Expression of Multiple Exogenous Insect Resistance and Salt Tolerance Genes in Populus nigra L.
Identifieur interne : 000404 ( Main/Exploration ); précédent : 000403; suivant : 000405Expression of Multiple Exogenous Insect Resistance and Salt Tolerance Genes in Populus nigra L.
Auteurs : Xinglu Zhou [République populaire de Chine] ; Yan Dong [République populaire de Chine] ; Qi Zhang [République populaire de Chine] ; Dandan Xiao [République populaire de Chine] ; Minsheng Yang [République populaire de Chine] ; Jinmao Wang [République populaire de Chine]Source :
- Frontiers in plant science [ 1664-462X ] ; 2020.
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.
DOI: 10.3389/fpls.2020.01123
PubMed: 32793270
PubMed Central: PMC7393212
Affiliations:
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wicri:level="1"><nlm:affiliation>Institute of Forest Biotechnology, Forestry College, Agricultural University of Hebei, Baoding, China.</nlm:affiliation><country xml:lang="fr">République populaire de Chine</country><wicri:regionArea>Institute of Forest Biotechnology, Forestry College, Agricultural University of Hebei, Baoding</wicri:regionArea><wicri:noRegion>Baoding</wicri:noRegion></affiliation><affiliation wicri:level="1"><nlm:affiliation>Hebei Key Laboratory for Tree Genetic Resources and Forest Protection, Baoding, China.</nlm:affiliation><country xml:lang="fr">République populaire de Chine</country><wicri:regionArea>Hebei Key Laboratory for Tree Genetic Resources and Forest Protection, Baoding</wicri:regionArea><wicri:noRegion>Baoding</wicri:noRegion></affiliation></author><author><name sortKey="Dong, Yan" sort="Dong, Yan" uniqKey="Dong Y" first="Yan" last="Dong">Yan Dong</name><affiliation wicri:level="1"><nlm:affiliation>Institute of Forest Biotechnology, Forestry College, Agricultural University of Hebei, Baoding, China.</nlm:affiliation><country xml:lang="fr">République populaire de Chine</country><wicri:regionArea>Institute of Forest Biotechnology, Forestry College, Agricultural University of Hebei, Baoding</wicri:regionArea><wicri:noRegion>Baoding</wicri:noRegion></affiliation><affiliation wicri:level="1"><nlm:affiliation>Hebei Key Laboratory for Tree Genetic Resources and Forest Protection, Baoding, China.</nlm:affiliation><country xml:lang="fr">République populaire de Chine</country><wicri:regionArea>Hebei Key Laboratory for Tree Genetic Resources and Forest Protection, Baoding</wicri:regionArea><wicri:noRegion>Baoding</wicri:noRegion></affiliation></author><author><name sortKey="Zhang, Qi" sort="Zhang, Qi" uniqKey="Zhang Q" first="Qi" last="Zhang">Qi Zhang</name><affiliation wicri:level="1"><nlm:affiliation>Institute of Forest Biotechnology, Forestry College, Agricultural University of Hebei, Baoding, China.</nlm:affiliation><country xml:lang="fr">République populaire de Chine</country><wicri:regionArea>Institute of Forest Biotechnology, Forestry College, Agricultural University of Hebei, Baoding</wicri:regionArea><wicri:noRegion>Baoding</wicri:noRegion></affiliation><affiliation wicri:level="1"><nlm:affiliation>Hebei Key Laboratory for Tree Genetic Resources and Forest Protection, Baoding, China.</nlm:affiliation><country xml:lang="fr">République populaire de Chine</country><wicri:regionArea>Hebei Key Laboratory for Tree Genetic Resources and Forest Protection, Baoding</wicri:regionArea><wicri:noRegion>Baoding</wicri:noRegion></affiliation></author><author><name sortKey="Xiao, Dandan" sort="Xiao, Dandan" uniqKey="Xiao D" first="Dandan" last="Xiao">Dandan Xiao</name><affiliation wicri:level="1"><nlm:affiliation>Institute of Forest Biotechnology, Forestry College, Agricultural University of Hebei, Baoding, China.</nlm:affiliation><country xml:lang="fr">République populaire de 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Chine</country><wicri:regionArea>Institute of Forest Biotechnology, Forestry College, Agricultural University of Hebei, Baoding</wicri:regionArea><wicri:noRegion>Baoding</wicri:noRegion></affiliation><affiliation wicri:level="1"><nlm:affiliation>Hebei Key Laboratory for Tree Genetic Resources and Forest Protection, Baoding, China.