几个杨树木质部特异启动子片段表达载体的构建与功能分析

Construction of Expression Vector and Functional Characterization of Xylem Specific Promoters from Poplar

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。

Expression of xylem-specific cellulose synthase gene（CesA） is confined in xylem cells during plant normal growth and can be induced in phloem cells by exogenous stress.The promoter of CesA gene has been used for tissue-specific expression in preparing transgenic poplar against mulberry longicorn.In present study,the previously isolated JCesAP,YCesAP and MDCesAP,all of which were upstream DNA segments of the CesA gene,were used to substitute the constitutive promoter CaMV35S in plant expression vector pCAMBIA1302 to construct novel plant tissue-specific expression vectors pJCesAP-GFP,pYCesAP-GFP and pMDCesAP-GFP,which were subsequently used to regenerate transgenic tobacco plants by Agrobacterium-mediated transformation.PCR analysis verified that the target genes have been integrated into tobacco genome.Fluorescence detection demonstrated that all JCesAP,YCesAP and MDCesAP segments could promote expression of GFP protein.Excitation by 395 nm light could emit yellowish green fluorescent light,indicating that these three segments had the xylem-specific promoter activity.