WO2014197951A2 - Cassette d'expression pour induire une résistance à de multiples espèces de nématoïdes dans des plantes, procédés et plantes l'utilisant - Google Patents

Cassette d'expression pour induire une résistance à de multiples espèces de nématoïdes dans des plantes, procédés et plantes l'utilisant Download PDF

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WO2014197951A2
WO2014197951A2 PCT/BR2013/000203 BR2013000203W WO2014197951A2 WO 2014197951 A2 WO2014197951 A2 WO 2014197951A2 BR 2013000203 W BR2013000203 W BR 2013000203W WO 2014197951 A2 WO2014197951 A2 WO 2014197951A2
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plant
plants
expression cassette
seq
gene
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WO2014197951A3 (fr
WO2014197951A8 (fr
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Maria Fátima GROSSI DE SÁ
Eduardo Romano De Campos Pinto
Rodrigo Da Rocha Fragoso
Maria Cristina Mattar Da Silva
André Vinícius Julio FERREORA
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Empresa Brasileira De Pesquisa Agropecuária - Embrapa
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Priority to BR112015024341-0A priority patent/BR112015024341B1/pt
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    • 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
    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • C12N15/8279—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance
    • C12N15/8285—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance for nematode resistance
    • 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]
    • 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

  • the present invention relates to the control of nematodes in plants. More specifically, the present invention relates to the control of multiple nematode species by gene silencing by expressing an RNA molecule in the plant capable of forming a double stranded RNA comprising a fragment of the plague target gene sequence.
  • Phytonematoids are among the main factors responsible for the fall in yield of several crops, particularly soybean, especially in tropical and subtropical regions.
  • the control of nematodes in scale crops is designed to integrate various methods and be cost effective.
  • the phytopathological principles of exclusion are considered (avoid infestation of uninhabited areas by species or new breeds on the property or in a larger geographical region); eradication (crop rotation with non-host summer and winter species); regulation (environmental modification and plant nutrition); and immunization (use of cultivars resistant to certain species or races).
  • Current strategies for nematode control are: nematicide use, crop rotation, natural genetic resistance and genetic engineering.
  • Nematicides have been widely used to control parasitic nematodes of sedentary and migratory plants, but these compounds are often associated with harmful effects on the environment.
  • Crop rotation an effective cultural practice for many biotic stresses, is also an important strategy for managing plant parasitic nematodes, although in many cases their effectiveness is limited.
  • Phytonematoid management is highly dependent on the resistance of host plant obtained by traditional breeding methods, often derived from a limited genetic base.
  • Conventional breeding has some limitations, such as the genetic link between genes of interest and undesirable genes and interspecific incompatibility.
  • Genetic engineering is an extremely useful tool because it allows the identification of genes of interest, manipulation of these genes, the construction and introduction of a single gene of interest directly into elite cultivars and the selection of plants that have this gene.
  • the gene to be introduced may come from the same species or from other species, thus breaking down the barriers imposed by sexual incompatibility between the different species and eliminating the effect of unwanted gene linkages.
  • RNA-mediated interference offers good results in nematode control (MCCARTER, J. Molecular approaches toward plant parasitic nematodes. In: (Ed.). Plant Celi Monographs v.15, 2008 .p.239-267.).
  • MCCARTER double-stranded RNA
  • dsRNA double-stranded RNA
  • Target genes of interest are genes essential to phytonematoid or genes involved with parasitism, migration, formation or maintenance of the feeding site.
  • nematode cycle During the nematode cycle, they ingest the cytoplasmic content of infected root giant cells, causing dsRNA absorption, which may result in silencing of the corresponding gene. Depending on the function of the silenced gene, several dysfunctions can be generated in the phytonematoid by silencing and / or reducing the expression of specific genes, so that the infection can be aborted.
  • RNA-mediated interference refers to the specific reduction of gene expression through the use of complementary sequence RNA molecules. This phenomenon, first reported in Caenorhabditis elegans by GUO & KEMPHUES (GUO.S. & KEMPHUES, K. J. par-1, a gene required for establishing polarity in C. elegans embryos, then a putative Ser / Thr kinase that is asymmetrically distributed. Cell, v. 81, no. 4, p. 611-620, 1995) and subsequently demonstrated by FIRE et al.
