Genetic transformation and expression of Cry1Ac–Cry3A–NTHK1 genes in Populus × euramericana “Neva”

Four exogenous genes, Cry3A , Cry1Ac, mtlD, and BADH , were inserted into the p1870 vector to obtain multigenic transgenic Populus nigra L. with improved insect resistance and salt tolerance. During vector construction, different promoters were used for each gene, the AtADH 5′-UTR enhancer was added between the Cry1Ac promoter and the target gene, and the matrix attachment region (MAR, GenBank: U67919.1) structure was added at both ends of the vector. It was then successfully transferred into the genome of European black poplar by Agrobacterium-mediated leaf disk transformation, and a total of 28 transgenic lines were obtained by kanamycin screening. Five events with the highest insect resistance were selected based on preliminary tests: nos. 1, 7, 9, 12, and 17. PCR, real-time PCR, and enzyme-linked immunosorbent assays (ELISA) were used to detect the expression of exogenous genes and to analyze the Bt protein toxin levels in transgenic lines from June to October. PCR results showed that all four genes were successfully introduced into the five selected lines. Fluorescence quantitative PCR showed no significant differences in the transcript abundance of the four exogenous genes between different lines. A Bt protein toxin assay showed that the Cry3A protein toxin content was significantly higher than the Cry1Ac protein toxin content by approximately three orders of magnitude. Levels of the two toxins were negatively correlated. Over the course of the growing season, Cry1Ac content raised and varied between 0.46 and 18.41 ng·g −1 . Cry3A content decreased over the same time period and varied between 2642.75 and 15775.22 ng·g −1 . Indoor insect feeding assay showed that the transgenic lines had high insect resistance, with mortality rates of 1–2-year-old Hyphantria cunea larvae reaching more than 80%, and those of Plagiodera versicolora larvae and nymphs reaching 100%. No. 17 and no. 12 lines had better insect resistance to Lepidoptera and Coleoptera pests. There was no clear improvement in salt tolerance of the transgenic lines, but comprehensive evaluation of 11 salt tolerance indicators showed that lines no. 17 and no. 7 had certain degrees of salt tolerance.

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“…Neither the transgenic strains of Yang et al. (2016) nor those of Liu et al. (2016) exhibited high salt tolerance.…”

Section: Discussionmentioning

confidence: 92%

Expression of Multiple Exogenous Insect Resistance and Salt Tolerance Genes in Populus nigra L.

et al. 2020

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“…Measurements of Bt protein levels and the results of insect feeding tests revealed a close positive relationship between protein expression level and pest-resistance; clones expressing high Bt levels exhibited high-level insect resistance (Tian et al, 2000; Dong et al, 2015; Liu et al, 2016). To date, Cry1Ac and Cry3A of the Bt family have been most widely used for genetic transformation of insect-resistant poplars in China.…”

Section: Expression and Transport Of Bt Proteins In Poplarsmentioning

confidence: 98%

“…The protein encoded by the BADH catalyzes the synthesis of glycinebetaine, an important quaternary ammonium compound produced in response to salt and other osmotic stressors by many organisms (Jia et al, 2002). Multigene plant transformation vectors carrying Cry1Ac + BADH , Cry1Ac + NTHK1 , Cry1Ac + Cry3A + NTHK1 (Du et al, 2014; Liu et al, 2014), and Cry1Ac + Cry3A + BADH (Yang et al, 2012, 2016; Liu et al, 2016) were created at the Hebei Key Laboratory for Tree Genetic Resources and Forest Protection, Hebei province. The genes were successfully transformed into tobacco and various poplars; regenerated plants exhibited both pest-resistance and an increase in salt tolerance compared with controls (Ren et al, 2015).…”

