WO2014197951A2 - Expression cartridge for inducing resistance to multiple nematoid species in plants, method and plants using said expression cartridge - Google Patents

Expression cartridge for inducing resistance to multiple nematoid species in plants, method and plants using said expression cartridge 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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PCT/BR2013/000203
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French (fr)
Portuguese (pt)
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WO2014197951A8 (en
WO2014197951A3 (en
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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 PCT/BR2013/000203 priority patent/WO2014197951A2/en
Publication of WO2014197951A2 publication Critical patent/WO2014197951A2/en
Publication of WO2014197951A8 publication Critical patent/WO2014197951A8/en
Publication of WO2014197951A3 publication Critical patent/WO2014197951A3/en

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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.

Abstract

The present invention provides expression cartridges for plants, capable of silencing genes of multiple nematoid species that feed on said plants, thus imparting resistance to said nematoid species. More specifically, the expression cartridges according to the present invention can express dsRNA molecules in tissues that are the target of nematoid infestation. Also provided are vectors comprising said expression cartridge, methods for producing a plant that is resistant to multiple nematoid species, and also methods for controlling nematoids in a plantation, plants that are resistant to multiple nematoid species, seed of these plants and products produced from material extracted from said plants.

Description

CASSETE DE EXPRESSÃO PARA INDUÇÃO DE RESISTÊNCIA A MÚLTIPLAS ESPÉCIES DE NEMATOIDES EM PLANTAS, MÉTODOS E PLANTAS QUE O UTILIZAM  EXPRESSION CASSETTE FOR INDUCTION OF RESISTANCE TO MULTIPLE NEMATOID SPECIES IN PLANTS, METHODS AND PLANTS USING IT
Campo da invenção  Field of the invention
A presente invenção refere-se ao controle de nematoides em plantas. Mais especificamente, a presente invenção refere-se ao controle de múltiplas espécies de nematoides por silenciamento gênico através da expressão de uma molécula de RNA na planta capaz de formar um RNA dupla fita compreendendo um fragmento da sequência de genes alvo da peste. Fundamentos da Invenção  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. Background of the Invention
Fitonematoides estão entre os principais fatores responsáveis pela queda do rendimento de diversas culturas, particularmente a da soja, especialmente nas regiões tropicais e subtropicais.  Phytonematoids are among the main factors responsible for the fall in yield of several crops, particularly soybean, especially in tropical and subtropical regions.
O controle de nematoides em culturas de escala, como a soja, é planejado de modo a integrar vários métodos e apresentar baixo custo. De um modo geral, são considerados os princípios fitopatológicos da exclusão (evitar a infestação de áreas indenes por espécies ou novas raças, na propriedade ou numa região geográfica maior); da erradicação (rotação de culturas com espécies de verão e de inverno não hospedeiras); da regulação (modificação do ambiente e nutrição das plantas); e da imunização (utilização de cultivares resistente a determinadas espécies ou raças). As atuais estratégias para controle de nematoides são: uso de nematicidas, rotação de culturas, resistência genética natural e engenharia genética.  The control of nematodes in scale crops, such as soybeans, is designed to integrate various methods and be cost effective. Generally, 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.
Nematicidas têm sido amplamente utilizados para controle de nematoides parasitas de plantas sedentários e migratórios, mas estes compostos são frequentemente associados a efeitos prejudiciais ao meio ambiente.  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.
A rotação de culturas, uma prática cultural efetiva para muitos estresses bióticos, também é uma estratégia importante para manejo de nematoides parasitas de plantas, embora, em muitos casos, sua eficácia é limitada.  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.
O manejo de fitonematoides depende muito da resistência da planta hospedeira obtida por métodos tradicionais de melhoramento, muitas vezes derivada de uma base genética limitada. O melhoramento convencional, no entanto, apresenta algumas limitações, como por exemplo, a ligação gênica entre genes de interesse e genes indesejáveis e a incompatibilidade interespecífica. 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, however, has some limitations, such as the genetic link between genes of interest and undesirable genes and interspecific incompatibility.
A engenharia genética é uma ferramenta extremamente útil, pois permite a identificação de genes de interesse, manipulação desses genes, a construção e introdução de um único gene de interesse diretamente em cultivares elite e a seleção das plantas que possuem esse gene. O gene a ser introduzido pode ser oriundo da mesma espécie ou de outras espécies, permitindo assim a quebra das barreiras impostas pela incompatibilidade sexual entre as diferentes espécies, além de eliminar o efeito das ligações gênicas indesejadas.  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.
A estratégia de silenciamento gênico, por interferência mediada por RNA dupla fita, oferece bons resultados no controle de nematoides (MCCARTER, J. Molecular approaches toward resistence to plant parasitic nematodes. In: (Ed.). Plant Celi Monographs v.15, 2008. p.239-267.). Tal estratégia se baseia na transformação de plantas para a expressão de RNA dupla fita (dsRNA) compreendendo um fragmento da sequência específica de genes-alvos do fitonematoide. Genes alvo de interesse são genes essenciais ao fitonematoide ou genes envolvidos com o parasitismo, migração, formação ou manutenção do sítio de alimentação. Durante o ciclo dos nematoides, estes ingerem o conteúdo citoplasmático das células gigantes de raízes infectadas, provocando a absorção do dsRNA, o que pode resultar no silenciamento do gene correspondente. Conforme a função do gene silenciado, diversas disfunções podem ser geradas no fitonematoide pelo silenciamento e/ou redução da expressão de genes específicos, de forma que a infecção pode ser abortada.  The gene silencing strategy by double-stranded 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.). Such a strategy is based on the transformation of plants to double-stranded RNA (dsRNA) expression comprising a fragment of the specific phytonematoid target gene sequence. Target genes of interest are genes essential to phytonematoid or genes involved with parasitism, migration, formation or maintenance of the feeding site. 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.
Interferência mediada por RNA (RNAi) refere-se à redução es- pecífica da expressão do gene através da utilização de moléculas de RNA de sequência complementar. Este fenómeno, primeiramente relatado em Caenorhabditis elegans por GUO & KEMPHUES (GUO.S. & KEMPHUES, K. J. par-l, a gene required for establishing polarity in C. elegans embryos, en- codes a putative Ser/Thr kinase that is asymmetrically distributed. Cell, v. 81 , n. 4, p. 611-620, 1995) e subsequentemente demonstrado por FIRE et al. (FIRE, A; XU.S.; MONTGOMERY.M. K.; KOSTAS.S. A; DRIVER, S. E. & MELLO, C.C. Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature, v. 391 , n. 6669, p. 806-11 , Feb 19 1998), que descobriram que a presença do RNA fita dupla (dsRNA), formado a partir do anelamento das fitas senso e antisenso presente nas preparações de RNA in vitro, é responsável pelo silenciamento gênico. RNA-mediated interference (RNAi) 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. (FIRE, A; XU.S .; MONTGOMERY.MK; KOSTAS.S.; DRIVER, SE & MELLO, CC. Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature, v. 391, No. 6669 , pp. 806-11, Feb 19 1998), who found that the presence of double stranded RNA (dsRNA), formed from the annealing of sense and antisense strands present in in vitro RNA preparations, is responsible for gene silencing.
Posteriormente, BERNSTEIN et al. (BERNSTEIN, E.; CAUDY.A. Subsequently, BERNSTEIN et al. (BERNSTEIN, E .; CAUDY.A.
A; HAMMOND,S. M. & HANNON, G. J. Role for a bidentate ribonuclease in the initiation step of RNA interference. Nature, v. 409, n. 6818, p. 363-6, Jan 18 2001 ), indicaram que a DICER ("dsRNA-specific RNase lll-type endonu- clease") uma enzima ribonuclease tipo III (RNase III) era responsável pelo processamento do dsRNA em sequências de -21 nucleotídeos (nt). O complexo DCR2/R2D2 liga-se a essas pequenas moléculas de RNA interferente (siRNA) (LIU, Q.; RAND, T. A.; KALIDAS.S.; DU, F.; KIM, H. E.; SMITH, D. P. & WANG, X. R2D2, a bridge between the initiation and effector steps of the DrosophiJa RNAi pathway. Science, v. 301 , n. 5641 , p. 1921 -5, Sep 26 2003), o siRNA é incorporado em um complexo chamado RISC (RNA Indu- ced Silencing Complex),que então determina a degradação de quaisquer moléculas de RNA que possuem sequência complementar (HAMMOND, S. M.; BERNSTEIN, E.; BEACH, D. & HANNON, G. J. An RNA-directed nuclease mediates post-transcriptional gene silencing in Drosophila cells. Nature, v. 404, n. 6775, p. 293-6, Mar 16 2000). Este fenómeno de silenciamento do gene ocorre em vários organismos eucariotos, incluindo os nematoides e as plantas superiores (BERNSTEIN, E.; CAUDY.A. A.; HAMMOND, S. M. & HANNON, G. J. Role for a bidentate ribonuclease in the initiation step of RNA interference. Nature, v. 409, n. 6818, p. 363-6, Jan 18 2001). THE; HAMMOND, S. M. & HANNON, G. J. Role for a bidentate ribonuclease in the initiation step of RNA interference. Nature, v. 409, no. 6818, p. 363-6, Jan 18 2001), indicated that DICER ("dsRNA-specific RNase lll-type endonuclease") a ribonuclease enzyme type III (RNase III) was responsible for processing dsRNA in -21 nucleotide sequences (nt ). 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. & HANNON, GJ. An 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).