  • dsRNA double stranded RNA
  • the DCR2 / R2D2 complex binds to these small interfering RNA (siRNA) molecules (LIU, Q .; RAND, TA; KALIDAS.S .; DU, F .; KIM, HE; SMITH, DP & WANG, X. R2D2, the bridge between the initiation and effector steps of the Drosophila RNAi pathway Science, v. 301, No. 5641, pp. 1921-5, Sep 26 2003), siRNA is incorporated into a complex called RISC (RNA Induc- ced Silencing Complex), which then determines the degradation of any complementary sequence RNA molecules (HAMMOND, SM; BERNSTEIN, E.; BEACH, D.
  • siRNA small interfering RNA
  • RNA-directed nuclease mediate post-transcriptional gene silencing in Drosophila Nature, v. 404, no. 6775, pp. 293-6, Mar 16 2000).
  • This gene silencing phenomenon occurs in a number of eukaryotic organisms, including nematodes and higher plants (BERNSTEIN, E.; CAUDY.AA; HAMMOND, SM & HANNON, GJ. Role for a bidentate ribonuclease in the initiation step of RNA interference. , see 409, No. 6818, pp. 363-6, Jan 18 2001).
  • KLINK & WOLNIAK KLINK, VP & WOLNIAK, SM Centrin is necessary for the formation of the motile apparatus in spermatids of Marsilea.
  • Mol Biol Celi, v. 12, no. 3, p. 761-76, Mar 2001 were able to silence centrin (centrin) mRNA using in vitro synthesized dsRNA, and for knockout effects, they demonstrated that dsRNA is at least ten times more effective than sense or antisense RNA. separately.
  • RNAi mechanism is partially executed by the plant and partly by the nematode. Plants express dsRNA from nematode genes, which are processed by DICER and generate siRNAs. When nematodes feed on these plants, both dsRNA and siRNA are ingested.
  • nematodes digest dsRNA into siRNA using their DICER complex. Ingested or processed by the nematode itself, siRNAs bind to RISC to induce degradation of specific nematode mRNA.
  • SiRNAs are amplified in nematodes by RNA-dependent RNA polymerase (RDRP) (CHAPMAN, EJ & CARRINGTON.JC Nature Reviews Genetics, v. 8, no. 11, p. 884-96, Nov 2007; ZA MORE, PD & .HALEY, B. Ribo-gnome: The Big World of Small RNAs, Science, v. 309, No. 5740, p. 1519-24, Sep 2 2005), where the mRNA serves as a template for the synthesis of more specific dsRNA, enhancing the effect of gene silencing.
  • RDRP RNA-dependent RNA polymerase
  • siRNA-mediated silencing is highly sequence-specific.
  • Tuschl and colleagues have shown that even a single base mismatch between siRNA and target mRNA influences gene silencing (ELBASHIR, SM; MARTINEZ, J .; PAT-KANIOWSKA, A.; LENDECKEL, W. & TUSCHL Functional anatomy of siRNAs for mediating efficient RNAi in Drosophila melanogaster embryo lysate The EMBO Journal, v. 20, no. 23, pp. 6877-88, Dec 3 2001).
  • gene silencing is highly specific, it is possible to silence homologous gene families that share conserved sequences (MIKI, D .; ITOH, R. & SHIMAMOTO, K.
  • RNA silencing of single and multi pie members in a gene family of rice Plant physiology, v. 138, no. 4, p. 1903-13, Aug 2005) or with the chimeric construction of various active genes (ALLEN, RS; MILLGA- ⁇ , G.) CH (TTY, JA; THISLETON, J .; MILLER, JA; FIST, AJ; GERLA-CH, WL & LARKIN, PJ RNAi-mediated replacement of morpine with the nonnarcotic reticulin in opium poppy Nature Biotechnology, v. 22, no. 12, p. 1559-66, Dec 2004). RNAi, is not only diffused from cell to cell (FAGARD, M. & VAUCHERET, H.
