摘要
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 heterologous expression in Saccharomyces cerevisiae of two genotypes： β-AS（A-T） genotype which is related to high content of glycyrrhizic acid and β-AS（G-C） genotype which is related to low content of glycyrrhizic acid,and compare two different genotypes on the impact of β-amyrin production in order to provide a foundation for licorice molecular breeding.Method： The 2 289 bp fragment in plasmid pMD-19T encoding β-amyrin synthase was subcloned into the yeast-Escherichia coli shuttle vector pY26,thus an expression recombinant plasmid PY-β-AS containing target gene was constructed.The PY-β-AS was introduced into defective mutant INVSc1 of S.cerevisiae by LiAc method,after induced by IPTG,the content of β-amyrin was determined by GC-MS.Result： GC-MS analysis demonstrates that the an occurring peak corresponding to β-amyrin standards was detected with the same retention time,which is absent in the cell transform with empty vector.Results showed the peak was β-amyrin and the percentage of β-amyrin in two genotypes： β-AS（A-T） genotype and β-AS（G-C） genotype were 19.08% and 1.40%,respectively.Thus the β-amyrin synthase exhibited the activity of catalyzing 2,3-oxidosqualene to β-amyrin.Conclusion： The catalytic efficiency of β-AS（A-T） genotype is higher than that of β-AS（G-C） genotype,which can lay the foundation for licorice molecular breeding.
出处
《中国中药杂志》
CAS
CSCD
北大核心
2010年第22期2941-2944,共4页
China Journal of Chinese Materia Medica
基金
国家自然科学基金项目(30672615)
