Molecular cloning and functional characterization of two squalene synthase genes in Atractylodes lancea.

MAIN CONCLUSION: Two squalene synthase genes AlSQS1 and AlSQS2 were isolated from Atractylodes lancea and functionally characterized using in vitro enzymatic reactions. Atractylodes lancea is a traditional herb used for the treatment of rheumatic diseases, gastric disorders, and influenza. Its major active ingredients include sesquiterpenoids and triterpenes. Squalene synthase (SQS; EC 2.5.1.21) catalyzes the first enzymatic step in the central isoprenoid pathway towards sterol and triterpenoid biosynthesis. In this study, we aimed to investigate two SQSs from A. lancea using cloning and in vitro enzymatic characterization. Bioinformatics and phylogenetic analyses revealed that the AlSQSs exhibited high homology with other plant SQSs. Furthermore, AlSQS1 was observed to be localized in both the nucleus and cytoplasm, whereas AlSQS2 was localized in the cytoplasm and endoplasmic reticulum. To obtain soluble recombinant enzymes, AlSQS1 and AlSQS2 were successfully expressed as glutathione S-transferase (GST)-tagged fusion proteins in Escherichia coli Transetta (DE3). Approximately 68 kDa recombinant proteins were obtained using GST-tag affinity chromatography and Western blot analysis. Results of the in vitro enzymatic reactions established that both AlSQS1 and AlSQS2 were functional, which verifies their catalytic ability in converting two farnesyl pyrophosphates to squalene. The expression patterns of AlSQS and selected terpenoid genes were also investigated in two A. lancea chemotypes using available RNA sequencing data. AlSQS1 and AlSQS2, which showed relatively similar expression in the three tissues, were more highly expressed in the stems than in the leaves and rhizomes. Methyl jasmonate (MeJA) was used as an elicitor to analyze the expression profiles of AlSQSs. The results of qRT-PCR analysis revealed that the gene expression of AlSQS1 and AlSQS2 plummeted at lowest value at 12 h and reached its peak at 24 h. This study is the first report on the cloning, characterization, and expression of SQSs in A. lancea. Therefore, our findings contribute novel insights that may be useful for future studies regarding terpenoid biosynthesis in A. lancea.

Comparison of the effects of fungal endophyte Gilmaniella sp. and its elicitor on Atractylodes lancea plantlets.

The effects of the endophytic fungus Gilmaniella sp. and its elicitor on the defense and metabolic responses of host plants Atractylodes lancea were investigated, in order to understand how to utilize endophytic fungi and their elicitor resources better. The results showed that the promotion effect of the fungus on the growth of host plantlets was much better than that of its elicitor. Both fungus and elicitor enhanced defense-related enzyme activities. In fungus-inoculated groups, phenylalanine ammonia lyase and polyphenol oxidase activities increased slowly, and reached a maximum level during the later stages, whereas peroxidase activity peaked in the first few days. Additionally, the activities of chitinase and ß-1,3-glucanase were significantly higher than those of the control plants. In elicitor-treated groups, however, most of the enzymes were activated during the early stage, and their highest levels were generally lower than those of the fungus-inoculated groups. Compared with the elicitor, fungal infection improved the photosynthetic rate of the host, and increased carbohydrate levels as well as chlorophyll content in host leaves. The total content of the four main components of volatile oil was also increased in elicitor-treated groups, but there was no particular pattern in this increase. Meanwhile, in the fungus-inoculated groups, the content of atractylone significantly increased with time, while the content of ß-eudesmol decreased. These results indicated that fungal elicitor could substantially improve the total content of volatile oil, while the fungus could more effectively enhance the quality of herbal medicines.

[Study on effect of drought stress on physiological adaptation of seedlings of Atractylodes lancea].

OBJECTIVE: To study the effect of drought stress on the changes of physiological adaptation of Atractylodes lancea seedlings. METHOD: Investigation was carried out on content changes of MDA, soluble protein, and activities of SOD, POD, CAT, APX in A. lancea seedlings under polyethylene glycol (PEG-6000)-simulated drought stress. RESULT: In A. lancea seedlings treated with 15% and 25% PEG, the content of MDA increased significantly with the stress time, and increased more significantly at a higher concentration of PEG. The content of soluble protein increased significantly after treatment on the day one and day three; activities of SOD, POD, CAT and APX increased at first and decreased later, increasing rates rose at high concentration of PEG moreover, activities of POD, APX reached the maximum after three days, and the time of maximum activities changed with concentration of PEG. CONCLUSION: A. lancea seedlings adapted to drought stress by increasing the content of soluble protein to decrease water potential, and by improving activities of protective enzymes to enhance anti-oxidative ability under drought stress.