过表达Dbtnbt基因提高中国红豆杉细胞的紫杉醇含量

Overexpression of a 3'-N-Debenzoyltaxol N-benzoyltransferase Gene Promotes Taxol Yield in Taxus chinensis Cells

本文通过克隆3'-N-去苯甲酰紫杉醇N-苯甲酰转移酶(3'-N-debenzoyltaxol Nbenzoyltransferase,DBTNBT)基因Dbtnbt,构建其表达载体p1303-SDbtnbtN,转化中国红豆杉细胞,经潮霉素抗性筛选获得转基因细胞系.转基因细胞分析结果表明,T-DNA及其包含的基因与宿主细胞染色体成功整合;转基因细胞中报告基因GusA-mgfp5正常表达;转基因细胞的Dbtnbt mRNA表达量是未转化细胞的1.33倍;转基因细胞的紫杉醇产量约为27.3μg/g,是未转化细胞的1.37倍.本研究结果表明,过表达Dbtnbt基因将中国红豆杉细胞的紫杉醇产量提高约37%.

In this study, one gene encoding 3＇-N-debenzoyltaxol N-benzoyhransferase （DBTNBT）, one key enzyme involved in taxol biosynthesis, was cloned and constructed into an expression vector pl303- SDbtnbtN. The pl303-SDbtnbtN was transformed into Taxus chinensis cells with an Agrobacterium mediated transformation method, and the Dbtnbt transgenic cells were obtained after hygromycin screening for generations. PCR and Southern blot analyses showed that the transgenes at the T-DNA region were integrated into the host chromosomes. Western blot analysis showed that a reporter gene GusA-mgfp5 could be detected. Quantitative real-time PCR analysis showed that the Dbtnbt expression level was 1.33- fold higher than that in non-transformed cells. I-IPLC analysis showed that the taxol yield from the transgenic cells was 27.3 pμg/g （ taxol/ dry weight of cells）, which was about 1.37 times of the non transformed cells. Our results showed that overexpression of the Dbtnbt gene increased taxol yield by 37% in the transgenic T. chinensis ceils.