Construction Of PKR Gene Expression Vector And Agrobacterium Tumefaciens Mediated Transformation Of Shoot Apex Of Maize

Maize (Zea mays L.) plays an importance role in agricultural production increasingly.But the quality and yield of maize is decreased by the viral disease during the regular (dwarf mosaic virus, maize rough dwarf virus,etc.) and affected by a large number of weed invasion and competition with soil moisture and nutrients.As maize germplasm has yet to find a suitable antigen gene, also the lack of conventional breeding and the antigen of genetic isolates between different species,therefore,the resistance breeding of maize has been extremely limited.The breeding process of disease resistance and herbicide have been accelerated by using genetic engineering techniques to combat the virus disease and herbicide resistance genes into the genome of corn and cultivating new varieties of resistance,.Anti-double-stranded RNA-dependent protein kinase PKR gene is the major anti-virus genes. Double-stranded RNA-dependent protein kinase by intracellular double-stranded RNA (dsRNA) or viral replication intermediates activated eukaryotic translation initiation factor 2 to make theαsubunit (elF-2α) phosphorylation, leading to viral protein can not start the synthesis. Thus, PKR antiviral genes in plants can play an effective role in inhibiting viral replication.In addition,the encoding phosphoenolpyruvate shikimic acid -3- phosphate synthase EPSPS gene has a high resistance to glyphosate herbicide. Therefore, plant expression vector of PKR and EPSPS gene constructed for transformation plants will enhance the plant's disease resistance and herbicide glyphosate complex traits.The acceptor system of genetic transformation in maize is an important link compared with Agrobacterium-mediated genetic transformation of immature embryos and the traditional embryogenic callus transformation system for the receptor,inbred seed directly into the seedling apical meristems was selected as a receptor for the advantages of simple, rapid, test short period, deriving from seasonal restrictions. Besides, It screened by spraying herbicides was improved efficiency, avoided the process of tissue culture, widened the receptor type of material for genetic transformation of corn,which has a high application value.In order to obtain disease-resistant diseases and toxic compound glyphosate herbicide trait GM maize germplasm, the bivalent PKR-EPSPS gene plant expression vector was constructed by application of In-Fusion cloning technique,and used for Agrobacterium-mediated maize shoot tips genetic transformation in the experiment . The results are as follows:1.The hygromycin gene (hpt) of based vector pCAMBIA5300 was cut by restriction enzyme of SacII and AatII,then the EPSPS gene expression cassette (containing the HSP90 intron sequence, leading signal to plastids sequence, CP4-EPSPS gene)was restructured,which driven by cauliffiower mosaic virus 35S(CaMV35S)promoter.Using the Agrobacterium tumefaciens LBA4404 carrying the middle of the vector pCAMBIA5300-EPSPS by electroporation method.2.The middle of the vector pCAMBIA5300-EPSPS was cut by restriction enzyme of BamHI,then the anti-double-stranded RNA-dependent protein kinase PKR is linked, which driven by maize Ubiqutin promoter.Using the Agrobacterium tumefaciens LBA4404 carrying the bivalent PKR gene plant expression vector pCAMBIA5300-Ubi-PKR-CaMV35S-EPSPS by electroporation method.3. The seedling apical meristems as a receptor of susceptible maize inbred lines Ye 478 seeds was studied the impact factors of the Agrobacterium, the EPSPS gene of the middle of the vector into corn,the results showed that appropriate bacterial concentration (OD600=0.6) by adding acetosyringone (150μmol/L) and surfactant (Silweet L-77) (0.04%) infection in the 50kPa vacuum treatment under 10min, and weeding with 1.5‰agent concentration on the conversion of glyphosate to filter into the most efficient plants.4.The Agrobacterium of containing PKR-EPSPS bivalent plant expression vector pCAMBIA5300-Ubi-PKR-CaMV35S-EPSPS was used transformation in maize inbred lines Ye 478 shoot tips.The 31 resistant plants obtained by PCR detection of 9 were positive, the one seed plant of positive resistant plants.5.The Agrobacterium of containing PKR-EPSPS bivalent plant expression vector pCAMBIA5300-Ubi-PKR-CaMV35S-EPSPS was used transformation in maize inbred lines Chang 7-2 shoot tips.The 27 resistant plants obtained by PCR detection of 14 were positive, the 3 seed plants of positive resistant plants.6.The T₁ Ye 478 transgenic seeds of 20 were received,and planted in the greenhouse to obtain 18 T₁ transgenic plants,which were the PCR detection untiling growed 3 to 4 leaf stage.Eight T₁ transgenic plants were positive,and it was indicated that 40% (8 / 20 ) individuals are genetically modified.7. The results of no come to the separation of the target gene 3:1 segregation ratio by the T₁ transgenic plants segregation ratio (3:1) testing,which may be due to T₀ transgenic plants were chimerism. But the T₁ generation of transgenic plants is no longer a chimeric individual, therefore, T₀ transgenic plants of chimeras obtained by genetic transformation of maize shoot apex of transgenic plants is not much of the breeding.