关键词: 灵芝 , 遗传转化 , 紫衫二烯合酶 , 紫衫二烯 , 紫杉醇前体

Abstract: 【Objective】The objective of the study is to lay a theoretical foundation for the expression and production of taxol or its precursor products by using Ganoderma lucidum as a novel bioreactor.【Method】 For preparation of the G. lucidum protoplast, mycelium were collected and washed twice with 0.6 mol•L-1 mannitol, and enzymed with 1% lywallzyme at 30℃ for 3 h. Protoplasts were collected and suspended with 0.6 mol•L-1 mannitol, and the final concentration was adjusted to 1.0×108 cells/mL. For transformation of the exogenous gene (ts), the plasmid pgGIgpd-TS (1.0 μg) and pBgGI-hph (1.0 μg) were added in G. lucidum protoplast culture. Incubation on ice was performed for 30 min. Subsequently 0.5 mL of PEG was added and incubated at room temperature for 20 min. The transformed protoplasts were inoculated on the medium without hygromycin and cultivated 5-7 days at 25℃. After the colonies grew up, they were covered with the PDA semisolid medium with hygromycin. Genomic DNA and RNA were extracted from the mycelium of regenerated G. lucidum for transformant analyses. PCR and RT-PCR analyses were carried out for detection of the ts gene in putative transformants. For analysis of taxa-4(5),11(12)-diene, samples were analyzed on GC linked to a mass spectrometer using split-less injection. Temperature of the column was initially set and maintained at 100℃ for 1 min and was then increased to 300℃ at a rate of 8.0℃•min-1. The column was then maintained at this temperature for 2 min. 【Result】The putative transformants were obtained on the selective plates with hygromycin at 25℃. No colonies appeared on the nontransformed plates. This result indicated that the hph gene was transformed into the G. lucidum and obtained preliminary expression. The results showed that 4 out of 20 putative transformants were integrated into both ts and hph genes，with a co-transformation efficiency was 20%. RT-PCR results verified that all the 4 transformants had obtained the transcription of taxadiene synthase gene. Crude extracts from the G. lucidum were analyzed. It was found a GC peak at 16.762 min which was absent in the wild type control. The mass fragmentation pattern of this peak had the same diagnostic ions with taxa-4(5),11(12)-diene. The taxadiene was detected in the mycelia of transformed G. lucidum strain by GC-MS analysis.【Conclusion】This is the first report of the production of taxa-4(5),11(12)-diene by engineering a novel transformation system G. lucidum with taxadiene synthase gene. The authors proposed that G. lucidum is a promising host for the biotechnological production of precursors of paclitaxel, and the results for the study of molecular biology of G. lucidum is important.

Key words: Ganoderma lucidum , genetic transformation , taxadiene synthase , taxa-4(5) , 11(12)-diene , paclitaxel precursor