Investigation Of The Integration Site Of Exogenous Gene In Transgenic Maize And Preliminary Study Of Grain Abortion

Maize?Zea mays L.?is one of the most important grain crops in China,which plays an important role in the agricultural and industrial production.Corn borers and other pests seriously harm corn production,resulting in an annual loss of about 10%of corn production.The wide application of insect-resistant transgenic maize can effectively control the pest of maize,reduce the loss of corn yield and the use of pesticides.It is necessary to identify the integration sites of exogenous genes in the plant genome,and the integration of exogenous genes should not adversely affect the production characteristics of maize.Transgenic maize event IE034 used in this study was generated by our laboratory.The preliminary study revealed that the transgenic maize event had high toxicity to corn borer and only single copy was integrated in the maize genome.In this study,we investigated the flanking sequences of insertion site by TAIL-PCR and Fosmid library.and the insertion site was confirmed by genetic mapping,and analyzed the abnormal kernel of the transgenic maize finally.The main results are as follows:1.The 5'-flanking sequence of exogenous gene was isolated by TAIL-PCR.We found that the exogenous gene was inserted into the repeating region of the maize genome.Some skeleton vector fragment was also integrated into the maize genome.2.Then we constructed a Fosmid library to identify the flanking sequence of IE034.A clone containing the exogenous gene was identified,and sequencing results reveald a 29 kb flanking sequence.Transgenic maize IE034 was crossed with inbred line B73 to construct F₂ population.The transgenic plants and non-transgenic plants had a segregation ratio of 3:1,indicating that there was single insertion site of exogenous gene.The localization of the exogenous gene was investigated by BSR-Seq method,and the results showed that there were significant peaks on chromosome 1 and chromosome 5.The insertion site was further narrowed in a range of 260 kb region by genetic mapping.3.In the process of field planting,it was found that the grain yield of IE034 is relatively low due to the kernel abortion.In order to further analyze the mechanism of kernel abortion in transgenic maize IE034,we performed a transcriptome analysis on the normal and abortive seeds of maize ears.It was found that there were 293 differentially expressed genes.KEGG enrichment analysis showed that the differentially expressed genes were mainly concentrated in the metabolic pathway of starch and sucrose metabolism,phenylpropanoid biosynthesis and plant hormone signal transduction.