A Novel Transgenic Maize With Resistance To Spodoptera Frugiperda By Transferring Vip3Aa Gene And Its Assessment

Maize is the largest crop in China,which is critically important source of food,feed,energy and forage.Pests and weeds are the important factors that seriously restrict the increase of maize yield.It has been reported that maize yield losses due to the insect pests could be up to 10%to 20%every year.Especially in recent years,Spodoptera frugiperda,invades China,and its"international,explosive,migratory and destructive"characteristics pose a major threat to maize production.Chemical insecticides are often used for pest control,which will cause great damage to the ecological environment,human health and other beneficial insects.Weeds is common in maize fields resulting in an average annual decrease of 12%to 16%in maize yield.Comprehensive use of tillage and artificial weeding is a common method to control weeds,which is laborious in actual production,and overuse of herbicides often poses a threat to human health and environmental safety.Therefore,rational use of herbicides is the most effective way to control weeds.In order to reduce the yield loss caused by Spodoptera frugiperda and weeds,we introduced the insect-resistant gene and herbicide-tolerant gene into maize plants through genetic engineering technology to cultivate a new maize material with insect resistance and herbicide tolerance,and meet the needs of agricultural production.1.To obtain a new insect resistant gene with insecticidal activity against the Spodoptera frugiperda,the vip3Aa11 gene from Bacillus thuringiensis（Bt）was modified through site mutation and sequence optimization,and four insect resistant genes vip3Aa11-m1,vip3Aa11-m2,vip3Aa11^S686R-m3 and vip3Aa11^M129I,K284Q-m4 were artificially synthesized.Four vip3Aa genes were expressed in Escherichia coli,and the purified proteins were used to perform the bioassay against Spodoptera frugiperda.It was found that the four proteins had insecticidal activity against Spodoptera frugiperda,and the four vip3Aa genes could be used as candidate genes for transgenic plant research.2.The tobacco expression vectors with four vip3Aa genes were constructed and were transformed to tobacco to obtain transgenic plants.Molecular detection and bioassay activity were performed and the results showed that all four proteins had insecticidal activity against Spodoptera frugiperda.3.Four plant expression vectors with four vip3Aa genes and herbicide-tolerant genes bar or cp4-epsps were constructed.951 transgenic maize were obtained by Agrobacterium tumefaciens-mediated transformation.Through successive generations of molecular detection,biological activity and herbicide tolerance of the transformed maize were conducted.The VP3C1 event transformed with vip3Aa11-m1 and cp4-epsps genes were determined to be highly resistant to Spodoptera frugiperda and glyphosate.4.The molecular characteristic analysis and target character function identification of VP3C1event were carried out.The results showed that the exogenous insertion sequence of VP3C1 event was inversely inserted into maize genome in the form of double copy in one insertion site,and the foreign insertion sequence was integrated into maize genome at 172147011-172147036 site（Zm-B73-REFERENCE-NAM-5.0）on chromosome 6.The insertion site was in the intergenic region,which did not affect the endogenous gene sequence.The foreign genes vip3Aa11-m1 and cp4-epsps of VP3C1 event were transcribed and translated normally,and the expression levels varied in different organs at different stages of growth and development,and the expression level of Vip3Aa11-m1 protein was the highest in leaf tissues during silking stage.Besides,VP3C1 transgenic plants with high resistance（HR）to Spodoptera frugiperda could tolerate 4 times the dose of glyphosate herbicide.The bioassay and glyphosate tolerance test of T₁～T₄ generation transgenic plants showed that insect resistance and herbicide tolerance could be stably inherited.5.The environmental safety assessment of VP3C1 event was carried out.The results showed that there was no significant difference in VP3C1 between transgenic maize and non-transgenic control in the pollen viability,tolerance to non-target herbicides and agronomic characters.In this study,a novel transgenic maize with glyphosate tolerance and resistance to Spodoptera frugiperda was created by using genetic engineering technology,which provided excellent germplasm resources for insect-resistant and herbicide-tolerant maize breeding and laid a foundation for subsequent industrial application.