Study On Construcion Of New Phytase Gene Expression Vector And Transformation Into Maize

Zea mays L is one of the most important food of the grain and feed crops, it also the most widely used chemical raw material crops. The maize industry occupies a very important part in the national economy. It’s a top priority to improve the output and quality of maize. The method of using genetic engineering techniques modified corn has an important significance in basic research and breeding practices, this technology can break the barrier that different species difficult to mating in nature, the genes of different species by the will of the people regrouped. Years of research and production that it have been brought enormous social and economic benefits.Phosphorus is one of the most important and necessary element in plants, it’s present in phytate form in the plant and is about70percent of total phosphorus in seed. Phytase is the general term of a class of enzyme which catalytic phytic acid and its salts hydrolysis into inositol and phosphoric acid. Phytate can be hydrolyzed to inorganic phosphorus and inositol hydrolysis and it can release inositol and some trace elements through the effects of phytase, which can improve the nutritional value of plants as a source of nutrition for the growth of plant seedlings. The concentration level of natural phytase in plants is very low, not enough to make the plant to release a sufficient amount of inorganic phosphorus from a large number of phytate. At present, the phytase used for plant breeding has been given considerable attention. The transgenic plants has been markedly improved in using organic phosphorus and plant growth by root-specific expression phytase gene, and has important potential applications in improving plant phosphorus uptake and biological detoxification.In this study, we use of genetic engineering techniques separated phytase gene phyAl from Aspergillus ficuum and combination with specific promoters of corn roots into highly efficient recombinant plant expression vector. The vector was transferred it to the maize inbred lines by Agrobacterium-mediated and pollen tube pathway to improve the maize’s ability of use the potential phosphorus of soil itself.Thereby enhancing the efficient use of nutrients, improve quality and yield of corn, laid a good foundation for high-quality high-yield transgenic plants research.The results of paper are as follows:1. We extraction genomic DNA as a PCR template form aspergillus ficuum materials and designed a pair of specific primers according to the the Known Genebank registration (of PhyA) gene (AY013315.1) sequences by software Primer Premier5.0. And we added two Restriction endonuclease enzyme Qieke Long-bit Xba I and Hind III to the upstream primer and downstream primer5’respectively and the target gene fragment was amplified with the enzyme Qieke Long-bit. Phytase gene was amplified by PCR (named:phyA1) and then cloned the target gene (phyA1) into the pMD18-T vector. The sequencing results showed that the fragment size of phyAl is1511bp, We have analysis this sequence with the DNASIS software and BLAST, and the homology is97%compared to the reported sequences.2. On this basis, The target gene (phyAl) was inserted into the plant expression vector (pCAMBIA3301-ZmGLU1P-Nos) downstream of the promoter which containing corn roots-specific promoter of the plant, and constructed recombinant plant expression vector pCAMBIA3301-ZmGLU1P-phyAl-Nos containing phyAl. Restriction endonuclease and target fragment PCR detection results show that the expression vector was constructed successfully. The pCAMBIA3301-ZmGLU1P-phyAl-Nos was transformed into Agrobacterium tumefaciens EHA105and preparation for engineering Agrobacterium tumefaciens.3. Plant expression vector pCAMBIA3301-ZmGLU1P-phyAl were introduced into maize inbred lines H99, Dan988by agrobacterium-mediated method. Using PCR to analysis TO transgenic plants which Primers were designed according to phyAl sequence and the template is the leaves of transgenic plants DNA. Test results show that the nine plants amplified a specific band (1511bp). The end obtained TO seeds of positive plants. We planted the seeds of positive plants of TO generation and then using PCR amplification to detection the T1generation of transformed plants, got the seven positive plants. Southern blot analysis was used to determine the exogenous gene integration of PCR positive plants. The results showed that the part transgenic plants have hybrid zones and the non-transformed plants not have hybridization signal. Such evidence suggests that foreign genes had been integrated into the genome.4. Phytase activity in transgenic plants were measured with the method of Acetone-ammonium phosphomolybdate, results show obviously increased in the enzyme activity of phytase in the phytase gene in maize plants compared with without conversion maize plants. Such evidence suggests that the phytase gene have already expressed in corn plants.