Using Cre/loxP System To Cultivate Insectresistant And Herbicide-resistant Transgenic Rice Without Exogenous Gene In Endosperm

Rice yield and quality are greatly affected by insects and weeds in the field.The use of genetic engineering techniques to breed insect-resistant and herbicide-resistant rice can reduce rice yield losses due to insect and weed infestation,However,there are concerns about the safety of genetic engineering technology.In order to enhance public acceptance of genetic engineering technology in production,in this study,transgenic rice lines were developed that specifically deleted the exogenous gene in the seed endosperm,with poten-tial production applications.This study utilized the Cre/loxP system from P1 phage and used the tissue-specific promoter glu C to drive Cre recombinase expression in endosperm.The effective resistance of transgenic rice to insect and herbicides was verified by feeding SSB and glyphosate spraying experiments,the specific deletion efficiency of this recombinant system in the endosperm of the transgenic rice was also tested in the expectation of producing insect and herbicide resistant transgenic rice with endosperm without exogenous genes.The main results of this study are as follows:We fused and ligated three gene expression cassettes,I.variabilis-EPSPS*,cry1C*and cre,into the PCAMBIA1300Z vector and transferred the artificial vector into the geng rice Zhonghua 11 using an agrobacterium-mediated genetic transformation system to obtain a total of 58 positive plants.The positive plants were sprayed with 3600 g/ha of glyphosate and 42 highly glyphosate-resistant transgenic plants were screened.two single-copy lines ML35 and ML70 were obtained at generation T₀.two single-copy homozygous lines ML35-1 and ML70-2 were screened at generation T₂.The deletion rates of each gene expression cassette in the endosperm of the homozy-gous transgenic lines ML35-1 and ML70-2 were examined at generation T_3.The results showed that in the seed endosperm of the ML35-1 line,the deletion rate of the I.variabilis-EPSPS*gene was 70%-92%,the deletion rate of the cry1C*gene was 80.8%-100%and the deletion rate of the cre gene was 70%-89%;in the seed endosperm of the ML70-2 line,the deletion rate of the I.variabilis-EPSPS*gene was 70%-88%,the deletion rate of the cry1C*gene was 73%-100%,the deletion rate of the cre gene was 70%-89%.Results from indoor insect resistance tests showed that the mortality rate of first instar larvae of SSB feeding on ML35-1 and ML70-2 was 39%and 44.5%respectively.In field resistance experiment of SSB,the whitehead rate was 10%and 9%for ML35-1 and ML70-2,respectively.Both transgenic lines showed significantly higher resistance to SSB com-pared to the control geng rice Zhonghua 11.Glyphosate resistance tests on ML35-1 and ML70-2 lines showed that the ML35-1line was tolerant to 5400 mg/L（6 fold recommended agricultural dose）of glyphosate and the ML70-2 line was tolerant to 3600 mg/L（4 fold recommended agricultural dose）of glyphosate.The results showed that the Cry1C*protein content in leaves of ML35-1 at tillering stage was 1.34μg/g;the Cry1C*protein content in leaves at heading and filling stage was0.43μg/g-0.44μg/g;the Cry1C*protein content in endosperm tissue at filling stage was0.0013μg/g.The Cry1C*protein content in leaves of ML70-2 at tillering stage was 1.15μg/g;the Cry1C*protein content in leaves at heading and filling stage was 0.39μg/g-0.37μg/g;the Cry1C*protein content in endosperm tissue at filling stage was 0.0014μg/g.The Cry1C*protein content in leaves at tillering stage was higher than at heading stage and filling stage,and the Cry1C*protein content in endosperm tissues was extremely low at filling stage.In conclusion,this study has produced two transgenic rice lines ML35-1 and ML70-2with high resistance to SSB and glyphosate without exogenous genes in the endosperm,and the target genes were deleted in the endosperm with high efficiency.