Insect-Resistant And Herbicide-Tolerant Transgenic Maize Harboring Bt Cry2Ah-vp And Bar Genes

Maize is not only a major food crop,but also an important feed and industrial raw material.Pests and weeds have been important factors limiting maize yield and quality.of maize.In order to reduce the harmfulness of pests and weeds on maize growth and development,people began to use chemical methods to control pests and weeds,but the resulting drawbacks gradually increased.With the development of biotechnology,scientists transferred insect-resistant gene and herbicide-tolerant gene into maize by means of genetic engineering and improved transgenic insect-resistant/herbicide-tolerant maize,which could effectively control pests and weed.It was beneficial to the environment and non-target insects and no adverse effects on human and livestock.Bt cry2Ah gene is an insecticidal protein gene cloned from Bacillus thuringiensis?Bt?strain.It has independent intellectual property rights.According to plant codon preference,codon optimization and modification of Bt cry2Ah gene were carried out in our laboratory.The expression vectors of Bt cry2Ah-vp and glufosinate resistance gene bar were constructed by genetic engineering method.The construct was transformed into maize inbred line B104 by Agrobacterium-mediated method to obtain T₀ generation of transgenic maize,and a high glufosinate-resistant strains were obtained through glufosinate screening.It was named VP1-5,and its molecular detection and insect resistance screening were carried out.In this study,VP1-5 of Bt cry2Ah-vp transgenic maize was detected at molecular level and investigated for agronomic traits.The transcription,copy number,transcription level and protein expression of cry2Ah-vp gene were detected by PCR,Southern blot,RT-PCR,fluorescence quantitative PCR?qPCR?and enzyme-linked immunosorbent assay?ELISA?.Resistance of transgenic maize VP1-5 to armyworm and corn borer was analyzed by laboratory and field bioassay.The growth period,plant height and yield of VP1-5 maize were studied.The results of PCR and Southern blot showed that cry2Ah-vp gene was inserted into maize genome in a single copy and inherited stably.;RT-PCR and q-PCR results showed that cry2Ah-vp gene could be normally transcribed in different tissues of VP1-5.The relative expression of cry2Ah-vp gene was leaf>filament>heart leaf>root>tassel>bract leaf>stem.ELISA results showed that Cry2Ah-vp protein was highly expressed in roots,stems and leaves of maize at 6-leaf stage,and the highest expression level in leaves was 2155+529.58 ng/g FW?fresh weight?.The results of bioassay showed that VP1-5 transgenic maize had good insecticidal activity against armyworm.The mortality rate of armyworm larvae reached 100%after 3 days of insecticidal inoculation.It also had obvious growth inhibition on Asian corn borer larvae,but had no obvious insecticidal effect on cutworm.By comparing the agronomic traits of VP1-5 and Zheng58,no significant differences were found in the phenological period,row number per ear,ear length and 1000-grain weight.The VP1-5 transgenic maize obtained in this study provides excellent germplasm materials for insect-resistant maize breeding and will have a good commercial prospect in the future.