高粱抗虫基因CYP71E1表达载体的Gateway技术构建

Gateway Technology for Expression Vector of Sorghum Insect-resistant Gene CYP71E1

[目的】探讨高梁HCN途径关键酶CYP71EI基因对玉米幼苗抗螟虫的影响，为培育抗玉米螟新品种奠定基础。[方法】以高梁自交系BTx623幼苗总RNA为模板，反转录成cDNA，利用聚合酶链式反应（PCR）扩增出1854bp的CYP71E1基因全长。利用基于位点特异性重组的Gateway技术，通过TOPO克隆将该基因克隆到入门克隆（vCRw8／GW／TOP00），在LR克隆酶作用下将入门克隆重组到表达载体（pMDC141）中。【结果】成功构建pMDC141-CYP71E1表达载体并将构建好的表达载体导人农杆菌EHA105，PCR和测序表明所构建的载体及获得的工程菌与预期的完全一致。【结论】Gateway克隆技术与传统的克隆方法相比具有阳性克隆率高、快速、灵活等优点。

[Aims] The effects of key enzyme of CYP71 E1 gene for sorghum HCN pathwayon the borer resistance of maize seedling were discussed, which lie the foundation for the cultivation of new anti-borer com. [Methods] The full length gene of CYP71 E1 of 1854 bp was amplified by polymerase chain reaction （PCR） with the total RNA of sorghum inbred BTx623 seedling as template. The Gateway technology was based on site-specific recombination. The interference fragment was cloned in entry clone via TOPO cloning. Entry clone （pCRTM 8/GW/TOPO） was recombination with expression vector ofpMDC14l by using the LR cloning enzyme. [Results] The expression vector of pMDC141-CYPT1E1 was obtained successfully, and the constructed expression vector was imported into Agrobacterium EHA105. PCR and sequencing showed that the constructed vector and engineering bacterium were agreement with expected. [Conclusions] Compared to traditional methods of cloning, Gateway cloning technology has the advantages of high positive clone rate, fast and flexible.