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目的:分析与高、低甘草酸含量密切相关的β-AS(A-T)基因型和β-AS(G-C)基因型在酿酒酵母中异源表达的情况,比较这2种基因型对所生成的β-香树酯醇产量的影响,从而为甘草分子育种奠定基础。方法:将克隆的甘草β-AS基因亚克隆到大肠杆菌和酿酒酵母的穿梭表达载体pY26中,从大肠杆菌中筛选出含有目的基因的重组质粒PY-β-AS,用醋酸锂法转化到酿酒酵母缺陷型菌株INVScI中,经半乳糖诱导后,收集菌体,用气相色谱/质谱(GC-MS)分析生成的β-香树酯醇的量。结果:甘草β-AS基因所编码的酶能催化酵母内源性2,3-氧化鲨稀生成β-香树酯醇,GC-MS分析显示甘草β-AS(A-T)基因型和β-AS(G-C)基因型所生成的β-香树酯醇的质量分数分别为19.08%,1.40%。结论:甘草β-AS(A-T)基因型的催化效率高于β-AS(G-C)基因型,可为甘草分子育种奠定基础。
OBJECTIVE: To analyze the heterologous expression of β-AS (AT) genotype and β-AS (GC) genotypes in Saccharomyces cerevisiae, which are closely related to the contents of high and low glycyrrhizic acid. β-amyrin alcohol production, which laid the foundation for licorice molecular breeding. Methods: The cloned gene of β-AS was subcloned into shuttle vector pY26 of Escherichia coli and Saccharomyces cerevisiae. The recombinant plasmid PY-β-AS containing the gene of interest was screened from Escherichia coli and transformed into wine by lithium acetate Yeast-deficient strain INVScI, induced by galactose, the cells were harvested and the amount of β-carnilate alcohol formed was analyzed by gas chromatography / mass spectrometry (GC-MS). Results: The enzyme encoded by β-AS gene of Glycyrrhiza uralensis could catalyze the endogenous 2,3-oxidized shark to produce β-amygdalin in yeast. The genotypes of β-AS (AT) and β-AS (GC) genotype generated β-amyrin alcohol mass fraction of 19.08%, 1.40%. Conclusion: The catalytic efficiency of β-AS (A-T) genotype is higher than that of β-AS (G-C) genotype, which may lay the foundation for licorice molecular breeding.