Pesquisadores têm sido capazes de adicionar RNA sintetizado in vitro diretamente nas células para se obter um silenciamento da expressão gênica. Por exemplo, KLINK& WOLNIAK (KLINK, V. P. & WOLNIAK, S. M. Centrin is necessary for the formation of the motile apparatus in spermatids of Marsilea. Mol Biol Celi, v. 12, n. 3, p. 761-76, Mar 2001 ) foram capazes de silenciar o mRNA de centrina (centrin) usando dsRNA sintetizado in vitro, e para os efeitos de nocaute, eles demonstraram que o dsRNA é pelo menos dez vezes mais eficaz do que o RNA senso ou antisenso separadamente. Para alcançar o controle de nematoides parasitas de plantas usando a estratégia de silenciamento gênico, o mecanismo de RNAi é parcialmente executado pela planta e parcialmente pelo nematoide. Plantas expressam dsRNA de genes do nematoide, que são processados pela DICER e geram os siR- NA. Quando os nematoides se alimentam dessas plantas, tanto dsRNA quanto siRNA são ingeridos. Researchers have been able to add in vitro synthesized RNA directly to cells to achieve silencing of gene expression. For example, 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. To achieve control of plant parasitic nematodes using the gene silencing strategy, the 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.
Como ocorrido na planta, os nematoides digerem o dsRNA em siRNA usando seu complexo DICER. Os siRNA ingeridos, ou processados pelo próprio nematoide, ligam-se a RISC para induzir a degradação do mR- NA específico no nematoide. Os siRNA são amplificados em nematoides pela RNA polimerase dependente de RNA (RDRP) (CHAPMAN, E. J. & CARRINGTON.J. C. Specialization and evolution of endogenous small RNA pathways. Nature Reviews Genetics, v. 8, n. 11 , p. 884-96, Nov 2007; ZA- MORE, P. D. &.HALEY, B. Ribo-gnome: the big world of small RNAs. Scien- ce, v. 309, n. 5740, p. 1519-24, Sep 2 2005), onde o mRNA serve de molde para a síntese de mais dsRNA específico, elevando o efeito do silenciamento gênico.  As in the plant, 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.
Este silenciamento mediado por siRNA é altamente sequência- específica. Por exemplo, Tuschl e colaboradores demonstraram que mesmo uma incompatibilidade de uma única base, entre o siRNA e o mRNA alvo influencia o silenciamento gênico (ELBASHIR, S. M.; MARTINEZ, J.; PAT- KANIOWSKA,A.; LENDECKEL,W. & TUSCHL.T. Functional anatomy of siR- NAs for mediating efficient RNAi in Drosophila melanogaster embryo lysate. The EMBO Journal, v. 20, n. 23, p. 6877-88, Dec 3 2001 ). Apesar do silenci- amento gênico ser altamente específico, é possível silenciar famílias de genes homólogos que compartilham sequências conservadas (MIKI, D.; ITOH, R. & SHIMAMOTO, K. RNA silencing of single and multi pie members in a gene family of rice. Plant physiology, v. 138, n. 4, p. 1903-13, Aug 2005) ou com a construção quimérica de vários genes-aivo (ALLEN, R. S.; MILLGA- ΤΕ,Α. G.; CH(TTY,J. A.;THISLETON, J.; MILLER, J. A.; FIST, A. J.; GERLA- CH,W. L.& LARKIN, P. J. RNAi-mediated replacement of morpl1ine with the nonnarcotic alkaloid reticuline in opium poppy. Nature Biotechnology, v. 22, n. 12, p. 1559-66, Dec 2004). Tem sido documentado que o efeito do RNAi, não é só difundido de célula para célula (FAGARD, M. & VAUCHERET, H. Systemic silencing signal(s). Plant Molecular Biology, v. 43, n. 2-3, p. 285-93, Jun 2000; KEHR, J. & BUHTZ.A. Long distance transport and movement of RNA through the phloem. Journal of Experimental Botany, v. 59, n. 1 , p. 85- 92, 2008), mas também em toda a planta (YOO, B. C; KRAGLER.F.; VAR- KONYI-GASIC,E.; HAYWOOD,V.;ARCHER-EVANS, S.; LEE, Y. M.; LOU- GH,T. J.& LUCAS.W. J.A systemic small RNA signaling system in plants. Plant Celi, v. 16, n. 8, p. 1979-2000, Aug 2004). This siRNA-mediated silencing is highly sequence-specific. For example, 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). Although 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-ΤΕ, 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. Systemic silencing signal (s). Plant Molecular Biology, v. 43, no. 2-3, p. 285-93, Jun 2000 KEHR, J. & BUHTZ.A Long distance transport and movement of RNA through the phloem. Journal of Experimental Botany, v. 59, no. 1, pp. 85-92, 2008), but also throughout the plant ( YOO, B.C.KRAGLER.F .; VAR-KONYI-GASIC, E.; HAYWOOD, V.; ARCHER-EVANS, S.; LEE, YM; LOUCH, TJ & LUCAS.WJA systemic small RNA signaling system in Plant Celi, v. 16, no. 8, pp. 1979-2000, Aug 2004).
Evidências indiretas indicam que o silenciamento de RNA move- se a longas distâncias através do floema e, no destino, espalha-se célula a célula através dos plasmodesmos em tecidos recipientes (JORGENSEN, R. A. RNA traffics information systemically in plants. Proceedings of the National Academy of Sciences, v. 99, n. 18, p. 11561-3, Sep 3 2002; MLOTSH- WA, S.; VOINNET, O.; METTE, M. F.; ΜΑΤΖΚΕ,Μ.; VAUCHERET, H.; DING, S. W.; PRUSS, G.& VANCE.V. B. RNA silencing and the mobile silencing signal. Plant Cell, v. 14 Suppl, p. S289-301 , 2002; VOINNET, O.; VAIN, P.; ANGELL,S. & BAULCOMBE,D. C. Systemic Spread of Sequence-Specific Transgene RNA Degradation in Plants Is Initiated by Localized Introduction of Ectopic Promoterless DNA. Celi, v. 95, n. 2, p. 177-187, 1998), também HIMBER et al (HIMBER, C; DUNOYER, P.; MOISSIARD, G.; RITZENTHA- LER, C.& VOINNET.O. Transitivity-dependent and -independent cell-to-cell movement of RNA silencing. The EMBO Journal, v. 22, n. 17, p. 4523-33, Sep 1 2003) demonstraram que o efeito RNAi pode ocorrer tanto localmente quanto a longas distâncias. 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 bo- tany, v. 55, n. 399, p. 983-92, May 2004) também constatou que o sinal de silenciamento foi transportado sistematicamente a partir de raízes de Arabidopsis thaliana até a parte aérea, embora o grau de silenciamento tenha si- do limitado e muito variável. Embora a transformação de nematoides parasitas de plantas represente uma abordagem para a implantação como estratégia de RNAi para controle de nematoides, não há relatos de sucesso com esta engenharia de nematoides, em parte pela sua natureza obrigatória de parasitismo. Além de inúmeros obstáculos regulatórios para liberar esse ne- matoide transgênico para o meio ambiente. Em C. elegans, RNAi tem sido utilizado transientemente para silenciar a expressão de quase todos os genes, induzindo diferentes efeitos fenotípicos incluindo letalidade (FRASER, A. G.;KAMATH,R. S.; ZIPPERLEN, P.; MARTI NEZ-CAMPOS, M.; SOHR- MANN, M. & AHRINGER, J. Functional genomic analysis of C. elegans c- hromosoll1e I by systematic RNA interference. Nature, v. 408, n. 6810, p. 325-30, Nov 16 2000; GONCZY.P.; ECHEVER- RI,C.;OEGEMA,K.;COULSON,A.; JONES, S. J.; COPLEY.R. R.; DUPERON, J.; OEGEMA.J.; BREHM, M.; CASSIN,E.;HANNAK, E.; KIRKHAM, M.; PIC- HLER, S.; FLOHRS, K.; GOESSENA; LEIDEL, S.;ALLEAUME,A. M.; MAR- TIN, C; OZLU, N.; BORK.P. & ΗΥΜΑΝ,Α. A. Functional genolnic analysis of cell division in C. elegans using RNAi of genes on cllrolHOsome 1 11. Nature, v. 408, n. 6810, p. 331-6, Nov 16 2000.; KAMATH, R. S.; FRASER.A. G.; DONG.Y.; POULIN, G.; DURBIN, R.; GOTTA, M.; KANAPIN, A.; LE BOT, N.; MORENO, S.; SOHRMANN,M.;WELCHMAN, D. P.; ZIPPERLEN, P. & A- HRINGER, J. Systematic functional analysis of the Caenorhabditis elegans genome using RNAi. Nature, v. 421 , n. 6920, p. 231 -7, Jan 16 2003; MAE- DA, 1.;KOHARA,V.;VAMAMOTO,M.