Abstract
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.
Highlights
Pests and soil salinization are key factors that restrict forestry production and development (Zhu, 2001; Dennis and Andrea, 2012; Li and Li, 2018)
Mortality was higher on all transgenic lines than on controls, and most lines showed significant differences in insect resistance compared with day 8
Poplar with the Cry3A gene is lethal to Coleoptera, producing close to 100% mortality in first instar larvae (Yang et al, 2003; Wang Y.P. et al, 2008; Niu et al, 2011)
Summary
Pests and soil salinization are key factors that restrict forestry production and development (Zhu, 2001; Dennis and Andrea, 2012; Li and Li, 2018). Cultivation of insect-resistant and salt-tolerant genetically modified superior varieties is an important development goal (Fladung et al, 2013; Movahedi et al, 2015; Yang et al, 2016), and use of the microbial insect-resistance gene Bt for the development of transgenic plants has received significant attention (Hou and Chen, 2000). The product of the BADH gene pathway, betaine, cannot be further metabolized to any great extent after synthesis It serves as a permanent or semi-permanent osmotic regulator and is considered as one of the most promising osmotic protectants (Shu et al, 1997). It has received increasing attention in salt and drought stress research, and has been used to generate transgenic plants with increased drought resistance and salt tolerance
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