紫杉二烯合酶基因在灵芝中的表达

Heterologous Expression of Taxadiene Synthase Gene in Ganoderma lucidum

【目的】论文旨在研究紫杉二烯合酶基因(ts)在灵芝中的表达情况,为利用灵芝作为生物反应器表达紫杉醇及其中间产物提供科学理论依据。【方法】制备灵芝菌株的原生质体:把培养好的灵芝菌丝用0.6 mol·L-1的甘露醇溶液洗涤两次后,加入1%溶壁酶溶液,30℃水浴酶解3 h,离心去上清液得到纯的灵芝原生质体,用0.6mol·L-1的甘露醇溶液把原生质体的浓度调整到108个/mL备用;外源基因(ts)的转化:在含有灵芝原生质体的缓冲液各加入含有ts基因和潮霉素抗性基因hph的真核表达载体pgGIgpd-TS和pBgGI-hph 1.0μg,冰浴30 min;加入0.5 mL PEG buffer,在室温下放置20 min;将离心收集的原生质体涂布在不含有潮霉素的再生平板上,25℃培养5—7 d后,待长出白色菌落后,往平板表层覆盖一层含有潮霉素的PDA半固体培养基,于25℃培养;转化子的鉴定:以灵芝的菌丝基因组DNA和RNA为模板,进行PCR和RT-PCR扩增,鉴定ts基因在灵芝中的表达情况;目标产物的检测:对提取的产物采用不分流进样的方式进行气相质谱联用(GC-MS)检测,进样温度250℃,初始温度100℃,保留1 min,每分钟升高8℃,加热到300℃,保留2 min。【结果】经潮霉素初步筛选后,在含有潮霉素抗性的平板上长出白色的菌落,灵芝原种作为阴性对照的平板上没有长出菌落,说明潮霉素基因被成功转入了灵芝并得到了初步的表达;PCR鉴定结果表明20个拟转化子中有4个转化子同时整合了ts基因和hph基因,两个基因的共转化率为20%;RT-PCR鉴定结果表明,4个灵芝转化子实现了外源ts基因的转录;提取转ts基因灵芝的菌丝产物;经过GC-MS测定分析表明,与原种相比,含有ts基因的灵芝工程菌株有一个明显的差异峰,对该峰进行离子碎片分析表明,该物质为紫衫二烯,说明在含有ts基因的灵芝菌丝体中含有目标产物紫衫二烯。【结论】灵芝作为大型真菌,首次被证实可以通过代谢工程生产合成紫杉醇过程中的中间产物紫杉二烯,为以后紫杉醇的生产提供一个潜在的可能,对今后开展灵芝的分子生物学研究具有重要意义。

【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.