& SUGIMOTO.A. Large-scale analysis of gene function in Caenorhabditis elegans by high-throughput RNAi. Current biology: CB, v. 1 1 , n. 3, p. 171-176, 2001 ). A interferência mediada por RNA pode ser realizada em C. elegans por ingestão de bactérias expressando dsRNA (TIMMONS, L. & FIRE, A. Specific interference by ingested dsRNA. Nature, v. 395, n. 6705, p. 854, Oct 29 1998), por absorção oral de dsRNA a partir da solução (TABARA, H.; GRISHOK, A. & MELLO, C. C. RNAi in C. elegans: soaking in the genome sequence. Science, v. 282, n. 5388, p. 430- 1 , Oct 161998) Ou por microinjeção (MELLO, C. C.& CONTE, D., JR. Revea- ling the world of RNA interference. Nature, v. 431 , n. 7006, p. 338-42, Sep 16 2004). Considerando os nematoides parasitas de plantas, o protocolo de ingestão de bactérias expressando dsRNA não é viável porque eles se alimentam apenas do conteúdo citoplasmático do sítio de alimentação. O protocolo inicialmente utilizado para administração de dsRNA para fitonematoi- des foi o da imersão de nematoides em solução de dsRNA para induzir in- gestão e/ou absorção. Desde 2006, moléculas de dsRNA têm sido produzidas e oferecidas diretamente pela planta hospedeira geneticamente modificada. Indirect evidence indicates that 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; ΜΑΤΖΚΕ, Μ; 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. & BAULCOMBE, DC Systemic Spread of Sequence-Specific Transgene RNA Degradation in Plants Is Initiated by Localized Introduction of Ectopic Promoterless DNA Celi, v. 95, no. 2, pp. 177-187, 1998), also HIMBER et al (HIMBER, C; DUNOYER, P.; MISSIARD, G .; RITZENTHER, C. & VOINNET.O Transitivity-dependent and -independent cell-to-cell movement of RNA silencing.The EMBO Journal, v. 22, no. 4523-33, Sep 1 2003) demonstrated that the RNAi effect can occur both locally and over long distances. 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. Although transformation of plant parasitic nematodes represents an approach to implantation as an RNAi strategy for nematode control, there are no reports of success with this nematode engineering, in part because of its obligatory nature of parasitism. In addition to numerous regulatory obstacles to releasing this transgenic nematode into the environment. In C. elegans, 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. elegans c-hromosol I by systematic RNA interference Nature, v. 408, no. 6810, pp. 325-30, Nov 16 2000; GONCZY.P .; ECHEVER-RI, C.; OEGEMA, K.; COULSON, A.; JONES, SJ; COPLEY.RR; DUPERON, J.; OEGEMA.J .; BREHM, M .; CASSIN, E.; HANNAK, E .; KIRKHAM, M .; PICHLER, S.; FLOHRS, K.; GOESSENA; LEIDEL, S.; ALLEAUME, AM; MARTIN, C; OZLU, N.; BORK.P. & A., A. Genetic analysis of cell division in C. elegans using RNAi of genes on chlrolHOsome 11 11. Nature, v. 408, No. 6810, pp. 331-6, Nov 16 2000 .; KAMATH, RS; FRASER.AG; DONG. Y ;; POULIN, G .; DURBIN, R.; GOTTA, M .; KANAPIN, A.; LE BOT, N .; MORENO, S.; SOHRMANN, M.; WELCHMAN, DP; ZIPPERLEN, P. & A- HRINGER, J. Systematic functional analysis of t he Caenorhabditis elegans genome using RNAi. Nature, v. 421, no. 6920, p. 231-7, Jan 16 2003; MAEDA, 1.; KOHARA, V.; VAMAMOTO, M. & SUGIMOTO.A. Large-scale analysis of gene function in Caenorhabditis elegans by high-throughput RNAi. Current biology: CB, v. 11, no. 3, p. 171-176, 2001). 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. 395, no 6705, p 854, Oct 29 By oral absorption of dsRNA at from the solution (TABARA, H .; GRISHOK, A. & MELLO, CC RNAi in C. elegans: soaking in the genome sequence. Science, v. 282, no. 5388, p. 430-1, Oct 161998) Or by microinjection (MELLO, CC & CONTE, D., JR. Reveal the world of RNA interference. Nature, v. 431, no. 7006, p. 338-42, Sep 16 2004). Considering plant parasitic nematodes, the ingestion protocol of bacteria expressing dsRNA is not viable because they feed only on the cytoplasmic content of the feeding site. The protocol initially used for 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.
Os nematoides formadores de galhas (NFGs) e nematoides formadores de cisto (NCs) são obrigatoriamente parasitas de raízes de plantas, tornando a oferta de dsRNA pelo hospedeiro uma estratégia ideal para silenciar genes de nematoides, bem como fornecer evidências diretas da função desses genes. Pesquisas anteriores, confirmam a viabilidade e efetividade do oferecimento de RNAi pelo hospedeiro para controle de nematoides. YA- DAV et al. (YADAV,B. C.;VELUTHAMBI,K.& SUBRAMANIAM.K. Host- generated double stranded RNA induces RNAi in plant-parasitic nematodes and protects the host from infection. Molecular and Biochemistry Parasito- logy, v. 148, n. 2, p. 219-22, Aug 2006) relataram que induziu RNAi utilizando dsRNAs de dois genes que codificam para integrase e fator de processamento de mRNA de M. incógnita, levando a proteção contra infecção por nematoides em plantas de fumo.  Gall-forming nematodes (NFGs) 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) reported that induced RNAi using dsRNAs of two genes encoding integrase and M. incognita mRNA processing factor, leading to protection against nematode infection in tobacco plants.
A expressão de dsRNA do gene de parasitismo 16D10 de NFG em plantas transgênicas de Arabidopsis, resultou em resistência contra quatro das principais espécies desse género (HUANG, G.;ALLEN, R. S.; DAVIS, E. L; ΒΑUΜ,Τ. J. & HUSSEY.R. S. Engineering broad root-knot resis- tance in transgenic plants by RNAi silencing of a conserved and essential root-knot nematode parasitism gene. n. 0027-8424 (Print), 20060927 DCOM- 20061030 2006), enquanto (SINDHU, A. S.; MAIER, T. R.; MITCHUM, M. G.; HUSSEY, R. S.; DAVIS, E. L. & BAUM, T. J. Effective and specific in planta RNAi in cyst nenlatodes: expression interference of four parasitism genes reduces parasitic success. Journal of Experimental Botany, v. 60, n. 1 , p. 315-24, 2009) obtiveram reduções de 23% a 64%, em fêmeas de H. schach- tii, em linhagens transgênica de Arabidopsis expressando o dsRNA de quatro genes de parasitismo. RNA de interferência parece ser igualmente eficaz contra H. glycines em linhagens de soja transformadas. RYAN et aí. (RYAN.M. S.; TIM, C. T.; JULIANE.S. E. & HAROLD.N. T. Transgenic soy- beans expressing siRNAs specific to a major spern1 protein gene suppress Heterodera glycines reproduction. Functional Plant Biology, v. 33, n. 1 1 , p. 991-991 , 2006) produziram com sucesso linhagens transgênicas de soja, usando a estratégia de RNAi específico de uma proteína principal do esperma de H. glycines. Dados do bioensaio indicaram plantas transgênicas com redução em até 68% de ovos/g de tecido da raiz. The expression of 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; ΒΑUΜ, Τ. 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. 0027-8424 (Print), 20060927 DCOM-20061030 2006), while (SINDHU, AS MAIER, TR; MITCHUM, MG; HUSSEY, RS; DAVIS, EL & BAUM, TJ Effective and specific in plant RNAi in cyst nenlatodes: expression interference of four parasitism genes decreases parasitic success. Journal of Experimental Botany, v. 60, no. 1, p. 315-24, 2009) achieved 23% to 64% reductions in H. schachtii females in transgenic Arabidopsis strains expressing the dsRNA of four parasitism genes. Interference RNA appears to be equally effective against H. glycines in transformed soybean strains. RYAN et al. (RYAN.MS; TIM, CT; JULIANE.SE & HAROLD.NT Transgenic soybeans expressing siRNAs specific to a major spern1 protein suppression gene Heterodera glycines reproduction. Functional Plant Biology, v. 33, no. 11, p. 991 -991, 2006) successfully produced transgenic soybean strains using the strategy of RNAi specific to a major protein of H. glycines sperm. Bioassay data indicated transgenic plants with up to 68% reduction in eggs / g of root tissue.
