Construction Of Plant Expression Vectors With Multi-Selectable Marker

Multi-selectable marker plant expression vector is used to monitor plant genetic transformation has a good potential in crop breeding with many excellent agronomic traits and the detection of target genes. Taking plant expression vector pCAMBIA1301 as recipients, fragments of exogenous soluble red shift green fluorescent protein (smRSGFP) gene, herbicide Basta resistance marker gene bar and downstream promoter of a plant CaMV35S were cloned to this vector. The vector contains smRSGFP, bar,β-glucuronidase (GUS) and Hygromycin resistance (Hpt) gene was constructed. Then we connected small heat shock protein (sHsp) cloned from the maize to CaMV35S promoter which is downstream of the bar gene, so the carrier has four marker gene and a functional gene. Not only in E. coli, Agrobacterium was detected in the smRSGFP gene, also detected a variety of corn in the preliminary and sHsp marker gene expression.The studying get to results:(1) Construction of green fluorescent protein gene to this vector, the resulting vector pCAMBIA 1301-smRSGFP in E. coli DH5a, Agrobacterium LBA4404, and the backbone of the parental corn Chang 7-2, inbred YQ7-96 in the group can express green fluorescent;(2) Constructing herbicide resistance gene bar into vector pCAMBIA 1301-smRSGFP. Then,using Agrobacterium that obtained the vector to infect corn Chang 7-2, YQ7-96; Next, maize were selected by herbicide and hygromycin. The survival plants, indicating that the carrier in the bar and the hygromycin resistance gene can be expressed in maize;(3) Continuing to add functions genes sHsp to vector pCAMBIA1301-smRSGFP-bar. By the heat stress screening and GUS staining corn Chang 7-2, YQ7-96 showed that the vector of these two genes can be successfully expressed.The successful construction of this vector provide markers for detecting and screening of multiple genes. By the use of this vector, screening steps could be more concise and widely applicable, what's more, it can reduce false positive rate in plant genetic transformation, and screening based on specific experimental detection conditions. The advantages of this Multi-selectable marker vector are low screening cost and little screening time, Besides the advantages in detection, under the premise of their own characteristics have not been damaged, the vector not only could be connected with the target gene by imported CaMV35S promoter which is in the downstream of the bar gene, but also could be used to the following fusion expression between the target gene and the fluorescent gene using the same promoter. Also according to the characteristics of each marker,with the principle of highly efficient, simple, easy, the detection method has strong originality. The vector with sHsp gene was successfully constructed and tested, not only makes the carrier the detection of marker genes have proven effective, but also demonstrate a functional gene. This vector is fitable of the widely use in practice application.