Production of Marker-Free Apple Plants Expressing the Supersweet Protein Gene Driven by Plant Promoter.
Identifieur interne : 000031 ( Main/Exploration ); précédent : 000030; suivant : 000032Production of Marker-Free Apple Plants Expressing the Supersweet Protein Gene Driven by Plant Promoter.
Auteurs : Vadim Timerbaev [Russie] ; Tatiana Mitiouchkina [Russie] ; Alexander Pushin [Russie] ; Sergey Dolgov [Russie]Source :
- Frontiers in plant science [ 1664-462X ] ; 2019.
Abstract
The presence of antibiotic resistance and other marker genes in genetically modified plants causes concern in society because of perceived risks for the environment and human health. The creation of transgenic plants that do not contain foreign genetic material, especially that of bacterial and viral origin, largely alleviates the tension and makes the plants potentially more attractive for consumers. To produce marker-free transgenic apple plants, we used the pMF1 vector, which combines Zygosaccharomyces rouxii recombinaseR and a CodA-nptII bifunctional selectable gene. The thaumatin II gene from the tropical plant Thaumatococcus daniellii, which is under the control of the plant E8 gene (a predominantly fruit-specific promoter) and rbsS3A terminator, was taken as the gene of interest for modification of the fruit taste and enhancing its sweetness. Exploitation of this gene in our laboratory has allowed enhancing the sweetness, as well as improving the taste characteristics, of fruits and vegetables of plants such as strawberry, carrot, tomato and pear. We have obtained three independent transgenic apple lines that have been analyzed by PCR and Southern blot analyses for the presence of T-DNA sequences. Two of them contained a partial sequence of the T-DNA. With one line containing the full insert we then used a delayed strategy for the selection of marker-free plants. After induction of recombinase activity in leaf explants on selective media with 5-fluorocytosine (5-FC) we obtained more than 30 sublines, most of which lost their resistance to kanamycin. Most of the apple sublines showed the expression of the supersweet protein gene in a wide range of levels as detected by RNA accumulation. The plants from the group with the highest transcript level were propagated and grafted onto dwarf rootstocks for early fruit production for future estimates of protein levels and organoleptic analyses. Thus, we developed a protocol that allowed the production of marker-free apple plants expressing the supersweet protein.
DOI: 10.3389/fpls.2019.00388
PubMed: 30984230
PubMed Central: PMC6449483
Affiliations:
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National Scientific Center, Russian Academy of Sciences, Yalta, Russia.</nlm:affiliation><country xml:lang="fr">Russie</country><wicri:regionArea>Laboratory of Plant Bioengineering, Nikita Botanical Gardens - National Scientific Center, Russian Academy of Sciences, Yalta</wicri:regionArea><wicri:noRegion>Yalta</wicri:noRegion></affiliation><affiliation wicri:level="3"><nlm:affiliation>Laboratory of Plant Genetic Engineering, All-Russia Research Institute of Agricultural Biotechnology, Russian Academy of Sciences, Moscow, Russia.</nlm:affiliation><country xml:lang="fr">Russie</country><wicri:regionArea>Laboratory of Plant Genetic Engineering, All-Russia Research Institute of Agricultural Biotechnology, Russian Academy of Sciences, Moscow</wicri:regionArea><placeName><settlement type="city">Moscou</settlement><region>District fédéral central</region></placeName></affiliation></author></analytic><series><title level="j">Frontiers in plant science</title><idno type="ISSN">1664-462X</idno><imprint><date when="2019" type="published">2019</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en">The presence of antibiotic resistance and other marker genes in genetically modified plants causes concern in society because of perceived risks for the environment and human health. The creation of transgenic plants that do not contain foreign genetic material, especially that of bacterial and viral origin, largely alleviates the tension and makes the plants potentially more attractive for consumers. To produce marker-free transgenic apple plants, we used the pMF1 vector, which combines <i>Zygosaccharomyces rouxii</i> recombinaseR and a <i>CodA-nptII</i> bifunctional selectable gene. The thaumatin II gene from the tropical plant <i>Thaumatococcus daniellii</i>, which is under the control of the plant E8 gene (a predominantly fruit-specific promoter) and rbsS3A terminator, was taken as the gene of interest for modification of the fruit taste and enhancing its sweetness. Exploitation of this gene in our laboratory has allowed