  • RNA silencing moves long distances through the phloem and, eventually, spreads cell-to-cell through tissue tissue plasmodes (JORGENSEN, RA RNA trafficking information systemically in plants.) of Sciences, v. 99, No. 18, pp. 11561-3, Sep 3 2002; MLOTSH-WA, S.; VOINNET, O .; METTE, MF; ⁇ , ⁇ ; VAUCHERET, H.; PRUSS, G. & VANCE.VB RNA silencing and the mobile silencing signal Plant Cell, v. 14 Suppl, pp. S289-301, 2002; VOINNET, O; VAIN, P.; ANGELL, S.
  • LIMPENS et al LIMPENS, E.; RAMOS, J .; FRANKEN, C.; RAZ.V .; COMPAAN.B.; FRANSSEN, H..; BISSELING, T. & GEURTS, R. RNA interference in Agrobacteriunl rhizogenes transformed roots of Arabidopsis and Medicago truncatula. Journal of Experimental Butterfly, v. 55, no. 399, p. 983-92, May 2004) also found that the silencing signal was systematically transported from Arabidopsis thaliana roots to the shoots, although the degree of silencing was limited and very variable.
  • RNAi has been used transiently to silence the expression of almost all genes, inducing different phenotypic effects including lethality (FRASER, AG; KAMATH, RS; ZIPPERLEN, P.; MARTI NEZ-CAMPOS, M .; SOHR- MANN, M. & AHRINGER, J. Functional genomic analysis of C.
  • RNA-mediated interference can be performed in C. elegans by ingestion of dsRNA-expressing bacteria (TIMMONS, L. & FIRE, A. Specific interference by ingested dsRNA. Nature, v.
  • dsRNA administration for phytononematoids was the immersion of nematodes in dsRNA solution to induce ingestion and / or absorption. Since 2006, dsRNA molecules have been produced and offered directly by the genetically modified host plant.
  • Gall-forming nematodes and cyst-forming nematodes (NCs) are necessarily plant root parasites, making host dsRNA an ideal strategy for silencing nematode genes as well as providing direct evidence of the function of these genes.
  • Previous research confirms the viability and effectiveness of host RNAi provision for nematode control.
  • YADV et al. YADAV, BC; VELUTHAMBI, K. & SUBRAMANIAM.K.
  • Host-generated double stranded RNA induces RNAi in plant parasitic nematodes and protects the host from infection.
  • Molecular and Biochemistry Parasitology v. 148, n. 2, p 219-22, Aug 2006
  • dsRNA from the NFG 16D10 parasitism gene in Arabidopsis transgenic plants resulted in resistance against four of the major species of this genus (HUANG, G.; ALLEN, RS; DAVIS, E. L; ⁇ U ⁇ , ⁇ . J. & HUSSEY.RS Engineering broad root-knot resistance in transgenic plants by RNAi silencing of a conserved and essential root-knot nematode parasitism gene No.
  • KLINK et al KLINK, VP; KIM, KH; MARTINS.V .; MACDONALD.MH; BEARD, HS; ALKHAROUF.NW; LEE, SK; PARK, SC & MATTHEWS, BFA correlation between host-mediated expression of parasite genes as tandem inverted repeats and abrogation of development of female Heterodera glycines cyst formation during infection of Glycine max. Plant, v. 230, no 1, pp. 53-71, Jun 2009) effectively inhibited the formation of H. glycines cysts using a construct with inverted repeats of several genes. Recently, LI et al.
  • RNAi delivery by host down-regulate nematode target gene expression, observed by real-time PCR (RT-qPCR) analysis of nematodes fed on transgenic roots (LI, J.; TODD, T. C; OAKLEV.TR; LEE, J. & TRICK, HN Host-derived suppression of reproductive nematode and fitness decreasing fecundity genes of Heterodera glycines Ichinohe Plant, v. 232, no. 3, pp 775-85, Aug 2010a; SINDHU, AS; MAIER, TR; MITCHUM, MG; HUSSEY, RS; DAVIS, E.
  • RT-qPCR real-time PCR
  • RNA constructs capable of forming dsRNA for inhibition of nematode genes by interfering RNA and consequent induction of resistance to such pests in the plant are available (EP2330203) .
  • WO2011060151 teaches vectors comprising constructs for nematode resistance induction, including Meloidogyne incognita and Heterodera glycines, encoding double-stranded RNA strands that inhibit expression of a target plague gene.
