| Maize(Zea mays l.)is not only the highest production of crops in the world,is also one of the important food crops in China.The application of genetic engineering breeding technology for the improvement and breeding new maize germplasm provides an important theoretical guidance,but conventional genetic engineering methods will cause people controversy about the safety of genetically modified food.Therefore,The use of an exogenous gene removal system "gene deletor" to create and cultivate safe transgenic maize has important theoretical and practical implications.In this study,the genetic transformation and screening of the corn cultivars T32 and QB506 were carried out with the LoxP/FRT plant expression vector I containing the new recombination site and the FPA plant expression vector II driven by the maize pollen seed specific promoter PMADS35-FLP.To drive exogenous gene removal using hybridization induction,Finally,it lays the foundation for screening and cultivating new genetically modified maize germplasm free of exogenous screening reporter genes.And to achieve the preservation of foreign genes in the seed production process of sexual reproduction,and the seeds for the human and animals that are harvested after the commercialized seeds are harvested do not contain any foreign genes.In this study,the effect of the deletion of the original gene deletion in maize was analyzed by the F1 hybrids of two transgenic plants,and the following results were obtained:1,A plant expression vector II containing a novel recombination site LoxP/FRT and plant expression vector I containing a specific promoter PMADS35-FLP-driven recombinase FLP was constructed,and the shoot apex transformation of maize T32 and QB506 varieties was performed.Thirty-two transgenic corn plants containing plant expression vector I and 29 transgenic corn plants containing plant expression vector II were screened by GUS histochemical staining and PCR amplification.2,Analysis of pollen reporter gene GUS encoded protein activity in hybrid F1 hybrids of two transgenic Plants,It was shown that the specific promoter MADS35 in the original expression before pollen maturation will not drive the expression of the recombinase FLP,but when the pollen matures,the specific promoter MADS35 drives expression of the recombinase FLP.Two maize plants with 100% deletion rate were screened from 51 F1 plants,and compared with the transgenic parents,the deletion rate of exogenous genes was 0-30% in 12 plants and 30-80% in 5 plants.80-100% were 34 strains.3,The morphological and physiological indexes of wild-type corn and the maize plants after the deletion of the reporter gene from F1 hybrids of two transgenic plants were determined.The investigation and analysis of yield traits in related maize showed that the average plant height,ear height,stem diameter,number of leaves,and single leaf area in the F1 generation of the trans-deletion system were: 112.98 cm,47.7 cm,1.375 cm,8.25,292.18 cm2,The average plant height,stem diameter,leaf number and single leaf area of wild-type corn plants were 111.95 cm,57.125 cm,1.725 cm,9.5 tablets,and 298.79 cm2.The data showed that there was no significant difference between wild-type maize and maize that was imported into the extermination system,indicating that the transfer of exogenous deletion system would not affect the agronomic traits.4,The morphological and physiological indexes of the wild-type maize and maize plants after the deletion of the exogenous gene in the F1 generation and the F2 generation of the two transgenic plants were compared.The moisture,oil content,protein content,starch content,lysine content,and glutamic acid average content of the F1 generation were: 13.1%,5.4%,11.9%,68.6%,0.33%,and 2.90%,respectively.The water,oil,protein content,starch content,lysine content,and glutamic acid average content of the F2 generation were: 13.8%,4.8%,11.3%,69.8%,0.22%,and 2.10%,respectively.The moisture,oil content,protein content,starch content,lysine content,and glutamic acid average content of wild-type corn plants were: 13.2%,4.1%,10.5%,70.86%,0.31%,and 2.41%,respectively.The average amylopectin content in F1 plants was 130.22 mg/g,the average amylopectin content in F2 plants was 169.75 mg/g,and the average amylopectin content in wild-type plants was 186.77 mg/g;The data showed that there was no significant difference in the agronomic traits between the transgenic plants with the original deletion and the wild type.Explain that the deletion of the original gene does not affect the quality and morphology of corn.However,in the index of amylopectin content reflecting the taste of corn,the content of amylopectin in the F1 generation was significantly lower than that in the wild type,but the F2 generation will rebound.It shows that the content of amylopectin will be restored in offspring5,Analysis of different space-time expression levels of GUS gene in F1 generation transgenic maize,It was found that the GUS gene expression level in the F1 generation maize ears decreased by 72.3% before maturation matured.The GUS gene expression level in F1 maize stems decreased by 48.4% before maturation matured.The GUS gene expression level in F1 maize leaves decreased by 7.9% before ripening after pollen maturation. |
PDF Full Text Request