Mao Pei-Hong, Ma Xiang-Dong, Jin Xiang, Yang Hong-Mei, Lou Kai
Ion Beam Biotechnology Center, Xinjiang University, Urumqi 830008, China.
Wei Sheng Wu Xue Bao. 2007 Oct;47(5):905-9.
The genome DNA from Ephedra glauca was randomly transferred into Hansenula anomala, respectively, by argon ion bombardment. Then, after screening by the motheds of bromothymol blue indicator selection, slant cultivation, l-ephedrine and d-pseudoephedrine copper chromic salt qualitative test and RP-HPLC determination, 3 strains, producing recombined yeasts were obtained, which can use glucose as a carbon source, NaNO3 as nitrogen source and be genetically stable. After cultivated in liquid medium for 72 hours and analyzed by the RP-HPLC, the recombined strains can produce l-ephedrine 11.87 mg/L and d-pseudoephedrine 4.11 mg/L excellular, d-pseudoephedrine 294.86 mg/g dry cell incellular, but l-ephedrine not detected incellular. The transformation efficiency of Ephedra genome DNA transferred into yeasts via argon ion bombardment was 0.65%. The effects of Ephedra genome DNA macromolecule integrity on yeast transformation system were discussed. The results shown that DNA macromolecule with integrated structure used as exogenous donor can obtain higher transformation efficiency than DNA macromolecule random fragments by ion implantation mediated DNA transformation. It was inferred that biosynthesis of l-ephedrine and the d-pseudoephedrin were controlled by linked together genes or gene clusters.
采用氩离子轰击法，将麻黄基因组DNA随机导入异常汉逊酵母中。然后，经过溴百里酚蓝指示剂筛选、斜面培养、l-麻黄碱和d-伪麻黄碱铜铬盐定性试验以及RP-HPLC测定等方法筛选，获得了3株能以葡萄糖为碳源、硝酸钠为氮源且遗传稳定的重组酵母菌株。在液体培养基中培养72小时后，经RP-HPLC分析，重组菌株胞外可产生l-麻黄碱11.87 mg/L和d-伪麻黄碱4.11 mg/L，胞内d-伪麻黄碱含量为294.86 mg/g干细胞，但未检测到胞内l-麻黄碱。通过氩离子轰击将麻黄基因组DNA导入酵母的转化效率为0.65%。探讨了麻黄基因组DNA大分子完整性对酵母转化系统的影响。结果表明，在离子注入介导的DNA转化中，具有完整结构的DNA大分子作为外源供体比DNA大分子随机片段能获得更高的转化效率。推测l-麻黄碱和d-伪麻黄碱的生物合成受连锁基因或基因簇控制。