  • WO2006045591 teaches, among others, methods for producing nematode-resistant transgenic plants comprising co-expression in the plant of double-stranded RNA molecules targeting genes from different species, including Meloidogyne incognita and Heterodera glycines. WO2006045591 further teaches that the transfer of dsRNA to the plague can be improved by the use of tissue-specific promoters for the most accessible plague parts, such as root-specific promoters or nematode-induced promoters.
  • Heterodera glycines splicing factor Prp-17 gene silencing by siRNA expression in transgenic soybean was effective induction of plague resistance (Li, J.; Todd, T. C.; Oaklev, TR; Lee, J. & Trick, HN. Host-derived suppression of reproductive nematode and fitness genes decreases fecundity of Heterodera glycines Ichinohe. Plant, v 232, No. 3, pp. 775-85, Aug 2010).
  • M. incognita splicing factor (FS) dsRNA expression obtained a 90% reduction of established nematodes (M. incognita) in the roots (Yadav, BC; Veluthambi, K. & Subramaniam, K. Host-generated double stranded RNA induces RNAi in plant parasitic nematodes and protects the host from infection Molecular and Bioehemistry Parasitolology, v. 148, no. 2, pp. 219-22, Aug 2006).
  • FS incognita splicing factor
  • dsRNA from an H. glycines FS-related gene reduced the number of females colonizing the roots to less than 20%.
  • PI0701172-5 describes a constitutive soybean promoter, the soybean ubiquitin conjugation protein gene promoter (UceS 8.3).
  • the UceS8.3 promoter is induced by root invasion by M. incognita (Miranda, VJ) Characterization of the expression of the ubiquitin-conjugating enzyme (E2) encoding gene in soybean inoculated with Melo-dogyne incognita and infested with Anticarsia gemmatalis. Magister Science, Cell Biology, University of Brasilia).
  • RNA expression constructs utilize constitutive promoters, generally isolated from viruses, such as CaMV 35S for constitutive dsRNA expression.
  • the differential of the present invention is that it is a target gene-specific dsRNA expression vector of more than one nematode species and is regulated by a nematode-induced soybean promoter.
  • the construct containing the target gene is under the control of a soybean promoter, isolated, characterized and patented by our group, and which has strong root expression (Grossi-de-Sa et al., 2010), especially when these they are parasitized by M. incognita (Miranda et al., 2013), specifically at feeding sites.
  • the UceS8.3 promoter was isolated from soybean plants by TAIL-PCR with oligonucleotides designed for the ubiquitin conjugation factor 2 (GmE2) gene. This promoter was later used in genetic transformation of the Arabidopsis thaliana model plant for its functional characterization due to the expression of the GUS reporter gene, demonstrating expression in all plant tissues, especially roots (Grossi-de-Sa et al., 2010 ).
  • Real-time PCR of M. incognita-inoculated soybean roots at 7, 14, 21 and 28 DAI demonstrate 2-6-fold increased transcriptional expression of the GmE2 gene, regulated by the UceS8.3 promoter (Miranda et al., 2013) .
  • In situ hybridization demonstrated the localization of UceS8.3-related expression in giant cells adjacent to the feeding site in the galls.
  • the present invention comprises plant expression cassettes capable of silencing genes of multiple nematode species that feed on said plants conferring resistance to said nematode species. More specifically, the expression cassettes of the present invention are capable of expressing dsRNA molecules in nematode infested target tissues.
  • the present invention further provides vectors comprising said expression cassette, methods for producing a plant resistant to multiple nematode species as well as for controlling nematodes in a plantation, plants resistant to multiple species of nematode. nematodes, their seeds and products made from material extracted from such plants.
  • Figure 1 Representative scheme of gene construction used for soybean transformation aiming resistance to Meloidogyne incognita and Heterodera glycines.
  • the black arrow indicates the position of the UceS8.3 soybean ubiquitin conjugation factor promoter, isolated and characterized in LIMPP and protected by Embrapa by patent.
  • the gene construct includes sequences from the target gene splicing factor of M. incognita and H. glycines totaling 440 bp in two palindromic positions, ie reverse and complementary, to allow the formation of double stranded RNA. This palindromic region is separated by the intronic region from the RNAi vector, pKannibal, originally isolated from Arabidopsis thaliana.
  • tNOS is the transcription terminator used.