Os efeitos das moléculas de dsRNA expresso pela planta, se mostraram contínuos, aparecendo nas próximas gerações. KLINK et al (KLINK, V. P.; KIM, K. H.; MARTINS.V.; MACDONALD.M. H.; BEARD, H. S.; ALKHAROUF.N. W.; LEE, S. K.; PARK, S. C.& MATTHEWS, B. F.A correla- tion 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. Planta, v. 230, n. 1 , p. 53-71 , Jun 2009) inibiram efetivamente a formação de cistos de H. glycines usando uma construção com repetições invertidas de vários genes. Recentemente, LI et al. (LI, J.;TODD, T. C; OAKLEV.T. R.; LEE, J.& TRICK, H. N. Host-derived suppression of nematode reproductive and fitness genes decreases fecun- dity of Heterodera glycines Ichinohe. Planta, v. 232, n. 3, p. 775-85, Aug 2010a); LI et al. (LI, J.; TODD, T. C. &TRICK, H. N. Rapid in planta evaluati- on of root expressed transgenes in chimeric soybean plants. Plant Celi Re- ports, v. 29, n. 2, p. 1 13-23, Feb 2010b) suprimiram com sucesso a reprodu- ção de H. glycines em plantas de soja por RNAi para três diferentes genes de H. glycines.  The effects of dsRNA molecules expressed by the plant were continuous, appearing in the next generations. 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. (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, n 3, pp 775-85, Aug 2010a); LI et al. (LI, J .; TODD, TC & TRICK, HN Rapid in plant evaluation of root expressed transgenes in chimeric soybean plants. Plant Celi Portions, v. 29, no. 2, pp. 13-23, Feb 2010b ) successfully suppressed H. glycines reproduction in soybean plants by RNAi for three different H. glycines genes.
Evidências moleculares diretas de oferecimento de RNAi pelo hospedeiro regulam para baixo a expressão de genes-alvo de nematoides, observado por análise de PCR em tempo real (RT-qPCR) de nematoides que se alimentaram de raízes transgênicas (LI, J.;TODD, T. C; OAKLEV.T. R.; LEE, J.& TRICK, H. N. Host-derived suppression of nematode reproducti- ve and fitness genes decreases fecundity of Heterodera glycines Ichinohe. Planta, v. 232, n. 3, p. 775-85, Aug 2010a; SINDHU, A. S.; MAIER, T. R.; MITCHUM, M. G.; HUSSEY, R. S.; DAVIS, E. L & BAUM, T. J. Effective and specific in planta RNAi in cyst nenlatodes: expression interference of four parasitism genes reduces parasitic success. Journal of Experimental Botany, v. 60, n. 1 , p. 315-24, 2009). Plantas transgênicas expressando dsRNAs de genes de nematoides têm demonstrado maior sucesso em NFGs do que em NCs. Uma provável razão pode ser a facilidade de ingestão de moléculas em NFGs devido ao seu maior limite de exclusão por tamanho do que em NCs. Direct molecular evidence of 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. L & BAUM, TJ Effective and specific in plant RNAi in cyst nenlatodes: expression interference of four parasitism genes, parasitic success Journal of Experimental Botany, v. 60, No. 1, pp. 315-24, 2009). Transgenic plants expressing nematode gene dsRNAs have been shown to be more successful in NFGs than in NCs. A likely reason may be the ease of ingestion of molecules in NFGs due to their larger size exclusion limit than in NCs.
Construções de RNA capazes de formar dsRNA para da inibição de genes de nematoides por meio de RNA de interferência e consequente indução de resistência a tais pragas na planta, bem como construções de DNA capazes de expressar tais construções de RNA na planta estão disponíveis (EP2330203).  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, as well as DNA constructs capable of expressing such plant RNA constructs are available (EP2330203) .
WO2011060151 ensina vetores compreendendo construções para a indução de resistência a nematoides, dentre eles Meloidogyne incógnita e Heterodera glycines, codificando fitas de RNA dupla-fita que inibem a expressão de um gene da peste alvo.  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 ensina, dentre outros, métodos para a produção de plantas transgênicas resistentes a nematoides compreendendo a co- expressão na planta de moléculas de RNA dupla-fita tendo como alvo genes de diferentes espécies, dentre elas Meloidogyne incógnita e Heterodera glycines. WO2006045591 ensina ainda que a transferência de dsRNA para a peste pode ser melhorada através do uso de promotores tecido-específicos para as partes mais acessíveis à peste, como, dentre outros, promotores específicos de raiz ou promotores induzidos por nematoides.  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.
O silenciamento do gene do fator de splicing Prp-17 de Heterodera glycines através da expressão de siRNAs em soja transgênica foi eficaz na indução de resistência à peste (Li, J.; Todd, T. C; Oaklev,T. R.; Lee, J.& Trick, H. N. Host-derived suppression of nematode reproductive and fitness genes decreases fecundity of Heterodera glycines Ichinohe. Planta, v. 232, n. 3, p. 775-85, Aug 2010). 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).
A expressão de dsRNA do fator de splicing (FS) de M. incógnita obteve uma redução de 90% de nematoides (M. incógnita) estabelecidos nas raízes (Yadav, B.C.; Veluthambi, K. & Subramaniam, K. Host-generated double stranded RNA induces RNAi in plant-parasitic nematodes and pro- tects the host from infection. Molecular and Bioehemistry Parasitolology, v. 148,n. 2, p. 219-22, Aug 2006).  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).
A expressão de dsRNA de um gene relacionado ao FS de H. glycines reduziu o número de fêmeas colonizando as raízes a menos de 20%. (Klink, V. P. & Matthews, B. F. Emerging Approaches to Broaden Re- sistance of Soybean to Soybean Cyst Nematode as Supported by Gene Ex- pression Studies. Plant Physiol. 2009 November; 151 (3): 1017-1022).  The expression of dsRNA from an H. glycines FS-related gene reduced the number of females colonizing the roots to less than 20%. (Klink, V.P. & Matthews, B.F. Emerging Approaches to Broaden Resistance of Soybean to Soybean Cyst Nematode as Supported by Gene Expression Studies. Plant Physiol. 2009 November; 151 (3): 1017-1022).
PI0701172-5 descreve um promotor constitutivo de soja, o promotor do gene da proteína de conjugação a ubiquitina de soja (UceS 8.3).  PI0701172-5 describes a constitutive soybean promoter, the soybean ubiquitin conjugation protein gene promoter (UceS 8.3).
O promotor UceS8.3 é induzido pela invasão de raízes por M. incógnita (Miranda, V. J. Caracterização da expressão do gene codificador da enzima de conjugação a ubiquitina (E2) em soja inoculada com Meloi- dogyne incógnita e infestada com Anticarsia gemmatalis. 201 1. Magister Science. Biologia Celular, Universidade de Brasília).  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).
No entanto, há no campo da promoção de resistência contra nematoides via RNA de interferência a necessidade de construções capazes de expressar moléculas de dsRNA que silenciem genes de nematoides apenas em tecidos alvo de infestação por nematoides.  However, in the field of promoting resistance against interfering RNA via nematodes there is a need for constructs capable of expressing dsRNA molecules that silence nematode genes only in nematode-infested target tissues.
As construções para expressão de RNA de interferência já conhecidas, utilizam promotores constitutivos, geralmente isolados de vírus, como o CaMV 35S para uma expressão constitutiva do dsRNA.  Known interference RNA expression constructs utilize constitutive promoters, generally isolated from viruses, such as CaMV 35S for constitutive dsRNA expression.
Há, portanto, a necessidade de construções eficientes no silenciamento de genes de nematoides para a indução de resistência a nematoides em culturas capazes de expressar as moléculas de dsRNA sob controle de um promotor específico de planta. There is therefore a need for efficient nematode gene silencing constructs for the induction of nematode resistance in cultures capable of expressing dsRNA molecules under control. of a plant specific promoter.
A presente invenção tem como diferencial ser um vetor de expressão de dsRNA específico para genes-alvo de mais de uma espécie de nematoide e por ser regulada por um promotor de soja induzido por nema- toides. A construção contendo o gene-alvo está sob controle de um promotor de soja, isolado, caracterizado e patenteado pelo nosso grupo, e que possui forte expressão na raiz da planta (Grossi-de-Sa et al.,2010), especialmente quando essas estão parasitadas por M. incógnita (Miranda et al., 2013), especificamente nos sítio de alimentação.  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.
Inicialmente o promotor UceS8.3 foi isolado de plantas de soja por TAIL-PCR com oligonucleotídeos desenhados para o gene do fator 2 de conjugação a ubiquitina (GmE2). Posteriormente esse promotor foi utilizado em transformação genética de planta modelo Arabidopsis thaliana para sua caracterização funcional devido à expressão do gene repórter GUS, de- monstrando expressão em todos os tecidos de planta, especialmente em raízes (Grossi-de-Sa et al.,2010). PCR em tempo real de raízes de soja inoculadas com M. incógnita em 7, 14, 21 e 28 DAI demonstram aumento de expressão transcricional de 2-6 vezes do gene GmE2, regulado pelo promotor UceS8.3 (Miranda et al., 2013). Hibridização in situ demonstrou a locali- zação da expressão relacionada à UceS8.3 em células gigantes e adjacentes ao sítio de alimentação nas galhas.  Initially 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.
Sumário da Invenção Summary of the Invention
A presente invenção compreende cassetes de expressão em plantas capazes se silenciar genes de múltiplas espécies de nematoides que se alimentam das referidas plantas conferindo resistência às referidas espécies de nematoides. Mais especificamente, os cassetes de expressão da presente invenção são capazes de expressar as moléculas de dsRNA em tecidos alvo de infestação por nematoides.  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.
A presente invenção fornece ainda vetores compreendendo o re- ferido cassete de expressão, métodos para produção de uma planta resistente a múltiplas espécies de nematoides bem como para o controle de nematoides em uma plantação, plantas resistentes a múltiplas espécies de nematoides, suas sementes e produtos produzidos a partir de material extraído das referidas plantas. 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.
