| Rice（Oryza sativa）is an important cereal crop in the world and is the staple food of nearly 3 billion worldwide people.However,rice is also one of the crops that is subjected to plant diseases and insect pest attacks seriously,the global annual production of rice caused by lepidoptera pests reached nearly 10 million tons.The long-term use of chemical pesticides,not only increased the cost of planting,cause pest resistance,but also make the environment pollution and destruction of ecological balance.Therefore,the use of transgenic technology to import foreign insect-resistant genes into the rice genome is the most effective way to solve pest problems.At present,the most commonly used insect resistance gene is the Bt gene,and also obtained a variety of transgenic Bt rice lines with insect-resistant.In this study,univalent cry2A*insect-resistant rice Kongyu 131 and Songjing 9were used as experimental materials,the differences of DNA level,mRNA level,protein level and soil microbes between transgenic plants and non-transgenic control were studied by using the method of Basta resistance test,PCR detection,colloidal gold immunochromatography,real time PCR,enzyme-linked immunosorbent assay（ELISA）,Southern blot hybridization,genome-wide sequencing and soil microbiological detection,the transgenic insect-resistant rice plants with low gene copy number,high genetic stability and high expression of exogenous protein,and no significant safety to soil environment were identified.The main results are as follows:1,Using Basta resistance detecting,colloidal gold immune chromatography and PCR detection methods,after T8T10 generation screening,successfully identified transgenic early japonica rice lines HD2 with cry2A*（Kongyu 131（cry2A/bar））and HD4（Songjing 9（cry2A/bar））.2,The mRNA expression of HD2 and HD4 gene in cry2A*transgenic rice was showed there were differences in the same growth period of the same tissues of different strains HD2-1>HD2-2,HD4-1>HD4-2;there were significant differences in the same growth period of the different tissues of the same strains,from high to low,followed by leaves>young spike>stem and sheath;and there were significant differences in the different growth period of the same tissues of the same strains,which showed heading stage>seedling stage.3,The expression of Bt protein in the transgenic rice lines HD2 and HD4 was showed there were differences in the same growth period of the same tissues of different strains,HD2-1>HD2-2,HD4-1>HD4-2;there were obvious differences in the same growth period of the different tissues of the same strains,the expression of Cry2A protein at heading stage,from high to low:leaf>young spike>stem sheath;and there were significant differences in the different growth period of the same tissues of the same strains,heading stage>seedling stage in leaf.4,The transgenic rice lines HD2 and HD4 with the cry2A*gene intergenerational stable inheritance and were single copy inserted into the rice genome.Among them,the cry2A*gene of HD2-1 was inserted into the endogenous gene of chromosome 7,and the target gene of HD4-1 was inserted into the endogenous gene intergenic region of chromosome 1.5,There were no significant differences in agronomic traits and grain quality of transgenic rice lines HD2 and HD4.Both HD2 and HD4 have commercial potential,among them HD2-1 and HD4-1 being better.6,Compared with the cultivation of common rice soil,there were no significantly difference in the number of bacteria,fungi and actinomycetes in rhizosphere and intercropping soil that cultivate the transgenic rice lines HD2 and HD4,and also found that no foreign genes in transgenic rice were transferred to soil microbial level. |
