铁皮石斛DoSMT2基因的功能分析

Functional Analyses of DoSMT2 Gene Isolated from Dendrobium officinale

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(1119 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P3>P1>P2(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。

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 1119bp 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.