Transcriptome sequencing and weighted correlation network analysis reveal the molecular mechanism of growth variation in highly resistant transgenic double Bt poplar

The Bt gene is widely used in insect-resistant transgenic plants, but its integration (especially dual Bt genes) may cause unintended growth effects. To explore the molecular mechanism of Bt genes regulating the growth of recipient plants, we used transgenic single Bt gene poplar 741, transgenic double Bt gene poplar 741 obtained through secondary transformation, and wild-type (741) as research subjects, combined with physiological determination and transcriptome sequencing. The results showed that, compared to 741 and the single Bt lines, double Bt lines exhibited noticeable variations, including reduced plant height and slow growth. Transcriptome analysis revealed that the numbers of differentially expressed genes (DEGs) in the three dual Bt transgenic lines (pc2, pc3, and pc9) relative to the wild-type were 1126, 920, and 1224, respectively. These values were notably higher than those detected in the transgenic single Bt lines pb29 (Integrated Cry1Ac) and CC84 (Integrated Cry3A), which exhibited 726 and 112 DEGs, respectively. More DEGs were observed in transgenic double Bt gene poplar 741 compared to 741 and CC84; the number of upregulated genes exceeded the number of downregulated genes. Fewer DEGs were identified relative to the pb29. The DEGs were mainly involved in metabolic processes such as starch and sucrose metabolism, pentose and glucuronate interconversions, plant hormone signal transduction, and flavonoid biosynthesis. Weighted gene co-expression network analysis (WGCNA) identified blue and brown modules significantly correlated with growth traits, and screened hub genes including CBL - interacting serine/threonine - protein kinase 11 and protein disulfide-isomerase A1 among others. These results indicated that the high expression and interaction of dual Bt genes disrupted the expression of growth - related genes in multiple metabolic pathways, such as plant hormone signal transduction, phenylpropanoid biosynthesis, and flavonoid biosynthesis, leading to metabolic disorders and phenotypic variation of poplar 741.