siRNA induced DNA methylation of the cry1Ac gene due to its inverted duplication in genetically modified cotton MON531

The application of insect-resistant cotton has yielded substantial benefits for both agriculture and the environment. Genetically modified insect-resistant cotton conversion MON531 represents one of the earliest instances of genetically modified cotton cultivated in China. The full-length insertion sequence of MON531 was elucidated through third-generation sequencing, revealing a total length of MON531 was 11,166 bp, which primarily comprises a complete cry1Ac gene reading frame alongside an incomplete counterpart. Notably, the incomplete cry1Ac gene reading frame and the complete one establish a head-to-head reverse repeat structure at the 3′ end of the full insertion sequence in MON531, with the repeat sequence measuring 1432 bp. The complete cry1Ac gene spans 3537 bp and encodes 1178 amino acids. Transcriptomic sequencing results indicated that only the nptII gene and the complete cry1Ac gene were transcribed. Methylation-sensitive restriction enzyme qPCR (MSRE-qPCR) and Bisulfite Sequencing (BS-seq) demonstrated elevated high levels of cytosine methylation within the 3′ terminal sequence of the cry1Ac gene and its terminus. Furthermore, siRNAs were found to be highly enriched in the 3′ terminal region of the cry1Ac gene, as determined by siRNA sequencing. The concurrent presence of DNA methylation and siRNA suggests that siRNA may facilitate DNA methylation in the reverse repeat region of the cry1Ac gene via the RdDM pathway. • The comprehensive characterization of the complete sequence of MON531. • The roles of DNA methylation and siRNA in regulating the expression of the cry1Ac gene are crucial. • The coexistence of DNA methylation and siRNA in the 3′ terminal region of the cry1Ac gene reveled the RdDM pathway.