Abstract
The genetic stability of exogenous genes in the progeny of transgenic trees is extremely important in forest breeding; however, it remains largely unclear. We selected transgenic birch (Betula platyphylla) and its hybrid F1 progeny to investigate the expression stability and silencing mechanism of exogenous genes. We found that the exogenous genes of transgenic birch could be transmitted to their offspring through sexual reproduction. The exogenous genes were segregated during genetic transmission. The hybrid progeny of transgenic birch WT1×TP22 (184) and WT1×TP23 (212) showed higher Bgt expression and greater insect resistance than their parents. However, the hybrid progeny of transgenic birch TP23×TP49 (196) showed much lower Bgt expression, which was only 13.5% of the expression in its parents. To elucidate the mechanism underlying the variation in gene expression between the parents and progeny, we analyzed the methylation rates of Bgt in its promoter and coding regions. The hybrid progeny with normally expressed exogenous genes showed much lower methylation rates (0–29%) than the hybrid progeny with silenced exogenous genes (32.35–45.95%). These results suggest that transgene silencing in the progeny is mainly due to DNA methylation at cytosine residues. We further demonstrated that methylation in the promoter region, rather than in the coding region, leads to gene silencing. We also investigated the relative expression levels of three methyltransferase genes: BpCMT, BpDRM, and BpMET. The transgenic birch line 196 with a silenced Gus gene showed, respectively, 2.54, 9.92, and 4.54 times higher expression levels of BpCMT, BpDRM, and BpMET than its parents. These trends are consistent with and corroborate the high methylation levels of exogenous genes in the transgenic birch line 196. Therefore, our study suggests that DNA methylation in the promoter region leads to silencing of exogenous genes in transgenic progeny of birch.
Highlights
Exogenous genes can be randomly integrated into the recipient chromosome through homologous or nonhomologous recombination using plasmid vectors
The Gus gene was normally expressed in transgenic birch pollen, not all pollen grains showed GUS enzyme activity, indicating that the exogenous genes were segregated in transgenic birch pollen during meiosis (Figures 1B,C)
The exogenous genes were successfully integrated in the hybrid progeny of TP73×WT (9), but the corresponding RT-PCR amplification bands were not obtained, indicating that the exogenous genes were silenced at the transcriptional level (Table 2)
Summary
Exogenous genes can be randomly integrated into the recipient chromosome through homologous or nonhomologous recombination using plasmid vectors. There are many studies on the genetic stability of transgenic plants (Mathieu et al, 2007; Prokopuk et al, 2015). Only a few studies reveal the genetic stability of exogenous genes in woody plants with a long growth cycle. A study on the development of secondary somatic embryos and genetic stability of regenerated Hevea brasiliensis plants indicates that the genome remained stable during multiplication (Wang et al, 2017). Cervera et al (1998) conducted a follow-up survey of genetically modified citrus for 5 years and find that the Gus gene could be stably expressed without exogenous gene silencing. Transgenes in genetically modified plants are often not stably expressed during development or in subsequent generations (Dietz-Pfeilstetter et al, 2016). A decrease in expression level with an increasing number of subcultures is reportedly associated with DNA methylation (Zeng et al, 2010a)
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