  • the red and green arrows indicate the ringing positions of oligonucleotides used for soy transformation diagnosis ( Figure 2A).
  • the sequence of oligonucleotides is in Table
  • FIG. 2 (A) Diagnosis of soybean transformation by PCR. The above image was obtained by ethidium bromide stained agarose gel electrophoresis of the PCR product with the GmFSMiHg-F and GmFSMiHg-R oligonucleotides (described in Table 1 and Figure 1). Leaf DNA was isolated and then used in PCR. The numbering corresponds to the third generation individuals (T3) obtained from the successive multiplication of the transformation event 4I T0. CN: negative control without template DNA. CP: positive control, DNA template UceS8.3 :: GmFSMiHg. (B) Acclimatization of genetically modified soybean plants.
  • the seedlings were kept in magenta for 15 days under selection with the herbicide imidazolinone. After growth, the plants were transferred to cups with organic substrate and clay and kept covered with plastic bag to maintain moisture for one week. Subsequently, the plants were transferred to 20 L plastic bags with the same substrate and kept until completing the life cycle. T1 seeds from TO were multiplied in a greenhouse until T2.
  • FIG. 3 Nematological bioassay.
  • the T3 plants were challenged with M. incognita for determination of nematode resistance induction.
  • A Graph of the outcome of the GM soy event challenge (4A, 4B, 4C, 4E, 4G and 41) and unprocessed control plant (BR-16).
  • the upper line denotes the number of plants individually tested in the resistance experiment (n) and the reduction percentage of eggs obtained (%).
  • the line above the bars denotes standard error of the experiment.
  • the letter above the bar demonstrates a statistically significant difference
  • Said cassette is capable of expressing dsRNA in plants comprising nematode gene fragments that silence conserved genes of said nematodes through the mechanism known as interference RNA (RNAi).
  • RNAi interference RNA
  • the sense and antisense sequences are separated by a separator sequence.
  • the separator region is an intron.
  • An "intron” is a nucleotide sequence that is transcribed and present in the pre mRNA, but It is removed by cleavage and re-binding of mRNA within the cell generating a mature mRNA that can be translated into a protein.
  • introns include, but are not limited to, pdk intron, castor bean intron catalase, cotton Delta 12 denaturase intron, Arabidopsis Delta 12 denaturase, maize ubiquitin intron, SV40 intron, malate synthase gene introns.
  • the spacer sequence of the present invention is a PDK intron.
  • Promoter refers to the DNA sequence in a gene, usually located upstream of the coding sequence, which controls expression of the coding sequence by promoting recognition by RNA polymerase and other factors required for transcription itself. In an artificial DNA construct, promoters may also be used to transcribe dsRNA. Promoters may also contain DNA sequences that are involved in the binding of protein factors which control the effect of transcription initiation in response to physiological or developmental conditions.
  • the promoter is a constitutive promoter.
  • promoter activity is stimulated by external or internal factors such as, but not limited to, hormones, chemical compounds, mechanical impulses, and biotic or abiotic stress conditions.
  • the promoter activity may also be regulated in a temporal and spatial manner (such as tissue-specific promoters and regulated promoters during development).
  • the promoter may contain enhancer elements.
  • An enhancer is a DNA sequence that can stimulate promoter activity. It may be an innate promoter element or a heterologous element inserted to increase the level and / or tissue specificity of a promoter.
  • Constutive promoters refers to those who drive gene expression in all tissues at all times.
  • tissue-specific or development-specific promoters are those that drive gene expression almost exclusively in specific tissues, such as leaves, roots, stems, flowers, fruits or seeds, or in stages of growth. development in a tissue, such as at the beginning or end of embryogenesis.
  • expression refers to the transcription and stable accumulation of dsRNA derived from the nucleic acid fragments of the invention which, together with the cell protein production apparatus, results in altered levels of myo-inositol 1-phosphate synthase.
  • inhibition by interference refers to the production of dsRNA transcripts capable of preventing expression of the target protein.
  • said promoter is specific to nematode-colonized or nematode-infested tissue-induced tissues.
  • the promoter has the sequence identified as SEQ ID NO: 1.
  • the sense and antisense sequences of (ii) and (iv) belong to a gene encoding a nematode splicing factor (FS).
