Full
Outline
To clone the full-length cDNA of CYP6620B1 gene, which is a key gene in the downstream pathway of triterpenoid synthesis in S. baumii and perform bioinformatics analysis and eukaryotic expression analysis of the gene, with a view to providing a theoretical basis for the elucidation of the mechanism of triterpenoid biosynthesis pathway of S. baumii and the enhancement of triterpenoid production.
The transcript level of CYP6620B1 gene was detected by qRT-PCR at different developmental stages of the mycelium, protocorm and zygote of S. baumii. The full-length cDNA sequence of the target gene was obtained by PCR amplification, and the physicochemical properties and spatial structure of the protein were analyzed by bioinformatics online software. The recombinant Saccharomyces cerevisiae strain INVSc/pYES2-CYP6620B1 was obtained by LiAC/PEG method, and the total triterpene content of it was detected by microplate reader.
The transcript level of CYP6620B1 gene in different developmental stages of S. baumii showed a trend of increasing and then decreasing, which coincided with the reported trend of total triterpenoids in S. baumii, suggesting that it might be involved in the regulation of triterpenoid synthesis, and the full-length cDNA of CYP6620B1 was cloned and obtained as 1 629 bp, encoding 542 amino acids, without signal peptide and transmembrane structure, and the sub The results of phylogenetic tree showed that the CYP6620B1 protein of Taiwanofungus camphoratus had the highest homology with that of S. baumii, and the molecular docking results showed that the CYP6620B1 protein had the lowest binding energy and good docking conformation with lanosterol; the CYP6620B1 gene was successfully expressed in the Saccharomyces cerevisiae strain INVSc, and the total triterpene content of recombinant Saccharomyces cerevisiae strain INVSc/pYES2-CYP6620B1 was significantly higher than that of the control strain.
In this study, the CYP6620B1 gene, which may be involved in the triterpene synthesis pathway of S. baumii, was successfully cloned and analysed, which lays the foundation for an in-depth investigation of the gene's function in the triterpene biosynthesis pathway of S. baumii.
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| 科 Family | 属数 Number of genus | 种数 Number of species | 占总种数比例 Percentage of total species (%) | 属 Genus | 种数 Number of species | 占总种数比例 Percentage of total species (%) |
|---|---|---|---|---|---|---|
| 鹅膏菌科Amanitaceae | 2 | 11 | 5.26 | 鹅膏菌属 Amanita | 10 | 4.78 |
| 小菇科 Mycenaceae | 2 | 12 | 5.74 | 丝盖伞属 Inocybe | 5 | 2.39 |
| 多孔菌科 Polyporaceae | 8 | 14 | 6.70 | 蜡蘑属 Laccaria | 5 | 2.39 |
| 红菇科 Russulaceae | 3 | 23 | 11.00 | 小皮伞属 Marasmius | 6 | 2.87 |
| 小菇属 Mycena | 11 | 5.26 | ||||
| 光柄菇属 Pluteus | 5 | 2.39 | ||||
| 红菇属 Russula | 17 | 8.13 | ||||
| 栓菌属 Trametes | 5 | 2.39 |
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