WO2018119501A1 - Procédé d'accélération de la croissance de plantes et plantes transgéniques - Google Patents

Procédé d'accélération de la croissance de plantes et plantes transgéniques Download PDF

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Publication number
WO2018119501A1
WO2018119501A1 PCT/BR2017/000150 BR2017000150W WO2018119501A1 WO 2018119501 A1 WO2018119501 A1 WO 2018119501A1 BR 2017000150 W BR2017000150 W BR 2017000150W WO 2018119501 A1 WO2018119501 A1 WO 2018119501A1
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WO
WIPO (PCT)
Prior art keywords
plant
gene
della
transgenic
plants
Prior art date
Application number
PCT/BR2017/000150
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English (en)
Inventor
Marcelo Menossi Teixeira
Rafael Garcia TAVARES
Prakash Lakshmanan
Anthony O'Connell
Original Assignee
Universidade Estadual De Campinas - Unicamp
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
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Application filed by Universidade Estadual De Campinas - Unicamp filed Critical Universidade Estadual De Campinas - Unicamp
Priority to BR112018013532A priority Critical patent/BR112018013532A2/pt
Publication of WO2018119501A1 publication Critical patent/WO2018119501A1/fr

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
    • C12N15/8218—Antisense, co-suppression, viral induced gene silencing [VIGS], post-transcriptional induced gene silencing [PTGS]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
    • Y02A40/10—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture
    • Y02A40/146—Genetically Modified [GMO] plants, e.g. transgenic plants

Definitions

  • This invention belongs to the field of biotechnology, in particular, as regards to the plant development / and describes a method for accelerating plant growth through gene silencing of the gene that encodes DELLA protein -
  • the invention has particular utility in connection with accelerating growth of sugar cane and to transgenic sugar cane plants obtained from the method, and will be described in connection with such utility, although other utilities are contemplated.
  • the invention may be used for accelerating growth of other plants and to transgenic plants obtained for the method* including but not. limited to sorghum, corn., eucalyptus and miscanthus sinensis.
  • Said gene that encodes a DELLA protein, that is responsible for. the control of plant development, and the sugar cane that comprises this gene silenced or with reduced expression feature a faster development without compromised sucrose levels.
  • the DELIA protein is a candidate for the regulation of plant growth, since the genes encoding this protein are widely known as the genes of the green revolution, being responsible for the semi-dwarf character in cereals, providing the increase in the world production during the decades of 60 and 70.
  • DELLA protein is a transcriptional nuclear regulator that suppresses the gibberellin hormone Signaling (GA) restricting the plant growth.
  • DELLA protein is characterized by presenting a specific domain in the N- terminal region, which, contains the DELLA amino acids (aspartic acid, D, glutamic acid, E, leucine, L, Leucine, L and Alanine, A) , from which its name is derived. Although this domain can present slight variations, it is highly conserved among the species. Therefore, DELLA proteins refer to proteins, containing the DELLA domain or related domains with small changes in the amino acid composition. For example, in sugarcane and sorghum, the first leucine is replaced by methionine (M) .
  • M methionine
  • ScRGA which encodes the DELLA growth repressor from sugarcane
  • the present invention relates to a method for accelerating plant growth comprising the steps of;
  • step (a) Phenotypic analysis of transgenic plants. wherein, in step (a) , the final construct produced is pUbi:hpRGAi and has at least 77% of DNA sequence identity and, in step (c), the plants phenotypically evaluated contain in its genome the pUbi:hpRGAi construct.
  • the plant is selected from the group comprising sugar cane, sorghum, corn, eucalyptus and miscanthus sinensis.
  • the invention further relates to a transgenic plant comprising the gene that encodes silenced DELLA protein, wherein the plant is selected from sugar cane, sorghum, corn,, eucalyptus and miscanthus sinensis,
  • FIG. 1 contains a ScRGA protein scheme, showing the different domains (A) and the alignment of the DELIA domain of the ScRGA protein with DELIA proteins from other species (BJ - In (A) , the DELLA and GRAS domains are indicated.
  • the DEMLA sequence similar to. the DELLA domain sequence, is indicated, as well as the TVHYNP sequence, the LH conserved domains, the nuclear ⁇ localization signal (NLS), and the VHIID, PFYRE and SAW regions that comprise the GRAS domain are found interlinked by the variable region poly S/T/V.
  • B shows, in gray background, identical amino acids in all. proteins analyzed. I and II indicate the two conserved regions of the DELLA domain, and the underlined amino acids DEMLA and TVHYNP in the protein alignment .
  • FIG. 2 is a schematic drawing of the gene silencing construct of ScRGA mediated by double-stranded RNA (A) and the hairpin sequence hpRSAi/ used for silencing the ScRGA gene (B) .
  • the hpRGAi hairpin is shown in detail, inserted into the expression cassette comprised by the ubiquitin promoter (maize Ubi w l gene) , the untranslated 5' region, the Ubi-1 gene intron and the gene transcription terminator of nopaline synthase (NOS) .
  • This complete vector was named pDbirhpRGAi.
  • SEQ ID NO: 6 shows the gene sequence of the hpRGAi hairpin, with the anti- sense gene of the ScRGA gene indicated in the nucleotides
  • E'igure 3 corresponds to the integration analysis of the construct pUbi:hpRGAx in the sugarcane genome.
  • A shows the expression cassette pUBI:hpScRGAi.
  • the site of the pair of primers used in the analysis of the polymerase chain reaction (PCR) is indicated by arrows. This pair of primers amplifies a fragment of 392-base pairs (BP) in transgenic plants.
  • B shows an agarose gel with the PCR products with the expected size of 392 bp. M - molecular weight marker "100 bp ladder" (Promega, Brazil); 1-water;
  • Figure 4 corresponds to the graphic that indicates the levels .of expression of the ScRGA gene in wild (WT, black bar) and silenced transgenic plants due to the expression of the hairpin hpRGAi (white bars) obtained by real-time PCR.
  • FIG. 5 demonstrates the phenotypic analysis of the transgenic plants silenced for the ScRGA gene (HpScRGAj .
  • HpScRGAj On the left, 3-month-old plants are represented, with WT being a wild plant,, and on the right, a HpScRGA plant, silenced for the ScRGA gene. By way of comparison, it is also shown in the central position a ScRGAOE plant, which over-expresses the scRGA gene and features a phenotype opposite of the HpScRGA plants.
  • height data. number of interno.des and plant tillers of the pl3 ⁇ 4nts of 3 months of age are shown.
  • This invention describes a method for accelerating plant growth through silencing of a DELLA gene, as well as the transgenic plants obtained from the method described, particularly sugarcane.
  • gibberellin (GA.) signaling has an important role in the plant growth, in which the defense is prioritized in relation to growth by increased levels of DELLA, a growth repressor protein.
  • the method for accelerating plant growth of this invention comprises the steps of:
  • the ScRGA gene (SEQ ID NO: 1) has homologous genes with high similarity at the DNA sequence level, and can be selected from the group consisting of sorghum gene ⁇ SEQ ID NO: 2) with 97% identity, corn gene (SEQ ID NO: 3) with 94% identity, Miscanthus sinensis gene (SEQ ID NO: 4), with 97% identity and eucalyptus gene (SEQ ID NO: 5), with 77% identity.
  • Step a) Construct production for silencing ScRGA gene
  • the. ScRGA cDNA clone was obtained and its encoding sequence was amplified via polymerase chain reaction (PCR) using specific primers using genomic DNA from the commercial sugarcane variety SP80-3280.
  • PCR polymerase chain reaction
  • the antisense and sense fragments of the ScRGA gene were amplified through specific primers and subcloned in the pGEMTEasy vector, generating the constructs pGEMTEasy:asDELLA and pGEMTEasy:sde11a.
  • the genomic DNA of the sugarcane variety RB92579 was used as a template for amplifying, the intron II of sugarcane ScRlMYBl gene.
  • the intron II. fragment was cloned in pGEMTEasy, generating the construct pGEMTEasy: IntronlI.
  • the gold particles were coated with a L:,l molar mixture of plasmids pUbi chpRGAi and pUKtS.
  • the helper plasmid pUKN provides the NPTII gene, which produces the neomycin phosphotransferase II protein regu.ired for the selection of transformed plants.
  • Spermidine and calcium chloride (CaCla) were used to precipitate DNA on gold.
  • RNA was treated with RQl RNase-Free DNAse (Promega, USA), at 37° C during 30 minutes to remove the genomic DNA contamination. After that, cDNAs were synthesized according to the kit protocol of Improm-II reverse transcriptase enzyme II ⁇ Promega ⁇ . A PCR was performed to verify the absence of genomic DNA contamination.
  • Step d Phenotypic analysis of transgenic plants.