Section: Multigene Transformationmentioning

confidence: 99%

“…Enzyme-linked immunosorbent assay (ELISA) analysis revealed significant differences in Bt protein levels; that of Cry3A was significantly higher than that of Cry1Ac, independent of the transformation method used (individual transfer of Cry1Ac and Cry3A ) (Tian et al, 2000; Yang et al, 2006a; Wang et al, 2008; Niu et al, 2011b), transformation of Cry3A into a plant already expressing Cry1Ac (Wang G.Y. et al, 2012a), or transfer of Cry1Ac and Cry3A together (two Bt genes were constructed in one vector and then inserted into the poplar genome) (Dong et al, 2015; Zhao et al, 2016; Liu et al, 2016). The toxic protein expression levels of the two Bt genes differed significantly between the acquired transgenic clones.…”

Section: Expression and Transport Of Bt Proteins In Poplarsmentioning

confidence: 99%

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The Current Status and Development of Insect-Resistant Genetically Engineered Poplar in China

et al. 2018

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Poplar is one of the main afforestation tree species in China, and the use of a single, or only a few, clones with low genetic diversity in poplar plantations has led to increasing problems with insect pests. The use of genetic engineering to cultivate insect-resistant poplar varieties has become a hot topic. Over the past 20 years, there have been remarkable achievements in this area. To date, nearly 22 insect-resistant poplar varieties have been created and approved for small-scale field testing, environmental release, or pilot-scale production. Here, we comprehensively review the development of insect-resistant genetically modified (GM) poplars in China. This review mostly addresses issues surrounding the regulation and commercialization of Bt poplar in China, the various insecticidal genes used, the effects of transgenic poplars on insects, toxic protein expression, multigene transformation, the stability of insect resistance, and biosafety. The efficacy of GM poplars for pest control differed among different transgenic poplar clones, larval instars, and insect species. The Bt protein analysis revealed that the expression level of Cry3A was significantly higher than that of Cry1Ac. Temporal and spatial studies of Bt protein showed that its expression varied with the developmental stage and tissue. The inheritance and expression of the exogenous gene were reviewed in transgenic hybrid poplar progeny lines and grafted sections. Biosafety issues, in terms of transgene stability and the effects on soil microorganisms, natural enemies of insects, and arthropod communities are also discussed.

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“…Although chemical insecticides can efficiently control this pest, their long-term application inevitably leads to undesirable and hazardous side effects to the environment, plants, and human [3]. Bacillus thuringiensis (Bt) toxin engineered to be expressed in plants is another method for P. versicolora control [4][5][6], but it would potentially encounter the problem of evolution of insect resistance [7,8].…”

Section: Introductionmentioning

confidence: 99%

Bacteria-Mediated RNA Interference for Management of Plagiodera versicolora (Coleoptera: Chrysomelidae)

Zhang

et al. 2019

Insects

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RNA interference (RNAi) has emerged as a novel and feasible strategy for pest management. Methods for cost-effective production and stable delivery of double-stranded RNA (dsRNA) to the target insects are crucial for the wide application of RNAi for pest control. In this study, we tested the expression of dsRNA in RNaseIII-deficient Escherichia coli HT115 which was then fed to Plagiodera versicolora larvae, an insect pest of Salicaceae plants worldwide. By targeting six potential genes, including actin (ACT), signal recognition particle protein 54k (SRP54), heat shock protein 70 (HSC70), shibire (SHI), cactus (CACT), and soluble N-ethylmaleimide-sensitive fusion attachment proteins (SNAP), we found that feeding bacteria-expressed dsRNA successfully triggered the silencing of the five target genes tested and the suppression of ACT and SRP54 genes caused significant mortality. Our results suggest that the oral delivery of bacteria-expressed dsRNA is a potential alternative for the control of P. versicolora, and that ACT and SRP54 genes are the potent targets.

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Genetic transformation and expression of Cry1Ac–Cry3A–NTHK1 genes in Populus × euramericana “Neva”

Cited by 15 publications

References 14 publications

Expression of Multiple Exogenous Insect Resistance and Salt Tolerance Genes in Populus nigra L.

Expression of Multiple Exogenous Insect Resistance and Salt Tolerance Genes in Populus nigra L.

The Current Status and Development of Insect-Resistant Genetically Engineered Poplar in China

Bacteria-Mediated RNA Interference for Management of Plagiodera versicolora (Coleoptera: Chrysomelidae)

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