| Maize(Zea Mays L.)is an important food crop,feed and industrial raw material in the world.Damage by insect pests and weeds is the main causes of the decline in maize yield and quality,which seriously threatens the global food security.By means of genetic engineering,different insect resistance genes and herbicide resistance genes are simultaneously transferred into maize to cultivate new transgenic maize varieties with the compound traits of insect resistance and herbicide tolerance,which can greatly alleviate the harm of insects and weeds,avoid the environmental pollution caused by the use of chemical pesticides and increase the economic benefits of agriculture.Previously cry2 Ab,vip3A and cp4 epsps genes were constructed in tandem into the plant expression vector,and the transgenic maize VA with insect resistance and herbicide tolerance was obtained by Agrobacterium-mediated transformation.In this study,the molecular characteristics including the exogenous gene integration,expression at transcriptional and translation levels in the transgenic maize were analyzed,the target traits including insect resistance and herbicide tolerance were evaluated,and the growth period,field agronomic traits,seed germination rates and pollen viability were investigated.The main findings are as follows:(1)The whole genomes of 12 transgenic maize VA lines were sequenced by means of the whole genome sequencing(WGS).Using the bioinformatics methods,all genome sequences was compared with the vector sequence and the maize reference genome B73sequence(Ref Gen＿v4),and the exogenous T-DNA in the three transformants VA-1,VA-2 and VA-3 were found to insert into the intergenic regions of the maize genome.Among them,the T-DNA insertion was located at Chr 8: 164 988 905–164 988 976 and caused a 72 bp deletion of chromosomal DNA,followed by a TT nucleotides insertion in the transgenic maize VA-1.The foreign T-DNA in VA-2 was inserted into the position Chr 1: 50 528 116–50 528 190 and 75 bp was missing in the genome sequence.The exogenous T-DNA in VA-3 were inserted into the position 239 251 591–239 251 625 on chromosome 4 and a 35 bp deletion of chromosomal DNA.Through the specific PCR detection and sequencing,it was further confirmed that the exogenous T-DNA and target genes cry2 Ab,vip3A and cp4 epsps had been integrated into the transgenic maize VA-1,VA-2 and VA-3 genome.(2)Fluorescence quantitative PCR(q RT-PCR)and enzyme-linked immunosorbent assay(ELISA)were used to identify the expression of exogenous target genes in transgenic maize at the transcriptional and translational levels,respectively.The successful expression of cry2 Ab,vip3A and cp4 epsps in VA-1 various tissues of the different developmental stages were confirmed,but their expression levels showed spatial and temporal differences.Cry2 Ab,Vip3A and CP4 EPSPS proteins were the most abundant in the R4 stage(dough stage),R1 stage(silking stage)and R5 stage(dent stage)leaves,with the content of 16.65±1.91 μg/g FW,19.61±3.43 μg/g FW and 149.74±6.41 μg/g FW,respectively.(3)The results of laboratory insect bioassay indicated that the mortality of the neonate larvaes of Mythimna separata feeding on VA-1,VA-2 and VA-3 maize leaves for2 days were 90.0%,86.3% and 88.8%,respectively.After 6 days,the mortality were98.8%,98.8% and 96.3%,respectively,while the mortality of 6 days were 10.0% for control maize Zheng 58.The neonate larvaes of Spodoptera frugiperda all died after feeding on maize VA-1,VA-2 and VA-3 leaves for 4 days,while the mortality of 6 days was lower than 10.0% for control maize Zheng 58.The neonate larvaes of Helicoverpa armigera were 100.0% dead after feeding on VA-1 and VA-2 maize leaves for 4 days,but all died after feeding on VA-3 leaves for 6 days.The mortality of 6 days was 52.1%for Zheng 58.There are significant differences between the mortality of M.separata,S.frugiperda and H.armigera feeding on the transgenic and control maize.Thus,transgenic maize VA-1,VA-2 and VA-3 have high insecticidal activity against M.separata,S.frugiperda and H.armigera larvaes.The further field insect resistance evaluation and herbicide spraying assays that VA-1 has excellent resistance to M.separata and H.armigera,and good tolerance to the target herbicide glyphosate.(4)The investigation of agronomic traits showed that compared with the control Zheng 58,VA-1 had no significant differences in the seedling emergence stage,tasseling stage,silking stage,growth period,plant height,ear position height,ear length,ear diameter,number of grains per rows,100-grain weight,bare tip length,seed germination rate,pollen viability and pollen germination rate.Taken together,these results show that the transgenic maize VA-1 obtained in this study is successfully integrated and expressed for the target genes cry2 Ab,vip3A and cp4 epsps,has insect resistance and herbicide tolerance,and agronomic traits are not affected by the insertion of exogenous T-DNA,which provide data support for the breeding of new transgenic maize varieties. |