  • FS nematode splicing factor
  • sequences of components (ii) and (iv) belong to the nematode species Heterodera glycines and Meloidogyne incognita, respectively, and more preferably the expression cassette contains the following composition:
  • the promoter terminator sequence having a sequence substantially similar to SEQ ID NO: 7.
  • the expression cassette sequence is substantially similar to SEQ ID NO: 8.
  • substantially similar refers to nucleic acid fragments in which changes in one or more nucleotide bases do not affect the ability of the nucleic acid fragment to mediate alteration of gene expression by gene silencing by antisense technology, co-suppression or interference RNA (RNAi).
  • RNAi co-suppression or interference RNA
  • Substantially similar nucleic acid fragments of the present invention may also be characterized by the percentage similarity of their nucleotide sequences to the nucleotide sequences of the nucleic acid fragments described herein (SEQ ID NO 1-8), as determined by common algorithms employed in the present invention. state of the art.
  • Preferred nucleic acid fragments are those whose nucleotide sequences have at least about 40 or 45% sequence identity, preferably about 50% or 55% sequence identity, more preferably about 60% or 65% identity. more preferably about 70% or 75% sequence identity, more preferably about 80% or 85% sequence identity, more preferably about 90%, 91%, 92%, 93%, 94 %, 95%, 96%, 97%, 98% or 99% sequence identity as compared to the reference sequence. Sequence alignment and percent similarity calculation of the present invention were performed using the DNAMAN for windows program (Lynnon Corporation, 2001) using sequences deposited with GenBank through Web browser integration.
  • dsRNA is by having the nucleotide sequence of the target gene in the sense orientation and a nucleotide sequence in the antisense orientation present in the DNA molecule, and there may or may not be a spacer region between the sense and antisense nucleotide sequences. .
  • the nucleotide sequences mentioned may consist of about 19nt to 2000nt or about 5000 nucleotides. or more, each having substantial total sequence similarity of about 40% to 100%. The longer the sequence, the less stringency is required for full substantial sequence similarity.
  • Fragments containing at least about 19 nucleotides should preferably have about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity when compared to the reference sequence, which may have about 2 distinct noncontiguous nucleotides. Preferably fragments above 60bp are used, more preferably fragments between 100 to 500bp.
  • the dsRNA molecule may comprise one or more regions having substantial sequence similarity to regions with at least about 19 consecutive nucleotides of the target gene sense nucleotides, defined as the first region, and one or more regions. regions having substantial sequence similarity to regions with about 19 consecutive nucleotides of the target gene sense nucleotide complement, defined as the second region, where these regions may have base pairs separating them from each other.
  • the invention further comprises vectors comprising the cassettes of the present invention as well as methods for producing a plant resistant to multiple nematode species, comprising inserting a cassette of the present invention into a plant cell and regenerating a plant from said plant cell.
  • the invention further comprises plants resistant to multiple species of nematodes having a cassette of the present invention integrated into their genome.
  • Plants refer to photosynthetic organisms, both eukaryotes and prokaryotes, where the term “developed plants” refers to eukaryotic plants.
  • the nucleic acids of the invention may be used to confer desired treatments on essentially any plant.
  • the invention has use over various plant species, including species of the genera Anacardium, Anona, Arachis, Artocarpus, Asparagus, Atropa, Avena, Brassica, Carica, Citrus, Citrullus, Capsicum, Carthamus, Coconuts, Coffea, Cucumis, Cucurbita, Daucus, Elaeis, Fragaria, Glycine, Gossypium, Helian- thus, Heterocallis, Hordeum, Hyoseyamus, Lactuca, Linum, Lupine, Lycopersicon, Malus, Manihot, Majorana, Medicago, Nicotiana, Olea, Oumza, Panea Pannesetum, Passiflora, Persea, Phaseolus, Pistachia, Pisum, Pyrus, Prunus, Psidium, Raphanus, Ricinus, Secale, Senecio, Sinapis, Solon, Sorghum, Trigonella, Triticum,
  • the invention further comprises seeds of a plant having a cassette of the present invention integrated with its genome as well as products produced from material extracted from a plant having a cassette of the present invention integrated with its genome, such as food and / or animal feed. .
  • the invention also comprises methods for controlling nematodes in a plantation, comprising cultivating a plant having a cassette of the present invention integrated into its genome.
