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.

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.

Sulfur dioxide limit standard and residues in Chinese medicinal materials / 中国中药杂志

The traditional sulfur fumigation processing method has been widely used in the initial processing and storage of traditional Chinese medicinal materials due to its economy, efficiency, convenience, high operability and effect on mold and insect prevention. However, excessive sulfur fumigation of traditional Chinese medicinal materials would lead to the changes in chemical compositions, and even endanger human health. This study showed that traditional Chinese medicinal materials were sulfur fumigated directly after being harvested for quick drying, or fumigated after being weted in the storage process for preventing mold and insects. We found that the sulfur dioxide limits for traditional Chinese medicinal materials were stricter than those for foods. Based on the existing limit standards, we obtained the data of sulfur dioxide residues for 35 types of traditional Chinese medicinal materials in a total of 862 batches. According to the limit standard in the Chinese Pharmacopoeia (150， 400 mg·kg⁻¹), the average over-standard rate of sulfur dioxide was as high as 52.43%, but it was reduced to 29.47% if calculated based on the limit for vegetable additive standard (500 mg·kg⁻¹). Sulfur fumigation issue shall be considered correctly: sulfur dioxide is a type of low toxic substance and less dangerous than aflatoxin and other highly toxic substances, and a small amount of residue would not increase the toxicity of traditional Chinese medicinal materials. However, sulfur fumigation might change the content of chemical substances and affect the quality of traditional Chinese medicinal materials. Furthermore, the exposure hazards of toxic substances are comprehensively correlated with exposure cycle, exposure frequency, and application method. In conclusion, it is suggested to strengthen the studies on the limit standard of traditional Chinese medicinal materials, formulate practical and feasible limit standard for sulfur dioxide residues in traditional Chinese medicinal materials that are consistent with the medication characteristics of traditional Chinese medicinal materials and can guarantee people's demand for safe medication.

Ecological agriculture: future of Good Agriculture Practice of Chinese materia medica / 中国中药杂志

Based on the ecological and economic problems in Good Agriculture Practice (GAP) of Chinese material medica, we introduced the origin, concept, features and operative technology of eco-agriculture worldwide, emphasizing its modes on different biological levels of landscape, ecosystem, community, population, individual and gene in China. And on this basis, we analyzed the background and current situation of eco-agriculture of Chinese materia medica, and proposed its development ideas and key tasks, including: (1) Analysis and planning of the production pattern of Chinese material medica national wide. (2) Typical features extraction of regional agriculture of Chinese materia medica. (3) Investigation of the interaction and its mechanism between typical Chinese materia medica in each region and the micro-ecology of rhizosphere soil. (4) Study on technology of eco-agriculture of Chinese materia medica. (5) Extraction and solidification of eco-agriculture modes of Chinese materia medica. (6) Study on the theory of eco-agriculture of Chinese materia medica. Also we pointed out that GAP and eco-agriculture of Chinese material medica are both different and relative, but they are not contradictory with their own features. It is an irresistible trend to promote eco-agriculture in the GAP of Chinese material medica and coordinate ecological and economic development.

Bioaccessibility of heavy metal in wild Artemisia annua and its health risk assessment / 中国中药杂志

In this study, we investigate the bioaccessibility of heavy metals (Cu, Pb, As, Cd and Hg) in wild Artemisia annua and use target hazard quotients (THQ) proposed by US Environmental Protection Agency to assess the health risk under the heavy metal exposure. The results showed that the bioaccessibility of Cu, Pb, As, Cd and Hg in A. annua are 0.77, 0.66, 0.46, 0.68 and 0, respectively, and that the value of THQ for adults and children were 0.030 and 0.025 calculated by risk assessment model. The results indicated that the heavy metals in A. annua were not able to be completely absorbed by human body and that their contents were in a safe range. In this study, by combining the bioavailability of heavy metal and health risk assessment, we assessed the security of heavy metals of wild A. annua, which will provide reference for the standard of heavy metals for medicinal materials.

Stability analysis of reference gene based on real-time PCR in Artemisia annua under cadmium treatment / 中国中药杂志

In this study, Actin, 18S rRNA, PAL, GAPDH and CPR of Artemisia annua were selected as candidate reference genes, and their gene-specific primers for real-time PCR were designed, then geNorm, NormFinder, BestKeeper, Delta CT and RefFinder were used to evaluate their expression stability in the leaves of A. annua under treatment of different concentrations of Cd, with the purpose of finding a reliable reference gene to ensure the reliability of gene-expression analysis. The results showed that there were some significant differences among the candidate reference genes under different treatments and the order of expression stability of candidate reference gene was Actin > 18S rRNA > PAL > GAPDH > CPR. These results suggested that Actin, 18S rRNA and PAL could be used as ideal reference genes of gene expression analysis in A. annua and multiple internal control genes were adopted for results calibration. In addition, differences in expression stability of candidate reference genes in the leaves of A. annua under the same concentrations of Cd were observed, which suggested that the screening of candidate reference genes was needed even under the same treatment. To our best knowledge, this study for the first time provided the ideal reference genes under Cd treatment in the leaves of A. annua and offered reference for the gene expression analysis of A. annua under other conditions.

Mechanism exploration on synthesis of secondary metabolites in Sorbus aucuparia cell cultures treated with yeast extract / 中国中药杂志

Suspension cultures cell of Sorbus aucuparia (SASC) was used as materials, the changes of physiological and biochemical indexes of SASC after treatment with yeast extract (YE) were detected, and the synthetic mechanism of secondary metabolites in SASC treated with YE was preliminarily explored. The results were as follows: under the assay conditions, SASC was induced to synthesize five biphenyl compounds, and these compounds content changed differently with induction time prolonging; YE treatment inhibited cell growth, the culture medium pH was gradually reduced after treatment; water-soluble protein content showed a trend of slow decline, which was significantly increased in YE treatment group (YE group) compared with the control group (CK group), the maximum relative content was 147.76% in contrast with CK group; both YE group and CK group were extracellular Ca2+ flow influx, but the YE group flow was significantly slow than CK group. The results indicate that YE induced the cells in a stress state, which was not conducive to the growth of cells and forced the cells to synthesize biphenyl compounds against external stress; water-soluble protein may serve as intracellular enzymes involved in the synthesis of compounds regulation; Ca2+ may as signal molecule mediate cell signal transduction respond to YE stress.