Integration Sites Of Photochrome Genes In Transgenic Maize Lines

Exogenous genes and expressions of the structure of the insertion site and integration methods, are not only the relation between the expressions of exogenous genes, but also may cause insertional locus mutation affecting the endogenous gene expression. It is the important content of genetically modified (GM) mechanism and safety evaluation. Compared with Arabidopsis thaliana and rice, maize genome is more complex, and there are a large number of repeat sequences and a variety of sequences. Therefore, in the study of genetically modified maize, the identification of insertion sites and integration method of exogenous genes is more important, and is also more difficult.In this experiment, we used six different generations and positive strains as materials by Agrobacterium-mediated transformation of maize endogenous phytochrome genes PhyA1, PhyA2 and PhyBl, though the specific PCR homozygosity testing the type of T-DNA flanking sequences with high-efficient thermal asymmetric interlaced PCR amplification, analyzing T-DNA the integration rule of maize genome, and discussing its effect on the phenotype. The results are as follows:1. With two sets of nested primers, the corresponding selection marker gene and its promoter sequence fragment was amplified respectively. The T7 generation strain of transformation by Expression vector pCAMBIA1390-Ubi-PhyA1-T-nos and pCAMBIA1390-Ubi-PhyB1-T-nos were all positive for plants, and its rate of homozygous was 100%. The T6 generation strain of transformation by Expression vector pTF101.1-Ubi-PhyA1-T-nos is mostly positive for plants, and its rate of homozygous was 90%. The T3 generation strain of transformation by Expression vector pTF101.1-Ubi-PhyA2-T-nos is partially positive for plants, and its rate of homozygous was 40%. In expression vector pTF101.1-Ubi-PhyB1-T-nos converting To generation plants, the detection of two independent positive plants was indentified, and its positive rate was 5%.2. The results of hiTAIL-PCR and genome amplification and sequence analysis. The T7 generation strain of transformation by pCAMBIA1390-Ubi-PhyA1-T-nos, the T7 generation strain of transformation by pCAMBIA1390-Ubi-PhyB1-T-nos, the T6 generation strain of transformation by pTF101.1-Ubi-PhyAl-T-nos, the T3 generation strain of transformation by pTF101.1-Ubi-PhyA2-T-nos, the To generation positive plants 1 and 2 by pTF101.1-Ubi-PhyA2-T-nos, located in maize genome chromosome 1 betweem 284557834 and 284557835 bases, chromosome 9 betweem 61944355 and 61944356 bases, chromosome 1 betweem 26981167 and 26981168 bases, chromosome 6 betweem 96491356 and 96491357, chromosome 3 betweem 125567445 and 125567446, chromosome 4 betweem 98934902 and 98934903, respectively.3. According to the T-DNA integration sites of above six GM strains or To generation positive strains, searching the function of the gene sequences, repetitive sequence and transposon sequences around its upstream and downstream in the maize genome sequence database within 10 KB. The results show that the T7 generation strain of transformation by pCAMBIA1390-Ubi-PhyA1-T-nos and pCAMBIA1390-Ubi-PhyBl-T-nos, the T6 generation strain of transformation by pTF101.1-Ubi-PhyAl-T-nos, the To generation positive plants 2 by pTF101.1-Ubi-PhyA2-T-nos,10 KB range found no function of upstream and downstream of the gene sequences, repetitive sequence and transposon sequences. The T-DNA integration sites from 1050 bp downstream of the T3 generation strain of transformation by pTF101.1-Ubi-PhyA2-T-nos found one coding WRKY40 transcription factor genes, and upstream 70 KB found an unknown functional gene. The T-DNA integration sites from 20 bp downstream of the To generation positive plants 1 by pTF101.1-Ubi-PhyB1-T-nos found one coding arginine tRNA genes, and in its upstream 200 bp as well as an unknown functional gene.4. The above six GM strains and positive strains To generation left and right boundary, has a total of 12 T-DNA integration sites, and a total of 4 kinds of integrated model:(1) the breaking point of T-DNA integration occurs within the sequence boundary, integrated into maize genome with not T-DNA expression vector skeleton and filling sequence. (2) the breaking point of T-DNA integration occurs outside of sequence boundary, integrated into maize genome expression vector with T-DNA part of the frame sequence. (3) there was a filler sequence between T-DNA boundary and the maize genome sequence, which is no homologous neither T-DNA boundary nor the maize genome sequence. (4) the breaking point of T-DNA integration occurs within the sequence boundary, causing the lack of T-DNA sequence boundary. In addition, in the integration of the 12 T-DNA sequence boundaries, the different degree of base substitution and mutations were found.To sum up, the integration sites of T-DNA in the maize genome and pattern is varied, every transformation event is not identical. For T-DNA integration sites near or overlap in functional gene sequence, should specialise in gene expression in the safety evaluation of GM detection. The character changes of plant height and ear higher of the T7 generation strain of transformation by pCAMBIA1390-Ubi-PhyA1-T-nos and pCAMBIA1390-Ubi-PhyB1-T-nos, are caused by excessive expression of endogenous genes ZmPhyAl and ZmPhyB1.