Expression Of Exogenous Gene Under The Induction Of Different Promoters And Their Combinations In Rice Suspension Cell Cultures

Promoters are important cis-elements which regulate gene expression, they are essential for the expression of the target gene. In the research and application of gene engineering, the selection of suitable promoters is significant for the efficient expression of exogenous genes. Plant suspension cell lines are an important material to study cell differentiation, metabolism and death. Therefore, the study of suitable promoters in plant suspension cell lines is of massive value in how to improve the expression level and prevent protein degradation and how to achieve efficient, induced, directional expression. Because the green fluorescent protein (GFP) is conveniently detected, fluorescent stable, non-toxic, easy to build, sharing and versatile, it had become one of the most common reporter genes in molecular biology.In this study, Nipponbare rice suspension cells were selected as material, we utilized cauliflower mosaic virus promoter (CaMV) 35S as a comparison, then cloned promoters of maize ubiquitin (Ubi), rice actin (Actin), rice Osccl in plant genome DNA by PCR, by linking up the promoters, we also structured ten expression vectors contained GFP reporter gene as follows:pCAMBIA1304 35S-GFP, Ubi-GFP, Actin-GFP, Osccl-GFP,35S/Ubi-GFP, 35S/Actin-GFP,35S/Osccl-GFP, Ubi/Actin-GFP, Ubi/Osccl-GFP, Actin/Osccl-GFP. Then by applying agrobacterium tumefaciens-mediated derivative method, transformed ten expression vectors mentioned above into Nipponbare rice suspension cells to obtain transgenic Nipponbare rice suspension cells. Fluorescence microscopy and microplate reader were used to detect GFP fluorescence intensity, western blot was operated to detect the level of GFP expression, we studied that the combination of different promoters had a different expression in Nipponbare rice suspension cells. This study is aimed to optimize promoters combination to enable efficient expression of exogenous genes in Nipponbare rice suspension cells by means of genetic engineering, and to lay the foundation for the study of genetic engineering in Nipponbare rice suspension cells.The results showed that:PCR result showed 10 kinds of transgenic Nipponbare rice suspension cells. The fluorescence microscopy revealed that the wild-type Nipponbare rice suspension cells (as a control) almost had no green fluorescence, other transgenic Nipponbare rice suspension cells had green fluorescence. It proved that GFP was expressed in different promoters-driven, and the expression of two promoter-driven GFP was significantly stronger than a single promoter-driven GFP. By quantitative detection of GFP in fluorescence microplate reader, it showed that GFP fluorescence had the highest value in pCAMBIA1304-Ubi/Osccl and the lowest value in the wild type. The expression of dual promoter-driven GFP is about two to four times higher than the expression of single promoter-driven GFP. The intensity of GFP expression in three dual promoters (pCAMBIA1304-Ubi/Oscc1, pCAMBIA1304-CaMV35S/Ubi, pCAMBIA1304-Ubi/Actin) is about three times higher than four single promoters (pCAMBIA1304-Ubi, pCAMBIA1304-Osccl, pCAMBIA1304-Actin, pCAMBIA1304-CaMV35S). Having Detected the expression level of GFP by western blot, we found that wild-type Nipponbare rice suspension cells do not have GFP bands, and other transgenic Nipponbare rice suspension cells all have GFP bands which were about the size of 26.9 kDa. By analyzing the immunoblotting bands of gray, we found that the expression of pCAMBIA1304-Ubi/Osccl, pCAMBIA1304-CaMV35S/Ubi, pCAMBIA1304-Ubi/Actin had higher expression level than other combinations. The result of western blot and expression intensity of GFP by fluorescence microscopy and fluorescence microplate reader were basically the same, so that we can select optimized promoter combinations. The above results indicated that three promoter combinations:pCAMBIA1304-Ubi/Osccl, pCAMBIA1304-CaMV35S/Ubi, pCAMBIA1304-Ubi/Actin are relatively more powerful to drive GFP, Ubi promoter was more suitable to drive a highly expression of foreign gene in Nipponbare rice suspension cells.