  • New target genes for RNAi are selected based on the following criteria: (a) your C. elegans orthologs must be target genes. and / or have lethal phenotypes when silenced or knocked out; (b) their sequences should have high identity only with nematode species; (c) their sequences should be available in the database (GenBank, http://www.ncbi.nlm.nih.gov/nuccore) and (d) to ensure that proposed dsRNAs are not a risk to non-target organisms.
  • sequences of the gene fragments in question used for dsRNA synthesis are compared with other sequences available from GenBank using the BLAST "n (version 2.2.15) tool (ALTSCHUL, SF; GISH.W .; MILLER.W. MYERS.EW & LIPMAN, DJ Basic Local Alignment Search Tool (Journal of Molecular Bioiogy, v. 215, no. 3, pp. 403-10, Oct 5 1990).
  • Block TM RNAi Designer http: rnaidesigner.invitrogen.com/rnai-express/
  • Block TM RNAi Designer The selected gene regions in Block TM RNAi Designer were submitted to the GenBank TM database by the BLASTn (nucleotide blast) and BLASTp (protein blast) programs at NCBI's website (http://www.ncbi.nlm.nih.gov/ biast / Blast.cgi) .afinl to identify possible RNAi effects on plants, hunlallOs and other non-target organisms.
  • oligonucleotide pairs were designed for each gene.
  • the design of the oligonucleotides was performed by the program primer3 v.0.4.0 (http://frodo.wi.mitt.edu/), which suggests the best pairs within the given sequence and for the desired product size.
  • the program also provides important parameters such as Tm (melting temperature),% GC, loop formation and homodimer.
  • Tungsten particle firing with adsorbed DNA was performed in the mehstatic region of embryos from BR-16 soybean seeds.
  • the co-transformation strategy was used with UceSB.3 :: GmFSMiHg (for phyto-nematode gene silencing) and pAC321 (herbicide resistance) plasmids using the biobalistics protocol (RECH, E. L; VIANNA.GR & ARAGON, FJL High-efficiency transformation by soybean biolistics, common bean and transgenic cotton Nature Protocols, v. 3, no. 3, pp. 410-418, 2008.
  • the seeds were first sterilized in 70% ethanol for 10 minutes, followed by soaking in 50% hypochlorite for twenty minutes, and then washed three times with autoclaved distilled water in a laminar flow chamber, remaining immersed in distilled water for approximately a period. 16 hours.
  • the seeds were then incised for removal of the embryos with the aid of sterile forceps and scalpels, and stored in a petri dish with distilled water to prevent desiccation. Then, the leaf primordia were removed with the aid of magnifying glass, to expose the apical meristem region.
  • the embryos were dried under exposure to the environment on laminar flow chamber filter paper and then 5 cm diameter Petri dishes containing 11 mL of MS medium (Murashige & Skoog 1962), 3% sucrose and 0.8% phytagel and pH 5.7. Arranged in line with a 16 mm diameter circle centered on the plate (death zone), with the apical meristem region directed upwards.
  • the DNA constructs were precipitated on tungsten microparticles as an aid of CaCl2 and spermidine.
  • the introduction of the gene constructions of interest occurred through the use of the particle accelerator developed in Brazil.
  • the embryos were transferred to plates containing benzylaminopurine supplemented MS medium (BAP - 5 mg / ml), 3% sucrose, 0.6% agar and pH 5.7. , where they remained approximately 18 hours in the dark at 28 ° C for multibrot induction.
  • the embryos were then transferred to magenta containing selective medium with MS, 3% sucrose, 0.15 ⁇ Imazapyr herbicide, 0.8% agar, and vitamin B5 pH 5.7, with 9 embryos in each. magenta, which were kept in a growth chamber at 28 ° C, with 16 hours of photoperiod and luminosity 350 ⁇ mols.m -2 .s -1 and relative humidity above 80% for approximately 45 days.
  • the InGmFSMiHg-F and InGmFSMiHg-R primers (SEQ ID NO 9 and SEQ ID NO 10 - Figure 1) amplify a 135 bp fragment at the intron.
  • GmFSMiHg-F and GmFSMiHg-R primers (SEQ ID NO 1 1 and SEQ ID NO 12 - Figure 1) amplify a 440 bp fragment in the sense region.
