Abstract
Bacillus thuringiensis (Bt) insecticidal protein genes are important tools in efforts to develop insect resistance in poplar. In this study, the Cry1Ac and Cry3A Bt toxin genes were simultaneously transformed into the poplar variety Populus × euramericana ‘Neva’ by Agrobacterium-mediated transformation to explore the exogenous gene expression and insect resistance, and to examine the effects of Bt toxin on the growth and development of Anoplophora glabripennis larvae after feeding on the transgenic plant. Integration and expression of the transgenes were determined by molecular analyses and the insect resistance of transgenic lines was evaluated in feeding experiments. Sixteen transgenic dual Bt toxin genes Populus × euramericana ‘Neva’ lines were obtained. The dual Bt toxin genes were expressed at both the transcriptional and translational levels; however, Cry3A protein levels were much higher than those of Cry1Ac. Some of the transgenic lines exhibited high resistance to the first instar larvae of Hyphantria cunea and Micromelalopha troglodyta, and the first and second instar larvae and adults of Plagiodera versicolora. Six transgenic lines inhibited the growth and development of A. glabripennis larvae. The differences in the transcriptomes of A. glabripennis larvae fed transgenic lines or non-transgenic control by RNA-seq analyses were determined to reveal the mechanism by which Bt toxin regulates the growth and development of longicorn beetle larvae. The expression of genes related to Bt prototoxin activation, digestive enzymes, binding receptors, and detoxification and protective enzymes showed significant changes in A. glabripennis larvae fed Bt toxin, indicating that the larvae responded by regulating the expression of genes related to their growth and development. This study lay a theoretical foundation for developing resistance to A. glabripennis in poplar, and provide a foundation for exploring the mechanism of Bt toxin action on Cerambycidae insects.
Highlights
Poplar is an economically important tree species, but its large-scale intensive monoculture has been accompanied by increasingly serious problems of disease and insect pests
There was a significant correlation coefficient of 0.789 (P < 0.05) between cellulase activity and body weight (Supplementary Table 2). These results suggest that Bacillus thuringiensis (Bt) toxin can indirectly affect the growth and development of A. glabripennis larvae by inhibiting cellulase activity in the midgut
Cry1Ac and Cry3A genes were simultaneously transformed into Populus × euramericana ‘Neva’ and 16 transgenic lines were obtained through kanamycin screening and confirmed by Polymerase Chain Reaction (PCR)
Summary
Poplar is an economically important tree species, but its large-scale intensive monoculture has been accompanied by increasingly serious problems of disease and insect pests. Poplar pests such as Hyphantria cunea (Lepidoptera), Micromelalopha troglodyta (Lepidoptera), and longicorn beetles can spread widely and cause substantial losses to forestry production. Many studies have shown that introduction of a Bt toxin gene into transgenic poplar provides a degree of resistance to target pests (Dong et al, 2014; Xu et al, 2019, 2020); the introduction of a single insect resistance gene has limitations, including a narrow insecticidal spectrum and development of tolerance in the pests. To expand the insecticidal spectrum, delay the development of tolerance in pests, and improve the insect resistance of transgenic poplar, recent research has focused on the introduction of bivalent or multivalent insect resistance transgenes (Zhang B. et al, 2011; Zhang et al, 2019; Yang et al, 2016; Zhou et al, 2020)
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