铁皮石斛DoSMT2基因的功能分析

为了揭示铁皮石斛(Dendrobium officinale)甾醇C-24甲基转移酶2基因(DoSMT2)在甾醇代谢过程的功能,该研究通过根癌农杆菌介导法将来源于铁皮石斛的DoSMT2基因转化烟草(Nicotiana tabacum),并采用qRT-PCR技术检测DoSMT2基因在转基因烟草叶片中的表达,采用气相色谱质谱法分析菜油甾醇和谷甾醇的含量。结果显示:(1)成功获得DoSMT2基因的开放阅读框(1 119 bp),并成功构建正义植物表达载体质粒pCXSN-DoSMT2,经农杆菌介导的烟草叶盘转化法转化烟草并鉴定,获得4株阳性转基因烟草植株。(2)Southern blot结果表明,4株转基因烟草植株都有1条杂交信号带,而非转基因烟草植株没有,说明外源DoSMT2基因都以单拷贝整合到4株转基因烟草基因组中。(3)qRT-PCR检测显示,非转基因烟草未检测到外源DoSMT2基因的表达,4株转基因烟草都能检测到DoSMT2基因的表达,且表达水平差异极显著,各株系表达量高低依次为P3P1P2(P4)。(4)气相色谱质谱分析显示,转DoSMT2基因烟草叶片的菜油甾醇含量均极显著低于非转基因烟草叶片,而谷甾醇含量均极显著高于非转基因烟草叶片。研究表明,DoSMT2具有催化24-亚甲基胆甾烯醇转化形成24-亚乙基胆甾烯醇活性。

Functional Analyses of DoSMT2 Gene Isolated from Dendrobium officinale

In order to reveal the function of the sterol C 24 methyltransferase 2 gene (DoSMT2) in the sterol metabolism process of Dendrobium officinale, we firstly transformed the DoSMT2 gene into tobacco (Nicotiana tabacum) by Agrobacterium tumefaciens mediated method. qRT PCR was then applied to detected the DoSMT2 gene expression in transgenic tobacco leaves, and the contents of brassinosterol and sitosterol were analyzed by GC MS. The results showed that: (1) a 1 119bp open reading frame (ORF) of DoSMT2 gene was successfully obtained, thereafter the sense plant expression vector pCXSN DoSMT2 was successfully constructed too. Four positive transgenic tobacco plants were obtained and identified by tobacco leaf disc transformation through A. tumefaciens mediated method. (2) Southern blot results showed that there was a hybridization signal band in all the four transgenic tobacco plants, while it was not found in non transgenic tobacco plants, which indicated that the foreign gene DoSMT2 was integrated into the genome of the transgenic tobacco plants in a single copy mode. (3) The DoSMT2 gene expression was not detected in non transgenic tobacco, while the expression of DoSMT2 gene could be detected in all four transgenic tobacco plants by qRT PCR, and the difference of expression level was significant. The expression level of each line was P3 > P1 > P2(P4), in turn. (4) The GC MS analysis results showed that the content of campesterol in transgenic tobacco leaves was significantly lower than that in non transgenic tobacco leaves, while the content of sitosterol in transgenic tobacco leaves was significantly higher than that in non transgenic tobacco leaves. This study indicated that the DoSMT2 protein had the catalytic activity in the transformation from 24 methylene lophenol to 24 ethylidene lophenol.