嗜水气单胞菌HBNUAh01 外膜蛋白A 基因在烟草叶片细胞中的瞬时表达

Transient expression of Aeromonas hydrophila HBNUAh01 outer membraneprotein A gene in tobacco leaf cells

摘要:

摘要:【目的】从嗜水气单胞菌(Aeromonas hydrophila)HBNUAh01 中克隆外膜蛋白A (outer membrane protein A，ompA)基因并在烟草(Nicotiana tabacum)叶片细胞中瞬时表达该蛋白。【方法】以嗜水气单胞菌HBNUAh01 为模板进行嗜水气单胞菌外膜蛋白A(AhompA)基因片段的PCR 扩增，并将其克隆到pEASYBlunt Simple 载体中以进行测序。测序正确的AhompA 基因序列与含有黄色荧光蛋白(yellow fluorescent protein，YFP)基因的表达载体pCAMBIA1300 构建重组表达载体。将该重组表达载体转化到农杆菌(Agrobacterium tumefaciens)GV3101 感受态细胞中，随后用阳性转化子转染烟草叶片细胞。使用激光扫描共聚焦成像系统(Confocal Laser Scanning Microscope)检测观察融合表达AhompA 基因的黄色荧光蛋白并采用RT-PCR 检测AhompA 基因在烟草叶片中的转录情况。【结果】从嗜水气单胞菌HBNUAh01中克隆出大小为1032 bp 的AhompA 基因序列，并在烟草叶片中成功表达AhompA 和YFP 的融合蛋白。【结论】AhompA 基因在烟草叶片细胞中的成功表达为进一步研究利用植物疫苗防治嗜水气单胞菌引起的水产动物疾病奠定了基础。

Abstract:

Abstract:［Objective］To clone outer membrane protein A (ompA) gene from Aeromonas hydrophila HBNUAh01 and transiently express this protein in tobacco (Nicotiana tabacum) leaf cells． ［Methods］ A． hydrophila outer membrane protein A (AhompA) gene was amplified by PCR using A． hydrophila HBNUAh01 cells as template，and cloned into the pEASY-Blunt Simple vector for sequencing． After sequence confirmation，AhompA gene was inserted into expression vector pCAMBIA1300 containing a yellow fluorescent protein (YFP) gene，to obtain a recombinant expression plasmid． The recombinant expression plasmid was introduced into Agrobacterium tumefaciens GV3101 competent cells，and the positive clones were transfected to tobacco leaf cells． The expression of yellow fluorescent protein fused with AhompA was observed by Confocal Laser Scanning Microscope，and the mRNA of AhompA was detected by RT-PCR． ［Results］The AhompA gene cloned from A． hydrophila HBNUAh01 was 1032 bp． The fusion protein of AhompA and YFP was successfully expressed in tobacco leaf cells． ［Conclusion］The successful expression of the AhompA gene in tobacco leaf cells has laid a foundation for further investigation of prevention of limnobios diseases caused by A． hydrophila with plant vaccine．