Expression of Multiple Exogenous Insect Resistance and Salt Tolerance Genes in Populus nigra L.
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 b...
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doaj-8f1372c37efe4a369313870984f0d7402020-11-25T03:45:02ZengFrontiers Media S.A.Frontiers in Plant Science1664-462X2020-07-011110.3389/fpls.2020.01123546447Expression of Multiple Exogenous Insect Resistance and Salt Tolerance Genes in Populus nigra L.Xinglu Zhou0Xinglu Zhou1Yan Dong2Yan Dong3Qi Zhang4Qi Zhang5Dandan Xiao6Dandan Xiao7Minsheng Yang8Minsheng Yang9Jinmao Wang10Jinmao Wang11Institute of Forest Biotechnology, Forestry College, Agricultural University of Hebei, Baoding, ChinaHebei Key Laboratory for Tree Genetic Resources and Forest Protection, Baoding, ChinaInstitute of Forest Biotechnology, Forestry College, Agricultural University of Hebei, Baoding, ChinaHebei Key Laboratory for Tree Genetic Resources and Forest Protection, Baoding, ChinaInstitute of Forest Biotechnology, Forestry College, Agricultural University of Hebei, Baoding, ChinaHebei Key Laboratory for Tree Genetic Resources and Forest Protection, Baoding, ChinaInstitute of Forest Biotechnology, Forestry College, Agricultural University of Hebei, Baoding, ChinaInstitute of Coastal Agriculture, Hebei Academy of Agriculture and Forestry Sciences, Shijiazhuang, ChinaInstitute of Forest Biotechnology, Forestry College, Agricultural University of Hebei, Baoding, ChinaHebei Key Laboratory for Tree Genetic Resources and Forest Protection, Baoding, ChinaInstitute of Forest Biotechnology, Forestry College, Agricultural University of Hebei, Baoding, ChinaHebei Key Laboratory for Tree Genetic Resources and Forest Protection, Baoding, ChinaFour 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.https://www.frontiersin.org/article/10.3389/fpls.2020.01123/fullmulti-resistance genemultigenic vectorPopulus nigra L.insect resistancesalt resistance |
collection |
DOAJ |
language |
English |
format |
Article |
sources |
DOAJ |
author |
Xinglu Zhou Xinglu Zhou Yan Dong Yan Dong Qi Zhang Qi Zhang Dandan Xiao Dandan Xiao Minsheng Yang Minsheng Yang Jinmao Wang Jinmao Wang |
spellingShingle |
Xinglu Zhou Xinglu Zhou Yan Dong Yan Dong Qi Zhang Qi Zhang Dandan Xiao Dandan Xiao Minsheng Yang Minsheng Yang Jinmao Wang Jinmao Wang Expression of Multiple Exogenous Insect Resistance and Salt Tolerance Genes in Populus nigra L. Frontiers in Plant Science multi-resistance gene multigenic vector Populus nigra L. insect resistance salt resistance |
author_facet |
Xinglu Zhou Xinglu Zhou Yan Dong Yan Dong Qi Zhang Qi Zhang Dandan Xiao Dandan Xiao Minsheng Yang Minsheng Yang Jinmao Wang Jinmao Wang |
author_sort |
Xinglu Zhou |
title |
Expression of Multiple Exogenous Insect Resistance and Salt Tolerance Genes in Populus nigra L. |
title_short |
Expression of Multiple Exogenous Insect Resistance and Salt Tolerance Genes in Populus nigra L. |
title_full |
Expression of Multiple Exogenous Insect Resistance and Salt Tolerance Genes in Populus nigra L. |
title_fullStr |
Expression of Multiple Exogenous Insect Resistance and Salt Tolerance Genes in Populus nigra L. |
title_full_unstemmed |
Expression of Multiple Exogenous Insect Resistance and Salt Tolerance Genes in Populus nigra L. |
title_sort |
expression of multiple exogenous insect resistance and salt tolerance genes in populus nigra l. |
publisher |
Frontiers Media S.A. |
series |
Frontiers in Plant Science |
issn |
1664-462X |
publishDate |
2020-07-01 |
description |
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. |
topic |
multi-resistance gene multigenic vector Populus nigra L. insect resistance salt resistance |
url |
https://www.frontiersin.org/article/10.3389/fpls.2020.01123/full |
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