  • PCR amplifications were performed in a Mycycler thermocycler (BioRad) with the program: initial denaturation of 1'30 "at 94 ° C, 35 denaturation cycles of 30" at 94 ° C, annealing of 30 "at 55 ° C and 45" extension at 72 ° C, followed by a final extension of 5 'at 72 ° C.
  • PCR products were separated by 1, 3% agarose gel electrophoresis, stained with bromide. etid and visualized in transluminator.
  • PCR-positive plants for both amplicons were transferred to 15L pots with soil and kept in a greenhouse.
  • the egg suspension was submitted to the Baernlann funnel technique, kept at room temperature, in a container containing distilled water to allow the eggs to hatch and subsequently to collect the nematodes.
  • the collection of hatched J2 was performed over a week every two days.
  • Second generation (T3) progenies at the 2-3 trifolium stage, were planted in pots containing 300 mL of soil, which were inoculated with a population of approximately 1,000 J2 M.incognita 1.
  • Plants of cultivar BR-16 (not transgenic), which has no resistance to M.incognita race 1, were planted and inoculated serving as a control. The plants remained in a greenhouse for 6 weeks and were irrigated when necessary.
  • Soybean roots were processed individually for egg extraction 45 days after inoculation (DAI). The roots were individually washed for soil removal, dried with paper towels, weighed, ground in a 0.5% NaCIO blender for 2 minutes, washed with a water jet and the eggs separated in a 500 Mesh sieve.
  • DAI inoculation
  • the final egg suspension volume was corrected to 30 mL, and three 1 mL aliquots were withdrawn. After counting eggs using the microscope and Peters slide, the count was normalized to root mass, determining the number of gl and root eggs.
  • Each plant was inoculated with approximately 1000 J2 obtained in hatching system. Each treatment consisted of 6 to 10 repetitions. The challenge was completely repeated twice at different times. Approximately six weeks after inoculation, the roots of the plants were individually extracted and processed to determine the number of eggs. root gl. All values of the bioassays were relative to the control treatment to normalize the data between the biological repetitions and to enable the statistical analysis. This normalization was necessary because the data related to phy- nonematoid infection vary greatly from one experiment to another and may cause misinterpretation of these data.

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Abstract

La présente invention concerne des cassettes d'expression dans des plantes pouvant opérer le silençage génique de multiples espèces de nématoïdes s'alimentant avec lesdites plantes, conférant une résistance auxdites espèces de nématoïdes. Plus particulièrement, les cassettes d'expression de la présente invention peuvent exprimer les molécules d'ARN double brin dans des tissus cibles d'infestation par des nématoïdes. L'invention concerne également des vecteurs comprenant ladite cassette d'expression, des procédés pour la production d'une plante résistante à de multiples espèces de nématoïdes et pour la lutte contre les nématoïdes dans une plantation, des plantes résistantes à de multiples espèces de nématoïdes, leurs graines et les produits obtenus à partir d'un matériel extrait desdites plantes.
PCT/BR2013/000203 2013-06-11 2013-06-11 Cassette d'expression pour induire une résistance à de multiples espèces de nématoïdes dans des plantes, procédés et plantes l'utilisant WO2014197951A2 (fr)

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BR112015024341-0A BR112015024341B1 (pt) 2013-06-11 2013-06-11 Cassete de expressão para indução de resistência a múltiplas espécies de nematoides em plantas, métodos e plantas que o utilizam

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CN106577796A (zh) * 2016-11-22 2017-04-26 山东省农业科学院蔬菜花卉研究所 蓖麻粕防治番茄根结线虫病方法

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US20080184391A1 (en) * 2007-01-29 2008-07-31 Kuppuswamy Subramaniam Pathogen resistant transgenic plants, associated nucleic acids and techniques involving the same
BRPI0701172B1 (pt) * 2007-02-05 2019-11-26 Empresa Brasileira De Pesquisa Agropecuaria Embrapa composições e métodos para modificar a expressão de genes usando o promotor do gene da proteína de conjugação à ubiquitina de plantas de soja

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CN106577796A (zh) * 2016-11-22 2017-04-26 山东省农业科学院蔬菜花卉研究所 蓖麻粕防治番茄根结线虫病方法

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