Descrição das Figuras Description of the Figures
Figura 1 : Esquema representativo da construção gênica utilizada para transformação de soja visando resistência a Meloidogyne incógnita e Heterodera glycines. A seta preta indica a posição do promotor do fator de conjugação a ubiquitina de soja UceS8.3, isolado e caracterizado no LIMPP e protegido pela Embrapa por patenteamento. A construção gênica inclui sequencias do gene-alvo fator de splicing de M. incógnita e H. glycines so- mando 440 pb em duas posições palindrômicas, ou seja, reverso e complementar, a fim de permitir a formação do RNA dupla fita. Tal região palindrô- mica está separada pela região intrônica oriunda do vetor de RNAi, pKanni- bal, originalmente isolado de Arabidopsis thaliana. tNOS é o terminador de transcrição utilizado. As setas vermelhas e verdes indicam as posições de anelamento dos oligonucleotídeos utilizados para diagnóstico de transformação de soja (Figura 2A). A sequencia dos oligonucleotídeos está na Tabela 1.  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 1.
Figura 2: (A) Diagnóstico de transformação de soja por PCR. A imagem acima foi obtida por eletroforese em gel de agarose corado com brometo de etídeo do produto de PCR com os oligonucleotídos GmFSMiHg- F e GmFSMiHg-R (descritos na Tabela 1 e Figura 1). O DNA de folhas foi isolado e depois utilizado na PCR. A numeração corresponde aos indivíduos de terceira geração (T3), obtidos da multiplicação sucessiva do evento de transformação 4I T0. CN: controle negativo sem DNA molde. CP: controle positivo, DNA molde do vetor UceS8.3::GmFSMiHg. (B) Aclimatação de plantas de soja geneticamente modificada. Após a transformação por bioba- lística as plântulas foram mantidas em magentas por 15 dias sob seleção com o herbicida imidazolinona. Após crescimento, as plantas foram transferidas para copos com substrato orgânico e argila e foram mantidas cobertas com saco plástico para manter a humidade por uma semana. Posteriormente, as plantas foram transferidas para sacos plásticos de 20 L com o mesmo substrato e mantidas até completarem o ciclo de vida. Sementes T1 oriundas de TO, foram multiplicadas em casa de vegetação até a T2. Figure 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. After biobalistic transformation 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.
Figura 3: Bioensaio nematológico. As plantas T3 foram desafiadas com M. incógnita para determinação de indução de resistência a nematoides. (A) gráfico do resultado do desafio de eventos de soja GM (4A, 4B, 4C, 4E, 4G e 41) e de planta controle não transformada (BR-16). A reta superior denota o número de plantas individualmente testadas no experimento de resistência (n) e o percentual de redução de ovos obtidos (%). A reta acima das barras denota erro padrão do experimento. A letra acima da barra demonstra diferença estatisticamente significante; (B) gráfico com detalhamen- to do bioensaio, sem a barra com contagem da planta controle e com escala apropriada. A análise estatística nesse caso não levou em conta a planta controle.  Figure 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; (B) detailed bioassay plot, without bar with control plant count and appropriate scale. Statistical analysis in this case did not take into account the control plant.
Descrição Detalhada da Invenção  Detailed Description of the Invention
Uma concretização da presente invenção é um cassete de ex- pressão compreendendo:  One embodiment of the present invention is an expression cassette comprising:
(i) um promotor funcional em planta, induzido por nematoides; (i) a nematode-induced plant functional promoter;
(ii) um fragmento sense substancialmente similar à SEQ ID NO: 2;(ii) a sense fragment substantially similar to SEQ ID NO: 2;
(iii) um fragmento sense substancialmente similar à SEQ ID NO: 3; (iii) uma sequência separadora; (iii) a sense fragment substantially similar to SEQ ID NO: 3; (iii) a separator sequence;
(iv) um fragmento anti-sense substancialmente similar à SEQ ID (iv) an antisense fragment substantially similar to SEQ ID
NO: 5; NO: 5;
(v) um fragmento anti-sense substancialmente similar à SEQ ID (v) an antisense fragment substantially similar to SEQ ID
NO: 6; NO: 6;
(vi) um terminador funcional em planta.  (vi) a plant functional terminator.
O referido cassete é capaz de expressar dsRNA em plantas compreendendo fragmentos de genes de nematoides que silenciam genes conservados dos referidos nematoides através do mecanismo conhecido como RNA de interferência (RNAi).  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).
Em uma concretização preferida da presente invenção, as se- quências sense e anti-sense estão separas por uma sequência separadora. Opcionalmente, da região separadora é um íntron. Um "íntron" é uma sequência de nucleotídeo que é transcrita e está presente no pré mRNA, mas é removida através de clivagem e a re-ligação do mRNA dentro da célula gerando um mRNA maduro que pode ser traduzido em uma proteína. Exemplos de íntrons incluem, mas não são limitados a, íntron pdk, íntron catalase da mamona, íntron Delta 12 desnaturase de algodão, Delta 12 desnaturase de Arabidopsis, íntron ubiquitina de milho, íntron de SV40, íntrons do gene da malato sintase. Preferencialmente a seqCiência separadora da presente invenção é um íntron PDK. In a preferred embodiment of the present invention, the sense and antisense sequences are separated by a separator sequence. Optionally, 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. Examples of 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. Preferably the spacer sequence of the present invention is a PDK intron.
"Promotor" refere-se à sequência de DNA em um gene, usualmente localizada a montante da sequência codificadora, a qual controla a expressão da sequência codificadora promovendo o reconhecimento pela RNA polimerase e outros fatores requeridos para a própria transcrição. Em uma construção de DNA artificial, promotores podem também ser utilizados para transcrever dsRNA. Promotores podem também conter sequências de DNA que estão envolvidas na ligação de fatores de proteínas as quais con- trolam o efeito do início da transcrição em resposta a condições fisiológicas ou de desenvolvimento.  "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.
Em um dos aspectos da invenção, o promotor é um promotor constitutivo. Em outro aspecto da invenção, a atividade do promotor é estimulada por fatores externos ou internos tais como, mas não limitado a, hor- mônios, compostos químicos, impulsos mecânicos, e condições de estresse biótico ou abiótico. A atividade do promotor também pode ser regulada de maneira temporal e espacial (como por exemplo, promotores tecido- específicos e promotores regulados durante o desenvolvimento).  In one aspect of the invention, the promoter is a constitutive promoter. In another aspect of the invention, 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).
O promotor pode conter elementos "enhancers". Um "enhancer" é uma sequência de DNA que pode estimular a atividade do promotor. Ela pode ser um elemento inato do promotor ou um elemento heterólogo inserido para aumentar o nível e/ou a tecido-especificidade de um promotor. "Promotores constitutivos" referem-se àqueles que dirigem a expressão gê- nica em todos os tecidos e durante todo tempo. Promotores "tecido- específicos" ou "desenvolvimento-específicos" são aqueles que dirigem a expressão gênica quase que exclusivamente em tecidos específicos, tais como folhas, raízes, caules, flores, frutos ou sementes, ou em estágios do desenvolvimento específicos em um tecido, como no início ou final da em- briogênese. O termo "expressão" refere-se a transcrição e acumulação estável do dsRNA derivado dos fragmentos de ácidos nucléicos da invenção que, em conjunto com a aparelhagem de produção de proteína da célula, resulta em níveis alterados de mio-inositol 1 -fosfato sintase. "Inibição por interferência" refere-se a produção de transcritos de dsRNA capazes de prevenir a expressão da proteína alvo. 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. "Constitutive 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. The term "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.
Em uma concretização preferida da presente invenção, o referido promotor é específico de tecidos colonizados por nematoides ou induzido por infestação por nematoides. Preferencialmente o promotor tem a sequência identificada como SEQ ID NO: 1 .  In a preferred embodiment of the present invention, said promoter is specific to nematode-colonized or nematode-infested tissue-induced tissues. Preferably the promoter has the sequence identified as SEQ ID NO: 1.
Ainda de acordo com uma concretização preferida da presente invenção, as sequências sense e anti-sense de (ii) e (iv) pertencem a um gene que codifica um fator de splicing (FS) do nematoide.  Still according to a preferred embodiment of the present invention, the sense and antisense sequences of (ii) and (iv) belong to a gene encoding a nematode splicing factor (FS).
