. SaPR1-like gene expression was significantly increased after harpin protein induced suspension cell of S. aucuparia for 24 h. Conclusion: A SaPR1-like gene derived from the PR1 family is found to induce significant responses to biotic elicitors in S. aucuparia. This study highlights a role for PR1 in immune signaling and suggests the potential application of PR1 in efforts to defeat biotic stress in plants.

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.