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biotechnology (AREA)
  • General Engineering & Computer Science (AREA)
  • Biomedical Technology (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Biophysics (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Physics & Mathematics (AREA)
  • Microbiology (AREA)
  • Plant Pathology (AREA)
  • Cell Biology (AREA)
  • Virology (AREA)
  • Botany (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Medicinal Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Breeding Of Plants And Reproduction By Means Of Culturing (AREA)

Abstract

La présente invention concerne un procédé d'accélération de la croissance des plantes par silençage du gène DELLA, ainsi que les plantes transgéniques obtenues à partir du procédé décrit, en particulier la canne à sucre. Ledit gène DELLA est impliqué dans la régulation du développement des plantes et, par conséquent, la canne à sucre qui comprend ce gène silencé ou à expression réduite présente un développement plus rapide sans altération des taux de saccharose.
PCT/BR2017/000150 2016-12-29 2017-12-19 Procédé d'accélération de la croissance de plantes et plantes transgéniques WO2018119501A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
BR112018013532A BR112018013532A2 (pt) 2016-12-29 2017-12-19 método para aceleração do crescimento vegetal e plantas transgênicas

Applications Claiming Priority (2)

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US201662440207P 2016-12-29 2016-12-29
US62/440,207 2016-12-29

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WO2018119501A1 true WO2018119501A1 (fr) 2018-07-05

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2019053725A1 (fr) * 2017-09-18 2019-03-21 Futuragene Israel Ltd. Contrôle d'expression spécifique au tissu de polypeptides della

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20050060773A1 (en) * 1997-08-13 2005-03-17 Pioneer Hi-Bred International, Inc. Genetic control of plant growth and development
US20150052634A1 (en) * 2011-12-09 2015-02-19 Ceres, Inc. Transgenic plants having altered biomass composition
US20160002648A1 (en) * 2013-03-11 2016-01-07 Mei Guo Genes for improving nutrient uptake and abiotic stress tolerance in plants

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20050060773A1 (en) * 1997-08-13 2005-03-17 Pioneer Hi-Bred International, Inc. Genetic control of plant growth and development
US20150052634A1 (en) * 2011-12-09 2015-02-19 Ceres, Inc. Transgenic plants having altered biomass composition
US20160002648A1 (en) * 2013-03-11 2016-01-07 Mei Guo Genes for improving nutrient uptake and abiotic stress tolerance in plants

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2019053725A1 (fr) * 2017-09-18 2019-03-21 Futuragene Israel Ltd. Contrôle d'expression spécifique au tissu de polypeptides della
US11555195B2 (en) 2017-09-18 2023-01-17 Futuragene Israel Ltd. Tissue-specific expression control of DELLA polypeptides

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