Molecular Characteristics And Functional Evaluation Of A Transgenic Insect-resistant And Herbicide-tolerant Maize

Maize is not only an important food crop in China,but also an important feed crop and industrial raw materials.The healthy development of maize industry is of great significance for ensuring national food security and effective supply of agricultural products.Insect pests and weeds seriously affect maize growth and development,leading to great losses in yield and quality.The development of insect-resistant and herbicide-tolerant maize using transgenic technology provides a new strategy for pest and weed control in maize field.The application of transgenic insect-resistant and herbicide-tolerant maize will bring remarkable economic,social and ecological benefits.In our previous study,we constructed and obtained a transgenic maize line BFC1-1containing lepidopteran resistance genes(cry1Ab,cry1F)and glyphosate resistance gene(cp4epsps)in tandem by using Agrobacterium-mediated transformation technology.In this thesis,the molecular characteristics and functional evaluation of BFC1-1 was conducted,and the main research results were as follows:(1)Analysis of the genetic stability of the exogenous target genes.The integration of exogenous target genes in maize genome was detected by target gene-specific PCR and transformant-specific PCR.The results showed that cry1 Ab,cry1F and cp4 epsps could be stably inherited in BC6F2 and BC6F3 generations of BFC1-1,and the exogenous gene expression cassettes was inserted in the intergenic region of maize genome.(2)Analysis of transcription levels of exogenous target genes.Real-time quantitative PCR(q RT-PCR)was used to detect the transcription levels of cry1 Ab,cry1F and cp4 epsps in BFC1-1 plants.The results showed that transcripts of cry1 Ab,cry1F and cp4 epsps can be detected in different tissues of BFC1-1 plants at different growth and development stages.The transcription levels of these genes were the lowest in the grains at mature stage.The transcription level of cry1 Ab was the highest in the root at the milk stage,and the transcription levels of cry1 F and cp4 epsps were the highest in the pollen at tasseling stage.(3)Analysis of the protein levels of exogenous target genes.The protein levels of Cry1 Ab,Cry1F and CP4 EPSPS were detected by enzyme-linked immunosorbent assay(ELISA).The results showed that Cry1 Ab,Cry1F and CP4 EPSPS were expressed in different tissues of BFC1-1 at different growth and development stages.The highest level of Cry1 Ab was detected in the leaves at elongation stage,the highest level of Cry1 F was detected in the leaves at milk stage,and higher levels of CP4 EPSPS protein were detected in the leaves at V6,V9,R1 and R3 stages.(4)Evaluation of insect resistance of BFC1-1.The larvae of Asian corn borer(Ostrinia furnacalis),armyworm(Mythimna separata)and fall armyworm(Spodoptera frugiperda)were used to test insect resistance of BFC1-1 at whorl stage in laboratory.The results showed that the leaves of BFC1-1 were intact after fed for 72 h to Asian corn borer and armyworm,and the adjusted mortality of both larvae was 100%.After fed for 72 h to fall armyworm,the leaves of BFC1-1 were slightly bitten and the adjusted mortality of fall armyworm was 82.8%.After fed for 96 h,all larvae were dead.These results indicated that BFC1-1 plants are highly resistant to Asiatic corn borer,armyworm and fall armyworm.The evaluation of insect-resistance of BFC1-1 in the field also showed that BFC1-1 plants are highly resistant to Asian corn borer and armyworm compared to Zheng 58 plants(non-transgenic control),and the damage level of BFC1-1 is between 1.0 and 2.0.(5)Evaluation of glyphosate tolerance of BFC1-1.In the whorl stage,glyphosate(41%)with different concentration(5 m L/L,10 m L/L,20 m L/L)was sprayed on BFC1-1and non-transgenic control Zheng 58 plants.The results showed that the growth of BFC1-1is consistent with that of the plants sprayed with water,whereas all Zheng 58 plants sprayed with glyphosate are dead.These results indicated that BFC1-1 is highly tolerant to glyphosate(at least 20 m L/L of 41% glyphosate).(6)Evaluation of the effect of BFC1-1 on non-target organisms including maize aphid,earthworm(Eisenia foetida)and Chrysoperla nipponensis.When maize aphids were fed with BFC1-1 and Zheng58 plants for 5 d,there was no significant difference in the number of corn aphid between BFC1-1 and Zheng 58 plants,indicating that BFC1-1 plants have no effect on maize aphid growth.When the earthworms were fed with artificial soil containing different concentration of Cry1 Ab or Cry1 F for 7 d,there were no significant differences in survival rate,body weight and body length of earthworms between insecticidal protein treatment and water treatment,indicating that the insecticidal proteins have no effect on earthworms growth and development.When the lacewing larvae of Chrysoperla nipponensis were fed with diets containing Cry1 Ab and Cry1 F,there were no significant differences in mortality,early oviposition,total oviposition and body weight of Chrysoperla nipponensis between the treatment group containing insecticidal proteins and the negative control group,indicating that insecticidal proteins have no effect on the growth and development of Chrysoperla nipponensis.(7)Evaluation of BFC1-1 tolerance to non-target herbicides.At the whorl stage,BFC1-1 and Zheng 58 plants were sprayed with herbicides containing atrazine,nicosulfuron and maize-sensitive herbicide quizalofop-p-ethyl,respectively.The results showed that BFC1-1 and Zheng 58 plants grow well and have similar tolerance to atrazine and nicosulfuron,whereas they are all sensitive to quizalofop-p-ethyl.Both BFC1-1 and Zheng 58 plants were dead after treatment with quizalofop-p-ethyl for 7 d.(8)Analysis of agronomic traits of BFC1-1.There were no significant differences between BFC1-1 and Zheng 58 on agronomic traits including heading stage,ear height,plant height,100 grain weight,ear weight.Both BFC1-1 and Zheng 58 plants had similar pollen grain size,pollen germination rate,seed germination potential,seed germination rate and germination index.In conclusion,we obtained a new transgenic line BFC1-1 with stacked insect resistance and glyphosate tolerance traits.