葫芦巴环阿屯醇合酶基因的分离及其对薯蓣皂素合成的影响

Molecular cloning of cycloartenol synthase from Trigonella foenum-graecum L. and its effect on diosgenin biosynthesis

以薯蓣皂素合成植物葫芦巴(Trigonella foenum-graecum L.)为材料,从中分离了环阿屯醇合酶基因Tf CAS,并对其序列特征、基因的表达及其对葫芦巴薯蓣皂素生物合成的影响进行了分析。结果显示,该基因全长2271 bp,共编码756个氨基酸;其氨基酸序列与蒺藜苜蓿(Medicago truncatula Gaertn.)、豌豆(Pisum sativum L.)及百脉根(Lotus japonicus L.)环阿屯醇合酶氨基酸序列的同源性分别为94%、91%和89%。利用酵母表达系统对Tf CAS蛋白的生物化学功能进行了验证,结果表明该蛋白能够催化环阿屯醇的合成。进一步利用葫芦巴发根遗传转化体系在葫芦巴中过量表达Tf CAS基因,发现该基因的过量表达大幅提高了Tf CAS的表达,且促进了葫芦巴中β-谷甾醇和薯蓣皂素的生物合成,但与对照相比差异不显著。研究结果表明Tf CAS基因参与了葫芦巴薯蓣皂素的生物合成,但其并非为该合成途径中的限速酶。

As a naturally occurring steroid sapogenin,diosgenin is an important intermediate for the synthesis of hundreds of steroid drugs.However,the biosynthetic pathway to diosgenin has not yet been completely elucidated and it remains unclear whether diosgenin biosynthesis originates from lanosterol or cycloartenol.In this study,the gene encoding cycloartenol synthase(CAS),TfCAS,was successfully cloned from a diosgenin-producing plant Trigonella f oenum-graecum L.The open reading frame of the TfCAS gene had 2 271 base pairs of nucleotides encoding 756 amino acids.TfCAS showed 94%,91%,and 89% of amino acid identities to the CASs from Medicago truncatula Gaertn.,Pisum sativum L.,and Lotus japonicas L.,respectively.Biochemical analysis using a yeast expression system confirmed that TfCAS encodes a functional CAS protein that can convert 2,3-oxidosqualene into cycloartenol.The TfCAS transcript level in Trigonella foenum-graecum was further genetically modified by overexpressing the TfCAS gene via the hairy root transformation system.Real-time PCR analysis showed that the TfCAS transcript level was significantly up-regulated in the TfCAS-expressed hairy roots,suggesting success of the transgene events.Compared with the empty vector-transformed lines,the overexpression of TfCAS led to a slight but non-significant improvement in the biosynthesis of diosgenin andβ-sitosterol.These data indicate that TfCAS may not be a rate limiting enzyme but may play a role in the diosgenin synthetic pathway in Trigonella foenum-graecum.