enhancing the sweetness, as well as improving the taste characteristics, of fruits and vegetables of plants such as strawberry, carrot, tomato and pear. We have obtained three independent transgenic apple lines that have been analyzed by PCR and Southern blot analyses for the presence of T-DNA sequences. Two of them contained a partial sequence of the T-DNA. With one line containing the full insert we then used a delayed strategy for the selection of marker-free plants. After induction of recombinase activity in leaf explants on selective media with 5-fluorocytosine (5-FC) we obtained more than 30 sublines, most of which lost their resistance to kanamycin. Most of the apple sublines showed the expression of the supersweet protein gene in a wide range of levels as detected by RNA accumulation. The plants from the group with the highest transcript level were propagated and grafted onto dwarf rootstocks for early fruit production for future estimates of protein levels and organoleptic analyses. Thus, we developed a protocol that allowed the production of marker-free apple plants expressing the supersweet protein.</div></front></TEI><pubmed><MedlineCitation Status="PubMed-not-MEDLINE" Owner="NLM"><PMID Version="1">30984230</PMID><DateRevised><Year>2020</Year><Month>09</Month><Day>29</Day></DateRevised><Article PubModel="Electronic-eCollection"><Journal><ISSN IssnType="Print">1664-462X</ISSN><JournalIssue CitedMedium="Print"><Volume>10</Volume><PubDate><Year>2019</Year></PubDate></JournalIssue><Title>Frontiers in plant science</Title><ISOAbbreviation>Front Plant Sci</ISOAbbreviation></Journal><ArticleTitle>Production of Marker-Free Apple Plants Expressing the Supersweet Protein Gene Driven by Plant Promoter.</ArticleTitle><Pagination><MedlinePgn>388</MedlinePgn></Pagination><ELocationID EIdType="doi" ValidYN="Y">10.3389/fpls.2019.00388</ELocationID><Abstract><AbstractText>The presence of antibiotic resistance and other marker genes in genetically modified plants causes concern in society because of perceived risks for the environment and human health. The creation of transgenic plants that do not contain foreign genetic material, especially that of bacterial and viral origin, largely alleviates the tension and makes the plants potentially more attractive for consumers. To produce marker-free transgenic apple plants, we used the pMF1 vector, which combines <i>Zygosaccharomyces rouxii</i> recombinaseR and a <i>CodA-nptII</i> bifunctional selectable gene. The thaumatin II gene from the tropical plant <i>Thaumatococcus daniellii</i>, which is under the control of the plant E8 gene (a predominantly fruit-specific promoter) and rbsS3A terminator, was taken as the gene of interest for modification of the fruit taste and enhancing its sweetness. Exploitation of this gene in our laboratory has allowed enhancing the sweetness, as well as improving the taste characteristics, of fruits and vegetables of plants such as strawberry, carrot, tomato and pear. We have obtained three independent transgenic apple lines that have been analyzed by PCR and Southern blot analyses for the presence of T-DNA sequences. Two of them contained a partial sequence of the T-DNA. With one line containing the full insert we then used a delayed strategy for the selection of marker-free plants. After induction of recombinase activity in leaf explants on selective media with 5-fluorocytosine (5-FC) we obtained more than 30 sublines, most of which lost their resistance to kanamycin. Most of the apple sublines showed the expression of the supersweet protein gene in a wide range of levels as detected by RNA accumulation. The plants from the group with the highest transcript level were propagated and grafted onto dwarf rootstocks for early fruit production for future estimates of protein levels and organoleptic analyses. Thus, we developed a protocol that allowed the production of marker-free apple plants expressing the supersweet protein.</AbstractText></Abstract><AuthorList CompleteYN="Y"><Author ValidYN="Y"><LastName>Timerbaev</LastName><ForeName>Vadim</ForeName><Initials>V</Initials><AffiliationInfo><Affiliation>Laboratory of Expression Systems and Modification of the Plant Genome "Biotron", Branch of the Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Pushchino, Russia.</Affiliation></AffiliationInfo><AffiliationInfo><Affiliation>Laboratory of Plant Bioengineering, Nikita Botanical Gardens - National Scientific Center, Russian Academy of Sciences, Yalta, Russia.</Affiliation></AffiliationInfo><AffiliationInfo><Affiliation>Laboratory of Plant Genetic Engineering, All-Russia Research Institute of Agricultural Biotechnology, Russian Academy of Sciences, Moscow, Russia.