实时荧光定量RT-PCR检测赖草LRC1基因表达研究

Studies on Expression of Gene LRC1 of Leymus sicalinus Detected by Real-time Fluorescence Quantitative RT-PCR

通过亚克隆技术获得赖草LRC1基因片段重组质粒,以其梯度稀释样品为标准,建立了实时荧光定量PCR技术检测LRC1基因绝对定量表达方法。进一步运用该技术和相对定量法对赖草初花期LRC1基因表达特性进行分析发现,该基因在根茎尖、根茎节间、根茎节、地上茎中的拷贝数分别为477.79、162.65、1368.00、53.86个拷贝/mg(组织鲜重);相对定量法检测结果显示,LRC1基因在赖草根茎节间中的表达量约为其在根茎节中表达量的10%,穗、叶片和茎中表达量则为其在根茎节中的1%、2%和1%。表明该基因在赖草根茎中的表达量显著高于地上组织,特别是其在根茎芽产生的根茎节内的表达量显著高于其他组织。

With sub-cloning technology,a combined plasmid was constructed,which contained LRC1 gene controlling rhizome tillering.The concentration of combined plasmid was detected by analyzing absorbance in 260 nm and then it was diluted to series as standard for real-time fluorescence quantitative RTPCR.LRC1 was amplified by real-time fluorescence quantitative RT-PCR from the plasmid DNA.The method of LRC1 mRNA real-time RT-PCR was well established,which helped to detect as low as 10-1 copies with the linear range from 10-1 to 10-（11） copies.The standard curves showed high correlations（r-2=0.988）.The expression level of LRC1 in the apical of rhizome,internode of rhizome,node of rhizome and stem of Leymus sicalinus at the stage of initial bloom reached 477.79,162.65,1368.00,53.86copies/mg respectively.The results of relative quantitative analysis showed that the expression level of LRC1 gene was approximately 10% ,1% ,2% and 1% in the rhizome internode,spike,blade and stem of that in the rhizome node respectively.Overall,the results showed that the expression level of the LRC1 gene was significantly higher than that in aboveground tissue,especially in rhizome buds,which was significantly higher than that in other tissues.The results laid a foundation for further study on how LRC1 gene regulates the differentiation of Leymus sicalinus underground rhizome buds.