</nlm:affiliation><country xml:lang="fr">République populaire de Chine</country><wicri:regionArea>Hebei Key Laboratory for Tree Genetic Resources and Forest Protection, Baoding</wicri:regionArea><wicri:noRegion>Baoding</wicri:noRegion></affiliation></author><author><name sortKey="Wang, Jinmao" sort="Wang, Jinmao" uniqKey="Wang J" first="Jinmao" last="Wang">Jinmao Wang</name><affiliation wicri:level="1"><nlm:affiliation>Institute of Forest Biotechnology, Forestry College, Agricultural University of Hebei, Baoding, China.</nlm:affiliation><country xml:lang="fr">République populaire de Chine</country><wicri:regionArea>Institute of Forest Biotechnology, Forestry College, Agricultural University of Hebei, Baoding</wicri:regionArea><wicri:noRegion>Baoding</wicri:noRegion></affiliation><affiliation wicri:level="1"><nlm:affiliation>Hebei Key Laboratory for Tree Genetic Resources and Forest Protection, Baoding, China.</nlm:affiliation><country xml:lang="fr">République populaire de Chine</country><wicri:regionArea>Hebei Key Laboratory for Tree Genetic Resources and Forest Protection, Baoding</wicri:regionArea><wicri:noRegion>Baoding</wicri:noRegion></affiliation></author></analytic><series><title level="j">Frontiers in plant science</title><idno type="ISSN">1664-462X</idno><imprint><date when="2020" type="published">2020</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en">Four exogenous genes, <i>Cry3A</i>, <i>Cry1Ac, mtlD,</i> and <i>BADH</i>, were inserted into the p1870 vector to obtain multigenic transgenic <i>Populus nigra</i> 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 <i>Cry1Ac</i> 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<sup>-1</sup>. Cry3A content decreased over the same time period and varied between 2642.75 and 15775.22 ng·g<sup>-1</sup>. Indoor insect feeding assay showed that the transgenic lines had high insect resistance, with mortality rates of 1-2-year-old <i>Hyphantria cunea</i> larvae reaching more than 80%, and those of <i>Plagiodera versicolora</i> larvae and nymphs reaching 100%. No. 17 and no. 12 lines had better insect resistance to <i>Lepidoptera</i> and <i>Coleoptera</i> 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.</div></front></TEI><pubmed><MedlineCitation Status="PubMed-not-MEDLINE" Owner="NLM"><PMID Version="1">32793270</PMID><DateRevised><Year>2020</Year><Month>09</Month><Day>28</Day></DateRevised><Article PubModel="Electronic-eCollection"><Journal><ISSN IssnType="Print">1664-462X</ISSN><JournalIssue CitedMedium="Print"><Volume>11</Volume><PubDate><Year>2020</Year></PubDate></JournalIssue><Title>Frontiers in plant science</Title><ISOAbbreviation>Front Plant Sci</ISOAbbreviation></Journal><ArticleTitle>Expression of Multiple Exogenous Insect Resistance and Salt Tolerance Genes in <i>Populus nigra</i> L.</ArticleTitle><Pagination><MedlinePgn>1123</MedlinePgn></Pagination><ELocationID EIdType="doi" ValidYN="Y">10.3389/fpls.2020.01123</ELocationID><Abstract><AbstractText>Four exogenous genes, <i>Cry3A</i>, <i>Cry1Ac, mtlD,</i> and <i>BADH</i>, were inserted into the p1870 vector to obtain multigenic transgenic <i>Populus nigra</i> 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 <i>Cry1Ac</i> 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<sup>-1</sup>. Cry3A content decreased over the same time period and varied between 2642.75 and 15775.22 ng·g<sup>-1</sup>. Indoor insect feeding assay showed that the transgenic lines had high insect resistance, with mortality rates of 1-2-year-old <i>Hyphantria cunea</i> larvae reaching more than 80%, and those of <i>Plagiodera versicolora</i> larvae and nymphs reaching 100%. No. 17 and no. 12 lines had better insect resistance to <i>Lepidoptera</i> and <i>Coleoptera</i> 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.</AbstractText><CopyrightInformation>Copyright © 2020 Zhou, Dong, Zhang, Xiao, Yang and Wang.</CopyrightInformation></Abstract><AuthorList CompleteYN="Y"><Author ValidYN="Y"><LastName>Zhou</LastName><ForeName>Xinglu</ForeName><Initials>X</Initials><AffiliationInfo><Affiliation>Institute of Forest Biotechnology, Forestry College, Agricultural University of Hebei, Baoding, China.</Affiliation></AffiliationInfo><AffiliationInfo><Affiliation>Hebei Key Laboratory for Tree Genetic Resources and Forest Protection, Baoding, China.