Construction of protoplast genetic transformation system for Mycena--symbiont of Gastrodia elata / 中国中药杂志

Mycena, a symbiont of Gastrodia elata, promotes seed germination of G. elata and plays a crucial role in the sexual reproduction of G. elata. However, the lack of genetic transformation system of Mycena blocks the research on the interaction mechanism of the two. In order to establish the protoplast transformation system of Mycena, this study analyzed the protoplast enzymatic hydrolysis system, screened the resistance markers and regeneration medium, and explored the transient transformation. After hydrolysis of Mycena hyphae with complexes enzymes for 8 h and centrifugation at 4 000 r·min~(-1), high-concentration and quality protoplast was obtained. The optimum regeneration medium for Mycena was RMV, and the optimum resistance marker was 50 mg·mL~(-1) hygromycin. The pLH-HygB-HuSHXG-GFP-HdSHXG was transformed into the protoplast of Mycena which then expressed GFP. The established protoplast transformation system of Mycena laid a foundation for analyzing the functional genes of Mycena and the molecular mechanism of the symbiosis of Mycena and G. elata.

Correlation analysis between continuous cropping obstacle of Gastrodia elata and Ilyonectria fungi and relieving strategy / 中国中药杂志

The continuous cropping obstacle of Gastrodia elata is outstanding, but its mechanism is still unclear. In this study, microbial changes in soils after G. elata planting were investigated to explore the mechanism correlated with continuous cropping obstacle. The changes of species and abundance of fungi and bacteria in soils planted with G. elata after 1, 2, and 3 years were compared. The pathogenic fungi that might cause continuous cropping diseases of G. elata were isolated. Finally, the prevention and control measures of soil-borne fungal diseases of G. elata were investigated with the rotation planting pattern of "G. elata-Phallus impudicus". The results showed that G. elata planting resulted in the decrease in bacterial and fungal community stability and the increase in harmful fungus species and abundance in soils. This change was most obvious in the second year after G. elata planting, and the soil microbial community structure could not return to the normal level even if it was left idle for another two years. After G. elata planting in soils, the most significant change was observed in Ilyonectria cyclaminicola. The richness of the Ilyonectria fungus in soils was significantly positively correlated with the incidence of G. elata diseases. When I. cyclaminicola was inoculated in the sterile soil, the rot rate of G. elata was also significantly increased. After planting one crop of G. elata and one to three crops of P. impudicus, the fungus community structure in soils gradually recovered, and the abundance of I. cyclaminicola decreased year by year. Furthermore, the disease rate of G. elata decreased. The results showed that the cultivation of G. elata made the Ilyonectria fungi the dominant flora in soils, and I. cyclaminicola served as the main pathogen of continuous cropping diseases of G. elata, which could be reduced by rotation planting with P. impudicus.

Identification and expression analysis of the DaNAC transcription factor family in Dipsacus asper / 药学学报

italic>NAC transcription factor genes play an important role in regulating plant adversity stress tolerance and secondary metabolism. To explore DaNAC transcription factor participation in the synthesis of asperosaponin Ⅵ in Dipsacus asper, we analyzed the expression of DaNAC genes based on full-length transcriptome data from different tissues (root, stem, leaf, flower, seed) to provide a theoretical foundation for regulating the metabolism of D. asper. RNA-seq data was used to identify open reading frames. Bioinformatic methods were used to identify the conserved domain motifs and construct an evolutionary tree. qRT-PCR was carried out to analyze tissue-specific and adversity-stressed expression. Twenty-nine DaNAC sequences were identified, all of which contain the conserved NAM domain and conserved motif 1 and motif 2 at the N terminal. Five DaNAC genes are closely related to the NAC genes in Arabidopsis thaliana and rice that are involved in adversity stress and are clustered in the Group Ⅰ subfamily. qRT-PCR revealed that DaNAC genes are differentially expressed between tissues. The expression levels were highest in leaves, followed by roots, stems and petioles, and the lowest in flowers and seeds. Compared with normal growth conditions, the expression of four NAC genes was up-regulated by treatment with low temperature (15 ℃). The expression of three genes (34564NAC2, 33883NAC48, 6727NAC14) was up-regulated and one gene (34480NAC22) was down-regulated by 150 μmol·L-1 MeJA. The results illustrate that the expression of NAC genes is induced by adversity stress, which provides a foundation for further study on the role of NAC family members in adversity stress in D. asper.