Study On The Transformation Of Maize With Glyphasate-resistant And High Efficient Phosphine Absorption

It is an urgent requirement in current maize production to increase the absorption and utilization of soil nutrient elements and lightly simplify cultivation.Glyphosate is one of the most widely used herbicides in the world.Phosphorus is a large amount of nutrients essential for the growth and development of maize.It is of great significance for corn production to cultivate complex glyphosate-tolerant compound traits with high-efficiency phosphorus absorption and utilization.In the current study,two bivalent plant expression vectors were constructed,which mainly contain two kinds of fusion genes comprising different chloroplast transit peptide and CP4 EPSPs driven by Ubiquitin1 promoter of and phytase-encoding gene(phyA2)driven by root-specific promoter GLU1 P,and used for genetic transformation of maize shoot tips mediated by Agrobacterium.This study lays a foundation for obtaining excellent transgenic corns seeds with high resistance to glyphosate and high absorption and utilization of phosphorus.The results of this study are as follows:1.In this study,Target P1.1 and ChloroP1.1 were used to predict chloroplast transport peptides containing rbcS sequences of 170 amino acids(AA).The cleavage sites may be located near forty-sixth residues.On this basis,the coding sequence of 74 AA at the N end of rbcS is identified as a candidate fragment with a base length of 222 bp.2.PCR technology and overlap extension PCR technology were used to get CEPSP,MEPSP,Ubiquitin,GLU1 P,phyA2 and Nos genes.Compared with GenBank database,the homology reached 98%,indicating that the target gene was successfully obtained.3.Using pCEPSP vector as the basic vector,Double enzyme digestion and InFusion homologous recombination methods were used to construct plant expression vectors pCE-NGC3 and pCE-NGM3,and then the plant expression vector was introduced into Agrobacterium EHA105 by freeze thaw transformation.Agrobacterium tumefaciens mediated transformation of shoot tip of maize was carried out,and 9 positive transgenic plants were obtained.Among them,there were 4 TY7-2 transgenic plants(2 CEPSP transgenic plants,2 transgenic MEPSP plants),and 5 TY4 CV strains(2 transgenic CEPSP genes and 3 transgenic MEPSP genes).4.The expression of EPSPs gene,using the determination of RT-qPCR in transformed plants showed that the plant and wild type(CK),EPSPs gene expression in transgenic plants leaves significantly increased in transgenic plants transformed in maize chloroplast transit peptide(CC)and EPSPs fusion gene(CEPSP)than the transit peptide fusion Barry according to(CM)the amount of gene expression was increased by 31%.The transgenic plants were sprayed with different concentration of glyphosate solution.The results showed that transgenic CEPSPS plants showed better resistance than MEPSPS transgenic plants.5.The results of phytase activity of transgenic plants showed that the phytase activity of the transformed plant was 15~23 times higher than that of the wild type plant.