紫杉烯合酶基因遗传转化金针菇的研究

Genetic transformation of Flammulina velutipes with taxadiene synthase gene

利用紫杉烯合酶基因(ts)和潮霉素抗性基因(hph),采用PEG转化法共转化金针菇的原生质体。研究结果表明,18个拟转化子中有8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44.44%;RT-PCR鉴定结果表明,7个金针菇转化子实现了外源ts基因的转录。金针菇转化子在不含潮霉素(HmB)的PDA平板上经5次继代培养后仍检测到有HmB抗性,说明外源基因在金针菇转化子的无性繁殖(即有丝分裂)过程中是稳定的。本研究为通过基因工程手段定向、快速改良金针菇品种以及利用金针菇作为生物反应器生产一些具有重大经济价值的外源基因产物奠定了基础。

The taxadiene synthase gene （ts） and hygromycin phosphotransferase gene （hph） were used in the co -transformation of Flammulina velutipes protoplasts by PEG -mediated method in this paper. The results showed that 8 out of 18 putative transformants were integrated into both ts and hph genes,its co-transformation eficiency was 44.44%. RT-PCR results indicated that 7 transformants obtained the transcription of taxadiene synthase gene. Those transformants still maintained the hgromycin B resistance after 5 generations of subcultures on PDA plates without hygromycin,suggesting that the exogenous gene hph was stable during asexual reproduction（mitosis） of transgenic F. velutipes. The research laid a solid foundation for the improvement of the quality of F. velutipes varieties and production of high value products by using F. velutipes as a novel bioreactor.