Em uma concretização preferida da presente invenção as sequências dos componentes (ii) e (iv) pertencem às espécies de nematoides Heterodera glycines e Meloidogyne incógnita, respectivamente, e mais preferivelmente o cassete de expressão contem a seguinte composição:  In a preferred embodiment of the present invention the 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:
(a) uma sequência promotora tendo uma sequência substanci- almente similar à SEQ ID NO: 1  (a) a promoter sequence having a sequence substantially similar to SEQ ID NO: 1
(b) a primeira sequência sense tendo uma sequência substancialmente similar à SEQ ID NO: 2;  (b) the first sense sequence having a sequence substantially similar to SEQ ID NO: 2;
(b) a segunda sequência sense promotora tendo uma sequência substancialmente similar à SEQ ID NO: 3;  (b) the second promoter sense sequence having a sequence substantially similar to SEQ ID NO: 3;
(c) a sequência separadora promotora tendo uma sequência substancialmente similar à SEQ ID NO: 4;  (c) the promoter splitter sequence having a sequence substantially similar to SEQ ID NO: 4;
(d) a primeira sequência anti-sense promotora tendo uma sequência substancialmente similar à SEQ ID NO: 5;  (d) the first promoter antisense sequence having a sequence substantially similar to SEQ ID NO: 5;
(e) a segunda sequência anti-sense promotora tendo uma se- quência substancialmente similar à SEQ ID NO: 6;  (e) the second promoter antisense sequence having a sequence substantially similar to SEQ ID NO: 6;
(f) a sequência terminadora promotora tendo uma sequência substancialmente similar à SEQ ID NO: 7. Em uma concretização preferida da presente invenção, a sequência do cassete de expressão é substancialmente similar à SEQ ID NO: 8. (f) the promoter terminator sequence having a sequence substantially similar to SEQ ID NO: 7. In a preferred embodiment of the present invention, the expression cassette sequence is substantially similar to SEQ ID NO: 8.
O termo "substancialmente similar" ou "similaridade substancial" refere-se a fragmentos de ácidos nucléicos nos quais mudanças em uma ou mais bases de nucleotídeos não afetam a habilidade do fragmento de ácido nucléico mediar a alteração da expressão gênica pelo silenciamento gênico através, por exemplo, da tecnologia antisense, co-supressão ou RNA de interferência (RNAi). Fragmentos de ácido nucléico substancialmente similares da presente invenção podem ser caracterizados também pela porcentagem de similaridade de suas sequências de nucleotídeos com as sequências de nucleotídeo dos fragmentos de ácidos nucléicos descritas aqui (SEQ ID NO 1-8), como determinada por algoritmos comuns empregados no estado da técnica. Os fragmentos de ácidos nucléicos preferidos são aqueles cujas sequências de nucleotídeos têm pelo menos cerca de 40 ou 45% de identidade de sequência, preferencialmente cerca de 50% ou 55% de identidade de seqQência, mais preferencialmente cerca de 60% ou 65% de identidade de sequência, mais preferencialmente cerca de 70% ou 75% de identidade de sequência, mais preferencialmente cerca de 80% ou 85% de identidade de sequência, mais preferencialmente ainda com cerca de 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% ou 99% de identidade de sequência quando comparada com a sequência de referência. O alinhamento de sequência e o cálculo de porcentagem de similaridade da presente invenção foram realizados utilizando-se o Programa DNAMAN for windows (Lynnon Corporati- on, 2001), utilizando sequências depositadas no GenBank, através da integração do Web browser.  The term "substantially similar" or "substantial similarity" 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). 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.
Uma das formas de se formar o dsRNA é estando presente na molécula de DNA a sequência de nucleotídeos do gene alvo na orientação sense, e uma sequência de nucleotídeos na orientação antisense, podendo haver ou não uma região espaçadora entre as sequências de nucleotídeos sense e antisense. As sequências de nucleotídeos mencionadas podem ser constituídas de cerca de 19nt a 2000nt ou ainda cerca de 5000 nucleotídeos ou mais, cada um tendo uma similaridade substancial de sequência total com cerca de 40% a 100%. Quanto mais longa for a sequência, menos es- tringência é requerida para similaridade substancial total da sequência. Os fragmentos contendo pelo menos cerca de 19 nucleotídeos devem ter prefe- rencialmente cerca de 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% ou 99% de identidade de sequência quando comparada com a sequência de referência, com possibilidade de ter cerca de 2 nucleotídeos distintos não contíguos. Preferencialmente são utilizados fragmentos acima de 60pb, mais preferencialmente ainda fragmentos entre 100 a 500pb. One way to form 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.
Em um dos aspectos da invenção, a molécula de dsRNA pode compreender uma ou mais regiões tendo uma similaridade substancial de sequência para as regiões com pelo menos cerca de 19 nucleotídeos consecutivos dos nucleotídeos sense do gene alvo, definida como primeira região e, uma ou mais regiões tendo uma similaridade substancial de sequência para as regiões com cerca de 19 nucleotídeos consecutivos do complemento dos nucleotídeos sense do gene alvo, definida como segunda região, onde essas regiões podem ter pares de bases separando-as uma da outra.  In one aspect of the invention, 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.
A invenção compreende ainda vetores compreendendo os cassetes da presente invenção assim como métodos para produção de uma planta resistente a múltiplas espécies de nematoides, compreendendo inserir em uma célula vegetal um cassete da presente invenção e regenerar uma planta a partir da referida célula vegetal.  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.
A invenção compreende ainda plantas resistentes a múltiplas espécies de nematoides possuindo um cassete da presente invenção inte- grado ao seu genoma.  The invention further comprises plants resistant to multiple species of nematodes having a cassette of the present invention integrated into their genome.
"Plantas" referem-se a organismos fotossintéticos, ambos euca- riotos e procariotos, onde o termo "plantas desenvolvidas" refere-se a plantas eucariotas. Os ácidos nucleicos da invenção podem ser utilizados para conferir tratos desejados em essencialmente qualquer planta. Então, a in- venção possui uso sobre várias espécies de plantas, incluindo espécies dos géneros Anacardium, Anona, Arachis, Artocarpus, Asparagus, Atropa, Avena, Brassica, Carica, Citrus, Citrullus, Capsicum, Carthamus, Cocos, Coffea, Cucumis, Cucurbita, Daucus, Elaeis, Fragaria, Glycine, Gossypium, Helian- thus, Heterocallis, Hordeum, Hyoseyamus, Lactuca, Linum, Lolium, Lupinus, Lycopersicon, Malus, Manihot, Majorana, Medicago, Nicotiana, Olea, Oryza, Panieum, Pannesetum, Passiflora, Persea, Phaseolus, Pistachia, Pisum, Pyrus, Prunus, Psidium, Raphanus, Ricinus, Secale, Senecio, Sinapis, Sola- num, Sorghum, Theobromus, Trigonella, Triticum, Vicia, Vitis, Vigna, e Zea. Preferivelmente a planta da presente invenção é uma planta de soja. "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. Thus, 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, Vicia, Vitis, Vigna, and Zea. Preferably the plant of the present invention is a soybean plant.
A invenção compreende ainda sementes de uma planta possuindo um cassete da presente invenção integrado ao seu genoma bem como produtos produzidos a partir de material extraído de uma planta possuindo um cassete da presente invenção integrado ao seu genoma, tais como produtos alimentícios e/ou ração animal.  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. .
A invenção compreende também métodos para o controle de nematoides em uma plantação, compreendendo cultivar uma planta possu- indo um cassete da presente invenção integrado ao seu genoma.  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.
EXEMPLOS EXAMPLES
A presente invenção é ainda definida nos seguintes Exemplos. Deve ser entendido que esses Exemplos, enquanto indicam parte da invenção, são colocados como forma de ilustração somente, não tendo, portanto, qualquer cunho limitante do escopo das presentes invenções.  The present invention is further defined in the following Examples. It is to be understood that these Examples, while indicating part of the invention, are provided by way of illustration only and therefore have no limiting scope on the scope of the present inventions.
Técnicas usuais de biologia molecular tais como transformação de bactérias e eletroforese em gel de agarose de ácidos nucleicos são referidos através de termos comuns para descrevê-los. Detalhes da prática dessas técnicas, bem conhecidos no estado da técnica, são descritos em Sam- brook, et al. (Molecular Cloning, A Laboratory Manual, 2nd ed. (1989), Cold Spring Harbor Laboratory Press). Várias soluções utilizadas nas manipulações experimentais são referidas por seus nomes comuns tais como "solução de lise", "SSC", "SDS", etc. As composições dessas soluções podem ser encontradas na referência Sambrook, et al. (supracitada).  Usual molecular biology techniques such as bacterial transformation and nucleic acid agarose gel electrophoresis are referred to by common terms to describe them. Details of the practice of these techniques, well known in the prior art, are described in Sambrook, et al. (Molecular Cloning, A Laboratory Manual, 2nd ed. (1989), Cold Spring Harbor Laboratory Press). Several solutions used in experimental manipulations are referred to by their common names such as "lysis solution", "SSC", "SDS", etc. Compositions of such solutions can be found in reference Sambrook, et al. (above).
Exemplo 1 - Seleção dos genes Example 1 - Gene Selection
Novos genes-alvo para RNAi são selecionados com base nos seguintes critérios: (a) seus ortólogos em C. elegans devem ser genes es- senciais e/ou possuir fenótipos letais quando silenciados ou nocauteados; (b) suas sequências devem apresentar alta identidade apenas com espécies de nematoides; (c) suas sequências devem estar disponíveis no banco de dados (GenBank, http://www.ncbi.nlm.nih.gov/nuccore) e (d) Para assegurar que os propostos dsRNAs não sejam um risco a organismos não-alvo, as sequências dos fragmentos dos genes em questão utilizados para a síntese de dsRNA são comparadas com outras sequências disponíveis no GenBank, utilizando a ferramenta BLAST"n(versão 2.2.15) (ALTSCHUL, S. F.; GISH.W.; MILLER.W.; MYERS.E. W. & LIPMAN, D. J. Basic local alignment search tool. Journal of Molecular Bioiogy, v. 215, n. 3, p. 403-10, Oct 5 1990). 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. , the 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).
