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木质部纤维素合酶基因(CesA)在植物正常生长期的木质部特异表达,受到外界压力时可诱导在韧皮部表达,因而被用作杨树抗桑天牛转基因研究中的特异性表达启动子。将前期分离的CesA上游DNA片段JCesAP、YCesAP和MDCesAP分别替换植物表达载体pCAMBIA1302中的组成型启动子CaMV35S,构建成新的植物组织特异性表达载体pJCesAP-GFP、pYCesAP-GFP和pMDCesAP-GFP,然后采用农杆菌介导法转化烟草,均得到完整的再生烟草植株。经PCR初步检测证明目的基因已整合到烟草基因组中。荧光检测JCesAP、YCesAP和MDCesAP片段均能启动GFP蛋白的表达,在395 nm激发光下出现黄绿色荧光,显示3个CesA片段均具有在木质部特异表达的启动子活性。
The xylem cellulose synthase gene (CesA) is expressed specifically in the xylem in normal growth stages of plants and is induced in the phloem when subjected to external pressure. Therefore, CesA was used as a specific promoter in the transgenic study of A. mongolica. The previously isolated CesA upstream DNA fragments JCesAP, YCesAP and MDCesAP were respectively replaced by the constitutive promoter CaMV35S in the plant expression vector pCAMBIA1302 to construct new plant tissue-specific expression vectors pJCesAP-GFP, pYCesAP-GFP and pMDCesAP-GFP, then Agrobacterium-mediated transformation of tobacco, have been completely regenerated tobacco plants. Initial PCR test proved that the target gene has been integrated into the tobacco genome. Fluorescence detection of JCesAP, YCesAP and MDCesAP fragments all induced the expression of GFP protein, and yellow-green fluorescence appeared under the excitation light of 395 nm, indicating that all three CesA fragments have the promoter activity specifically expressed in xylem.