华南象草PpCAD基因的亚细胞定位及其在转基因烟草中的异源表达

Subcellular Localization of PpCAD from Elephant Grass (Pennisetum purpureum cv.Huanan) and Its Heterologous Expression in Transgenic Tobacco

该研究根据已克隆的华南象草(Pennisetum purpureum cv.Huanan)肉桂醇脱氢酶(CAD)基因PpCAD的cDNA序列,构建亚细胞定位载体pAN580-PpCAD,用PEG介导法转化象草原生质体,以探究PpCAD蛋白在细胞内的定位;同时构建植物过表达载体pBA002-PpCAD,通过农杆菌介导法在烟草中异源表达,以研究PpCAD基因与植物木质素合成的关系。结果显示:(1)PpCAD定位在象草原生质体的细胞质内;(2)过表达载体pBA002-PpCAD转化烟草后获得27株转基因烟草,其中25株PCR鉴定为阳性;(3)半定量RT-PCR检测6株转基因烟草后发现,PpCAD基因在不同植株的表达量存在差异,通过Southern杂交检测后发现该差异与目的基因插入的拷贝数有关;(4)6株转基因烟草和野生型烟草表型上没有明显差异,除目的基因多拷贝插入的植株OEC6外,木质素含量有不同程度的提高,最高比野生型提高了56.50%。研究表明,PpCAD是一个细胞质蛋白,在烟草中过表达PpCAD能够提高植株木质素含量,表明PpCAD基因参与了植物的木质素合成,可用于象草的木质素调控研究。

In the present study,a vector of pAN580-PpCAD was constructed based on the cDNA sequence of PpCAD gene from elephant grass （Pennisetum purpureum cv.Huanan）,and transformed to elephant grass protoplasts by PEG-mediated method,attempting to figure out the subcellular localization of PpCAD protein.At the same time,the sense vector pBA002-PpCAD was constucted and overexpressed in tobacco by Agrobacterium-mediated method,aiming to study the relationship between PpCAD and plant lignin biosynthesis.The results showed that：（1）PpCAD was located in the cytoplasm of elephant grass protoplasts.（2）27 transgenic tobacco plants were obtained after transformed with overexpression vector pBA002-PpCAD,of which,25 plants were identified positive by PCR.（3）The expression levels of 6 transgenic plants were different after analyzed by semiquantitative RT-PCR.The Southern blot indicated the expression level was related to the insert copy number of target gene.（4）There was no obvious phenotype difference between PpCAD overexpressed plants and wild type,but the lignin contents were increased in varying degrees among 6 transgenic plants except OEC6.Compared with wild type,the maxium amplification rate of lignin content was 56.50%.This study demonstated that PpCAD was a cytoplasmic protein.Overexpression of PpCAD in tobacco would lead to a increase of lignin content,which indicated that PpCAD was involve in plant lignin biosynthesis and it could be available for regulation of elephant grass lignin biosynthesis.