Composite Transformation Of Different Bt Insect-resistant Genes Into Rice And Detection And Validation Of Green Tissue-specific Expressive Insect-resistant Rice

Rice is one of the most important food crops in the world,and lepidopteron insects are the main pests in rice production.Conventional pesticide controlling not only increases the cost of investment,but also easily induces insect resistance.Based on the fact that a single insect-resistant gene can easily cause insect resistance and the public's excessive concern about the food safety of genetically modified rice,this article carries out the research from the following two aspects:pyramiding two different insect-resistant genes to breed new persistent Insect-resistant rice materials;detecting and validating the existing transformants to obtain the expected new endosperm zero-expression Insect-resistant rice materials as soon as possible;and the following results have been obtained.:?.Identification and analysis of insect-resistant rice with null-expression in endosperm8 homozygous transgenic lines were obtained from 12 different transformants by PCR analysis,and 5 lines having single copies were determined by Southern blotting analysis.Further quantitative analysis showed that the Cry1Ab/1Ac protein were detected in these 5 lines in the leaves and stems at tillering and filling stage.The CrylAb/lAc expression level in the leaves was significantly higher than that in the Stems.The content of Cry1Ab/1Ac protein in these 5 lines was higher than that of the positive control Zheyou 3,and the Cry1Ab/1Ac expression level of some transformed lines were even 10 times higher than that of the control in the leaves.In the endosperm,the detected level of Cry1Ab/1Ac in each of transgenic lines was no significant difference from the negative control,but of course it was significantly lower than 0.23 ?g/g of Zheyou 3.In addition,the transgenic plants were tested for insect resistance in the field.The results showed that 68.04%tiller per plant of the wild type control was damaged by manual infestation of stem borers,and number of leaffolder-damaged leaves per plant under the natural outbreaks was reached 24,whereas the damaged tillers of the transgenic plants by the stem borer were less than 2.88%,and the number of damaged leaves by leaffolders was not more than 0.37.These results therefore indicated that the transgenic plants were highly resistant to lepidopteran insects.To conclude,CrylAb/lAc protein can be expressed efficiently and steadily in green tissues such as leaves and stems in new green tissue-specific expression-resistant rice materials,while only a very small amount of CrylAb/lAc protein can be expressed in non-green endosperm or even not at all,which achieves the desired effect,?.Pyramiding of two different Bt insect-resistant genes Cry and Vip by composite transformationThe Vip3ArLrl gene was transferred into the photoperiod-and thermo-sensitive male sterility 208S harboring the Cry1Ab/1Ac gene,and a total of 40 green plantlets were obtained.Southern blotting analysis showed that only one plant carried the Vip3ArLrl gene,representing a transformant,and the corresponding phenotypic identification needs to wait until the backcross improvement is successful.