Abstract
BackgroundThe molecular characterization information of T-DNA integration is not only required by public risk assessors and regulators, but is also closely related to the expression of exogenous and endogenous genes. At present, with the development of sequencing technology, whole-genome resequencing has become an attractive approach to identify unknown genetically modified events and characterise T-DNA integration events.ResultsIn this study, we performed genome resequencing of Pb29, a transgenic high-resistance poplar 741 line that has been commercialized, using next-generation and Nanopore sequencing. The results revealed that there are two T-DNA insertion sites, located at 9,283,905–9,283,937 bp on chromosome 3 (Chr03) and 10,868,777–10,868,803 bp on Chr10. The accuracy of the T-DNA insertion locations and directions was verified using polymerase chain reaction amplification. Through sequence alignment, different degrees of base deletions were detected on the T-DNA left and right border sequences, and in the flanking sequences of the insertion sites. An unknown fragment was inserted between the Chr03 insertion site and the right flanking sequence, but the Pb29 genome did not undergo chromosomal rearrangement. It is worth noting that we did not detect the API gene in the Pb29 genome, indicating that Pb29 is a transgenic line containing only the BtCry1AC gene. On Chr03, the insertion of T-DNA disrupted a gene encoding TAF12 protein, but the transcriptional abundance of this gene did not change significantly in the leaves of Pb29. Additionally, except for the gene located closest to the T-DNA integration site, the expression levels of four other neighboring genes did not change significantly in the leaves of Pb29.ConclusionsThis study provides molecular characterization information of T-DNA integration in transgenic poplar 741 line Pb29, which contribute to safety supervision and further extensive commercial planting of transgenic poplar 741.
Highlights
The molecular characterization information of T-DNA integration is required by public risk assessors and regulators, but is closely related to the expression of exogenous and endogenous genes
This study provides molecular characterization information of T-DNA integration in transgenic poplar 741 line Pb29, which contribute to safety supervision and further extensive commercial planting of transgenic poplar 741
The BAM file generated by comparing all junction reads with the P. trichocarpa reference genome was imported into Integrative Genomics Viewer (IGV) software for visual analysis
Summary
The molecular characterization information of T-DNA integration is required by public risk assessors and regulators, but is closely related to the expression of exogenous and endogenous genes. China, like most other countries and regions in the world, is still very cautious about the application and supervision of transgenic technology, requiring that the research and experiment, environmental release and commercial production of genetically modified organisms (GMOs) all require safety certificates provided by relevant departments [6]. Inheritance and expression stability of exogenous genes is a prerequisite for commercial application of transgenic plants, which depends on the molecular characteristics of T-DNA integration into the host genome [7]. Clarifying the molecular characterization data of T-DNA integration such as T-DNA copy number and insertion site locations is important for risk assessors and regulators of transgenic plants
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