</Affiliation></AffiliationInfo></Author><Author ValidYN="Y"><LastName>Mitiouchkina</LastName><ForeName>Tatiana</ForeName><Initials>T</Initials><AffiliationInfo><Affiliation>Laboratory of Expression Systems and Modification of the Plant Genome "Biotron", Branch of the Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Pushchino, Russia.</Affiliation></AffiliationInfo><AffiliationInfo><Affiliation>Laboratory of Plant Bioengineering, Nikita Botanical Gardens - National Scientific Center, Russian Academy of Sciences, Yalta, Russia.</Affiliation></AffiliationInfo><AffiliationInfo><Affiliation>Laboratory of Plant Genetic Engineering, All-Russia Research Institute of Agricultural Biotechnology, Russian Academy of Sciences, Moscow, Russia.</Affiliation></AffiliationInfo></Author><Author ValidYN="Y"><LastName>Pushin</LastName><ForeName>Alexander</ForeName><Initials>A</Initials><AffiliationInfo><Affiliation>Laboratory of Expression Systems and Modification of the Plant Genome "Biotron", Branch of the Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Pushchino, Russia.</Affiliation></AffiliationInfo><AffiliationInfo><Affiliation>Laboratory of Plant Genetic Engineering, All-Russia Research Institute of Agricultural Biotechnology, Russian Academy of Sciences, Moscow, Russia.</Affiliation></AffiliationInfo></Author><Author ValidYN="Y"><LastName>Dolgov</LastName><ForeName>Sergey</ForeName><Initials>S</Initials><AffiliationInfo><Affiliation>Laboratory of Expression Systems and Modification of the Plant Genome "Biotron", Branch of the Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Pushchino, Russia.</Affiliation></AffiliationInfo><AffiliationInfo><Affiliation>Laboratory of Plant Bioengineering, Nikita Botanical Gardens - National Scientific Center, Russian Academy of Sciences, Yalta, Russia.</Affiliation></AffiliationInfo><AffiliationInfo><Affiliation>Laboratory of Plant Genetic Engineering, All-Russia Research Institute of Agricultural Biotechnology, Russian Academy of Sciences, Moscow, Russia.</Affiliation></AffiliationInfo></Author></AuthorList><Language>eng</Language><PublicationTypeList><PublicationType UI="D016428">Journal Article</PublicationType></PublicationTypeList><ArticleDate DateType="Electronic"><Year>2019</Year><Month>03</Month><Day>29</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">Agrobacterium-mediated transformation</Keyword><Keyword MajorTopicYN="N">Malus × domestica</Keyword><Keyword MajorTopicYN="N">R/RS recombination system</Keyword><Keyword MajorTopicYN="N">cytosine deaminase</Keyword><Keyword MajorTopicYN="N">marker-free plants</Keyword><Keyword MajorTopicYN="N">thaumatin II</Keyword></KeywordList></MedlineCitation><PubmedData><History><PubMedPubDate 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IdType="pubmed">9107046</ArticleId></ArticleIdList></Reference></ReferenceList></PubmedData></pubmed><affiliations><list><country><li>Russie</li></country><region><li>District fédéral central</li></region><settlement><li>Moscou</li></settlement></list><tree><country name="Russie"><noRegion><name sortKey="Timerbaev, Vadim" sort="Timerbaev, Vadim" uniqKey="Timerbaev V" first="Vadim" last="Timerbaev">Vadim Timerbaev</name></noRegion><name sortKey="Dolgov, Sergey" sort="Dolgov, Sergey" uniqKey="Dolgov S" first="Sergey" last="Dolgov">Sergey Dolgov</name><name sortKey="Dolgov, Sergey" sort="Dolgov, Sergey" uniqKey="Dolgov S" first="Sergey" last="Dolgov">Sergey Dolgov</name><name sortKey="Dolgov, Sergey" sort="Dolgov, Sergey" uniqKey="Dolgov S" first="Sergey" last="Dolgov">Sergey Dolgov</name><name sortKey="Mitiouchkina, Tatiana" sort="Mitiouchkina, Tatiana" uniqKey="Mitiouchkina T" first="Tatiana" last="Mitiouchkina">Tatiana Mitiouchkina</name><name sortKey="Mitiouchkina, Tatiana" sort="Mitiouchkina, Tatiana" uniqKey="Mitiouchkina T" first="Tatiana" last="Mitiouchkina">Tatiana Mitiouchkina</name><name sortKey="Mitiouchkina, Tatiana" sort="Mitiouchkina, Tatiana" uniqKey="Mitiouchkina T" first="Tatiana" last="Mitiouchkina">Tatiana Mitiouchkina</name><name sortKey="Pushin, Alexander" sort="Pushin, Alexander" uniqKey="Pushin A" first="Alexander" last="Pushin">Alexander Pushin</name><name sortKey="Pushin, Alexander" sort="Pushin, Alexander" uniqKey="Pushin A" first="Alexander" last="Pushin">Alexander Pushin</name><name sortKey="Timerbaev, Vadim" sort="Timerbaev, Vadim" uniqKey="Timerbaev V" first="Vadim" last="Timerbaev">Vadim Timerbaev</name><name sortKey="Timerbaev, Vadim" sort="Timerbaev, Vadim" uniqKey="Timerbaev V" first="Vadim" last="Timerbaev">Vadim Timerbaev</name></country></tree></affiliations></record>Pour manipuler ce document sous Unix (Dilib)
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