Baseado nas revisões de literatura, bem como sequências disponíveis no banco de dados (GenBank,) foi selecionado um gene de processamento de mRNA (FS) em M. incógnita (AW828516) e um gene também envolvido no processamento de mRNA (FS) em H. glycines (AF1 13915). Exemplo 2 - Seleção das sequências complementares aos mRNAs dos genes de processamento de mRNA em M. incógnita e H. glycines  Based on literature reviews as well as sequences available in the database (GenBank), a mRNA (FS) processing gene in M. incognita (AW828516) and a gene also involved in mRNA (FS) processing in H were selected. glycines (AF1 13915). Example 2 - Selection of mRNA Complementary Sequences of the mRNA Processing Genes in M. incognita and H. glycines
Uma vez selecionados os genes-alvo, suas sequências foram submetidas ao Block™ RNAi Designer (http: rnaidesigner.invitrogen.com/ rnai- express/), para a escolha da região de cada gene com maior probabilidade de produção de siRNAs eficientes. Foram escolhidas regiões contendo entre 120 e 250 pares de base.  Once the target genes were selected, their sequences were submitted to Block ™ RNAi Designer (http: rnaidesigner.invitrogen.com/rnai-express/) to choose the region of each gene that is most likely to produce efficient siRNAs. Regions containing between 120 and 250 base pairs were chosen.
As regiões gênicas selecionadas no Block™ RNAi Designer foram submetidas ao banco de dados GenBank™ pelos programas BLASTn (nucleotide blast) e BLASTp (protein blast, no endereço eletrônico do NCBI (http://www.ncbi.nlm.nih.gov/ biast/Blast.cgi).afinl de identificar possíveis e- feitos de RNAi em plantas, hunlallOs e outros organismos não-alvo.  The selected gene regions in Block ™ RNAi Designer were submitted to the GenBank ™ 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.
Exemplo 3 - Oligonucleotídeos para análise de expressão e de silenciamento gênico Example 3 - Oligonucleotides for Expression and Gene Silencing Analysis
Para avaliar a eficiência do silenciamento gênico após ensaios de infestação em linhagens de soja GM, foram desenhados pares de oligonucleotídeos (primers) para cada gene.  To evaluate gene silencing efficiency after GM soybean infestation assays, oligonucleotide pairs (primers) were designed for each gene.
O desenho dos oligonucleotídeos foi realizado pelo programa primer3 v.0.4.0 (http://frodo.wi.mitt.edu/), que sugere os melhores pares dentro da sequência fornecida e para o tamanho do produto desejado. O programa também fornece parâmetros importantes como Tm (melting tempera- ture), %GC,formação de loop e de homodímero. 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.
Os oligos escolhidos estão descritos na Tabela 1  The chosen oligos are described in Table 1
Figure imgf000022_0001
Figure imgf000022_0001
Exemplo 4 - Obtenção das plantas transqênicas por biobalística  Example 4 - Obtaining transgenic plants by biobalistics
Os disparos de partículas de tungsténio com DNA adsorvido foram efetuados na região mehstemática de embriões provenientes de semen- tes da cultivar de soja BR-16. Foi utilizada a estratégia de co-transformação com os plasmídios UceSB.3::GmFSMiHg (para silenciamento gênico de fito- nematoides) e pAC321 (resistência a herbicida), utilizando o protocolo de biobalística (RECH, E. L; VIANNA.G. R.& ARAGÃO, F. J. L. High-efficiency transformation by biolistics of soybean, common bean and cotton transgenic plants. Nature Protocols, v. 3, n. 3, p. 410-418, 2008.  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.
As sementes foram primeiramente esterilizadas em etanol 70% por 10 minutos, seguida por imersão em hipoclorito 50% por vinte minutos, e então lavadas três vezes com água destilada autoclavada em câmara de fluxo laminar, permanecendo imersas em água destilada por um período a- proximadamente de 16 horas.  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.
As sementes foram então incisadas para a retirada dos embriões com o auxílio de pinças e bisturis estéreis, sendo armazenadas em placa de Petri com água destilada para evitar a dessecação. Em seguida, os primórdios foliares foram retirados com o auxílio de lupa, para exposição da região do meristema apical.  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.
Posteriormente, os embriões foram secos sob exposição ao ambiente em papel filtro em câmara de fluxo laminar, e posteriormente posicio- nados em placas de Petri de 5 cm de diâmetro, contendo 11 mL de meio MS (Murashige & Skoog 1962), 3% de sacarose e 0,8% de phytagel e pH 5,7. Dispostos na linha de um círculo de 16 mm de diâmetro centralizado a placa (zona de morte), tendo a região do meristema apical direcionada para cima. Subsequently, 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.
As construções de DNA foram precipitadas sobre micropartícu- las de tungsténio como auxílio de CaCI2e espermidina. A introdução das construções gênicas de interesse ocorreu pelo uso do acelerador de partículas desenvolvido no Brasil.  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.
Após a co-transformação com os genes de interesse, os embri- ões foram transferido para placas contendo meio MS suplementado com benzilaminopurina (BAP - 5 mg/ml), 3% de sacarose, 0,6% de ágar e pH 5,7, onde permaneceram aproximadamente 18 horas ao abrigo da luz a 28° C para indução ao multibrotamento. Os embriões foram então transferidos para magentas contendo meio seletivo com, MS, 3% de sacarose, 0,15 μΜ de herbi- cida Imazapyr, 0,8% de ágar e, vitamina B5, pH 5,7, sendo 9 embriões em cada magenta, os quais foram mantidos em câmara de crescimento a temperatura de 28° C, com 16 horas de fotoperíodo e luminosidade 350 μmols.m-2.s-1e umidade relativa acima de 80% por aproximadamente 45 dias. After co-transformation with the genes of interest, 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 μΜ 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 μmols.m -2 .s -1 and relative humidity above 80% for approximately 45 days.
Após esse período foram transferidos dois embriões multibrota- dos por copo contendo areia:vermiculita (1 :1) autoclavadas e umedecidas com solução nutritiva e cobertos com saco plástico para aclimatação. Os embriões multibrotados foram irrigados com solução nutritiva a cada 7 dias e mantidos por mais 28 dias em câmara climatizada, até a transferência para a casa de vegetação.  After this period, two multibrotted embryos were transferred per cup containing sand: autoclaved (1: 1) vermiculite moistened with nutrient solution and covered with plastic bag for acclimatization. Multibrotted embryos were irrigated with nutrient solution every 7 days and kept for 28 days in a climate chamber until transfer to the greenhouse.
Após esse período de 28 dias, foram transferidos para casa de vegetação em vasos contendo uma mistura de terra e areia esterilizada (5:3), cobertos com saco plástico durante sete dias para aclimatação, sendo substituído por saco plástico furado por mais cinco dias. Após esse período de aclimatação os sacos plásticos foram retirados para o desenvolvimento normal das plantas até o início das análises moleculares para a identificação de plantas positivas pela técnica de PCR.  After this 28-day period, they were transferred to a greenhouse in pots containing a sterile (5: 3) mixture of earth and sand, covered with a plastic bag for seven days for acclimatization, and replaced with a punctured plastic bag for another five days. After this acclimatization period, the plastic bags were removed for normal plant development until the beginning of the molecular analysis to identify positive plants by PCR.
No período de 3 meses foram transformados 2946 embriões por biobalística, que foram selecionados por resistência ao herbicida Imazapir, cuja resistência é conferida pelo gene Ahas. Os 1495 embriões remanescentes foram aclimatados e transferidos para a casa de vegetação. Desses anteriores, 673 foram capazes de se regenerarem e se desenvolverem. Des- ses, 350 plantas foram testadas por PCR resultando em 31 plantas (8,85% do total analisado) que amplificaram o transgene (Tabela 2). Considerando todas as etapas realizadas, foi obtido quase 1 % de plantas GM PCR-positivo em relação ao número de embriões transformados. Within 3 months 2946 embryos were transformed by biobalistics, which were selected for resistance to the herbicide Imazapir, whose resistance is conferred by the Ahas gene. The remaining 1495 embryos were acclimatized and transferred to the greenhouse. Of these earlier, 673 were able to regenerate and develop. Of these, 350 plants were tested by PCR resulting in 31 plants (8.85% of the total analyzed) that amplified the transgene (Table 2). Considering all steps performed, almost 1% of PCR-positive GM plants were obtained in relation to the number of transformed embryos.
Os resultados foram confirmados por diferentes amplificações por PCR utilizando pares de primers específicos (Figura 1) para construção UceS8.3::GmFSMiHg.  Results were confirmed by different PCR amplifications using specific primer pairs (Figure 1) for UceS8.3 :: GmFSMiHg construction.
Foram realizadas 3150 amplificações, partindo de 1050 amostras de DNA, para caracterização das 350 plantas regeneradas (Tabela 2).  3150 amplifications were performed from 1050 DNA samples to characterize the 350 regenerated plants (Table 2).
