A Study On Constructing Marker-free Expression Vector Of Phytase Gene For Plant Genetic Transformation

Phosphorus is an important constituent of cereals, legumes, and oilseed crops, which serve as a major source of nutrients for humans and animals. Accounting for about 60-80% of the total phosphorus presenting in these crops is in the form of phytic acid. Monogastric animals are unable to metabolize phytic acid and largely excrete it in their mature, causing environmental pollution in areas of intensive livestock production. Furthermore, in order to compensate for the low available phosphorus, inorganic phosphate has to be supplemented to the feed, which increases feed cost. In addition, phytic acid can chelate various metal ions, such as calcium, copper, zinc, etc., and proteins with positive ligands forming insoluble chelates, which decrease mineral nutrients availability.Phytases can catalyze the hydrolysis of phytase into inorganic phosphates. Genetically engineered crops including rice that contain phytase gene derived from microorganisms will resolve the above problems. The phytase originated from Aspergillus Nigar has a high intrinsic resistance to heat and acid inactivation. Adding phytase to the feed for monogastric animals will reduce the amount of phosphate excreted in the mature and to circumvent supplementation of the feed with inorganic phosphate.During plant genetic transformation, selectable marker is used to identify transgenic plants. However, it is not a necessity for plant growth and it causes public concern about bio-safety, interferes with multiple transformations and hampers the combination of superior characters.The research established a marker-free phytase gene expression vector p1300UbiPHYTASE via co-transformation system with selectable marker gene that can be easily excised. pCAMBIA1300, a member of high-efficient plant genetic transformation expression vector family pCAMBIA, was appliedto construct the target vector, hpt gene, which is a selectable marker gene with T-DNA of pCAMBIA1300 vector, and CaMV 35S promoter were excised. Thus the T-DNA zone contained only phytase gene of interest. High-efficient monocot promoter of Ubiquitin was used and results of enzyme digests and PCR analysis showed that the vector was well established and suitable for the genetic transformation of cereal crops.In this study, expression vector pCAMBIA1300 was transformed to Agrobacterium EHA105 through liquid nitrogen freezing and co-transformation of plant cell conducted via two-plasmid-mediated method, which aimed to obtain marker-free transgenic plants.