</Affiliation></AffiliationInfo></Author><Author ValidYN="Y"><LastName>Dong</LastName><ForeName>Yan</ForeName><Initials>Y</Initials><AffiliationInfo><Affiliation>Institute of Forest Biotechnology, Forestry College, Agricultural University of Hebei, Baoding, China.</Affiliation></AffiliationInfo><AffiliationInfo><Affiliation>Hebei Key Laboratory for Tree Genetic Resources and Forest Protection, Baoding, China.</Affiliation></AffiliationInfo></Author><Author ValidYN="Y"><LastName>Zhang</LastName><ForeName>Qi</ForeName><Initials>Q</Initials><AffiliationInfo><Affiliation>Institute of Forest Biotechnology, Forestry College, Agricultural University of Hebei, Baoding, China.</Affiliation></AffiliationInfo><AffiliationInfo><Affiliation>Hebei Key Laboratory for Tree Genetic Resources and Forest Protection, Baoding, China.</Affiliation></AffiliationInfo></Author><Author ValidYN="Y"><LastName>Xiao</LastName><ForeName>Dandan</ForeName><Initials>D</Initials><AffiliationInfo><Affiliation>Institute of Forest Biotechnology, Forestry College, Agricultural University of Hebei, Baoding, China.</Affiliation></AffiliationInfo><AffiliationInfo><Affiliation>Institute of Coastal Agriculture, Hebei Academy of Agriculture and Forestry Sciences, Shijiazhuang, China.</Affiliation></AffiliationInfo></Author><Author ValidYN="Y"><LastName>Yang</LastName><ForeName>Minsheng</ForeName><Initials>M</Initials><AffiliationInfo><Affiliation>Institute of Forest Biotechnology, Forestry College, Agricultural University of Hebei, Baoding, China.</Affiliation></AffiliationInfo><AffiliationInfo><Affiliation>Hebei Key Laboratory for Tree Genetic Resources and Forest Protection, Baoding, China.</Affiliation></AffiliationInfo></Author><Author ValidYN="Y"><LastName>Wang</LastName><ForeName>Jinmao</ForeName><Initials>J</Initials><AffiliationInfo><Affiliation>Institute of Forest Biotechnology, Forestry College, Agricultural University of Hebei, Baoding, China.</Affiliation></AffiliationInfo><AffiliationInfo><Affiliation>Hebei Key Laboratory for Tree Genetic Resources and Forest Protection, Baoding, China.</Affiliation></AffiliationInfo></Author></AuthorList><Language>eng</Language><PublicationTypeList><PublicationType UI="D016428">Journal Article</PublicationType></PublicationTypeList><ArticleDate DateType="Electronic"><Year>2020</Year><Month>07</Month><Day>24</Day></ArticleDate></Article><MedlineJournalInfo><Country>Switzerland</Country><MedlineTA>Front Plant Sci</MedlineTA><NlmUniqueID>101568200</NlmUniqueID><ISSNLinking>1664-462X</ISSNLinking></MedlineJournalInfo><KeywordList Owner="NOTNLM"><Keyword MajorTopicYN="N">Populus nigra L.</Keyword><Keyword MajorTopicYN="N">insect resistance</Keyword><Keyword MajorTopicYN="N">multi-resistance gene</Keyword><Keyword MajorTopicYN="N">multigenic vector</Keyword><Keyword MajorTopicYN="N">salt resistance</Keyword></KeywordList></MedlineCitation><PubmedData><History><PubMedPubDate PubStatus="received"><Year>2020</Year><Month>03</Month><Day>28</Day></PubMedPubDate><PubMedPubDate PubStatus="accepted"><Year>2020</Year><Month>07</Month><Day>07</Day></PubMedPubDate><PubMedPubDate 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Wang</name><name sortKey="Wang, Jinmao" sort="Wang, Jinmao" uniqKey="Wang J" first="Jinmao" last="Wang">Jinmao Wang</name><name sortKey="Xiao, Dandan" sort="Xiao, Dandan" uniqKey="Xiao D" first="Dandan" last="Xiao">Dandan Xiao</name><name sortKey="Xiao, Dandan" sort="Xiao, Dandan" uniqKey="Xiao D" first="Dandan" last="Xiao">Dandan Xiao</name><name sortKey="Yang, Minsheng" sort="Yang, Minsheng" uniqKey="Yang M" first="Minsheng" last="Yang">Minsheng Yang</name><name sortKey="Yang, Minsheng" sort="Yang, Minsheng" uniqKey="Yang M" first="Minsheng" last="Yang">Minsheng Yang</name><name sortKey="Zhang, Qi" sort="Zhang, Qi" uniqKey="Zhang Q" first="Qi" last="Zhang">Qi Zhang</name><name sortKey="Zhang, Qi" sort="Zhang, Qi" uniqKey="Zhang Q" first="Qi" last="Zhang">Qi Zhang</name><name sortKey="Zhou, Xinglu" sort="Zhou, Xinglu" uniqKey="Zhou X" first="Xinglu" last="Zhou">Xinglu Zhou</name></country></tree></affiliations></record>Pour manipuler ce document sous Unix (Dilib)
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