Figure imgf000024_0001
Figure imgf000024_0001
Exemplo 5 - Análises de PCR  Example 5 - PCR Analyzes
A seleção das plantas transformadas foi feita por análises de PCR.Usando o kit Extract-N-Amp™ Tissue PCR Kit (Sigma-Aldrich, Saint Louis, MO, EUA), o DNA de folhas foi purificado e a PCR amplificou fragmentos dos transgenes no genoma de cada evento gerado. Cada planta teve seu DNA extraído independentemente três vezes, cada qual partindo de diferentes trifólios, sendo que cada amostra de DNA serviu de molde para três P- CRs também independentes. Foram consideradas como plantas PCR- positivas aquelas que amplificaram o transgene ao menos uma vez para cada extração de DNA, ou seja, para cada trifólio. A confirmação da inserção da construção UceS8.3::GnlFSMiHg foi feita separadamente com dois pares de primers que amplificam diferentes regiões dentro da construção. Os pri- mers InGmFSMiHg-F e InGmFSMiHg-R (SEQ ID NO 9 e SEQ ID NO 10 - Figura 1) amplificam um fragmento de 135 pb, no intron. Já os primers GmFSMiHg-F e GmFSMiHg-R (SEQ ID NO 1 1 e SEQ ID NO 12 - Figura 1) amplificam um fragmento de 440 pb na região senso. Utilizando 400 nM de cada primer, as amplificações por PCR foram realizadas em termociclador Mycycler (BioRad), com o programa: desnaturação inicial de 1 '30" a 94° C, 35 ciclos de desnaturação de 30" a 94° C, anelamento de 30" a 55° C e extensão de 45" a 72° C, seguido de uma extensão final de 5' a 72° C. Os produtos de PCR foram separados por eletroforese em gel de agarose 1 ,3%, corados com brometo de etídeo e visualizados em transluminador. Selection of transformed plants was done by PCR analysis. Using the Extract-N-Amp ™ Tissue PCR Kit (Sigma-Aldrich, Saint Louis, MO, USA), leaf DNA was purified and PCR amplified transgenic fragments in the genome of each generated event. Each plant had its DNA extracted three times independently, each from different trifoliums, and each DNA sample served as a template for three independent P-CRs. PCR-positive plants were those that amplified the transgene at least once for each DNA extraction, ie for each trifoliate. Confirmation of insertion of the UceS8.3 :: GnlFSMiHg construct was made separately with two pairs of primers that amplify different regions within the construct. 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. Using 400 nM of each primer, 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.
Plantas PCR-positivas para os dois amplicons foram transferidas para vasos de 15L com solo e mantidas em casa de vegetação.  PCR-positive plants for both amplicons were transferred to 15L pots with soil and kept in a greenhouse.
Exemplo 6 - Bioensaio dos eventos transformados com a construção U- ceS8.3::GmFSMiHg contra M. incógnita Example 6 - Bioassay of Events Transformed with Construct Ucs8.3 :: GmFSMiHg against M. incognita
Para averiguar o efeito do silenciamento do gene de processa- mento do mRNA (FS) nos nematoides foram realizados bioensaios com inoculação de J2 de M. incógnita nas plantas transformadas com a construção a ser estudada.  To investigate the silencing effect of the mRNA processing gene (FS) on nematodes, bioassays were carried out with inoculation of M. incognita J2 in plants transformed with the construct to be studied.
Cultura de M. incógnita M. incognita culture
A cultura de M. incógnita, raça 1 , foi mantida em plantas de toma- teiro (Solanum lycopersicum), da variedade suscetível Santa Cruz e cultivar Kada Gigante. A coleta de nematoides em seus diferentes estádios de desenvolvimento foi realizada a partir de 28 dias após a inoculação (DAI). Extração de ovos The culture of M. incognita, race 1, was kept in tomato (Solanum lycopersicum) plants of susceptible variety Santa Cruz and cultivar Kada Gigante. The collection of nematodes at their different stages of development was performed from 28 days after inoculation (DAI). Egg extraction
Os ovos foram extraídos de acordo com HUSSEY & BARKER 1973 (HUSSEY, R. S. & BARKER, A. A. Comparation methods of colleting inocula of Meloigogyne spp. including a new technique. The Plant Disease Repórter v. 57, p. 1025-1028, 1973). Brevemente, as raízes foram trituradas em liquidificador por dois minutos, em hipoclorito de sódio (NaOCI)0,5%. A contagem dos ovos foi feita em lâmina de Peters, utilizando microscópio óptico de luz (VRAIN, T. CA technique for the collection of larvae of Meloi- dogyne spp. and a comparison of eggs and larvae as inocula. Journal Nema- tology, v. 9, p. 249-251 , 1977.).  Eggs were extracted according to HUSSEY & BARKER 1973 (HUSSEY, R. S. & BARKER, A. A. Comparison of colleting inoculations of Meloigogyne spp. Including a new technique. The Plant Disease Reporter v. 57, p. 1025-1028, 1973). Briefly, the roots were ground in a blender for two minutes in 0.5% sodium hypochlorite (NaOCI). Egg counts were performed on a Peters slide using light optical microscopy (VRAIN, T. CA. Technique for the collection of larvae of Melo- dogyne spp. And a comparison of eggs and larvae as inocula. Journal Nematology, v 9, pp. 249-251, 1977.).
Extração de J2 pré-parasíticos Pre-parasitic J2 extraction
Para a coleta de juvenis em estádio J2, a suspensão de ovos foi submetida à técnica do funil de Baernlann, mantidas a temperatura ambiente, em um recipiente contendo água destilada para permitir a eclosão dos ovos e subsequentemente coleta dos nematoides. A coleta de J2 eclodidos foi realizada ao longo de uma semana, a cada dois dias.  To collect juveniles at stage J2, 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.
Bioensaio Bioassay
A fim de avaliar a indução de resistência a M. incógnita nos eventos de soja GM foi montado um bioensaio em casa de vegetação. Progénies da segunda geração (T3), no estádio de 2-3 trifólios, foram plantadas em vasos que continham 300 mL de solo, que foram inoculados com uma população aproximadamente 1.000 J2 de M.incognita 1. Plantas da cultivar BR-16 (não transgênica), que não possui resistência a M.incognita raça 1 , foram plantadas e inoculadas servindo como controle..As plantas permaneceram em casa de vegetação durante 6 semanas, sendo irrigadas quando necessário. As raízes de soja foram processadas individualmente para a extração dos ovos aos 45 dias após inoculação (DAI). As raízes foram individualmente lavadas para retirada de solo, secas com papel toalha, pesadas, trituradas em liquidificador com 0,5% NaCIO por 2 minutos, lavadas com jato d'água e os ovos foram separados em peneira de 500 Mesh.  In order to evaluate the induction of resistance to M. incognita in GM soybean events, a greenhouse bioassay was set up. 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.
Considerando cada planta, o volume final da suspensão com ovos obtidos foi corrigido para 30 mL, sendo que três alíquotas de 1 mL fo- ram retiradas. Após contagem de ovos usando microscópio e lâmina de Peters, a contagem foi normalizada com a massa da raiz, determinando o número de ovos g-l de raiz. Considering each plant, 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.
Todos os dados obtidos do bioensaio foram avaliados estatisti- camente por meio do procedimento de modelos lineares generalizados (GLM) utilizando pacotes estatísticos do Programa R (R:A ianguage and en- vironment for statistical computing. 2005. Disponível em: http://www.R- project.org ).  All data obtained from the bioassay were statistically evaluated using the generalized linear model (GLM) procedure using R program statistical packages (R: A ianguage and environment for statistical computing. 2005. Available at: http: // www.r-project.org).
As sementes dos eventos transformados foram germinadas e as plantas que se desenvolveram foram transplantadas para vasos em casa de vegetação. Quando atingiram o estádio de 2-3 trifólios, as plantas foram ge- notipadas por PCR para a detecção do transgene (Figura 2A). As plantas que não apresentaram o fragmento correspondente foram descartadas, sendo que apenas as plantas positivas foram utilizadas nos bioensaios (Figura 2B).  The seeds of the transformed events were germinated and the plants that developed were transplanted to pots in a greenhouse. When they reached the stage of 2-3 trifolium, the plants were genotyped by PCR for transgene detection (Figure 2A). Plants that did not have the corresponding fragment were discarded, and only positive plants were used in the bioassays (Figure 2B).
Cada planta foi inoculada com aproximadamente 1000 J2 obtidos em sistema de eclosão. Cada tratamento foi composto de 6 a 10 repetições. O desafio foi completamente repetido duas vezes em períodos diferentes. Aproximadamente seis semanas após a inoculação, as raízes das plan- tas foram individualmente extraídas e processadas para determinação do o número de ovos. g-l de raiz. Todos os valores dos bioensaios foram relativa- dos em relação ao tratamento controle para normalizar os dados entre as repetições biológicas, e possibilitar a análise estatística. Esta normalização foi necessária porque os dados referentes à infecção de fitonematoides vari- am muito de um experimento para outro, podendo causar erro de interpretação destes dados. Estas variações são inerentes tanto do experimento com nematoides, pois não se sabe quantos realmente penetram na raiz, como da técnica de RNAi, pois não se sabe o quanto o nematoide vai ingerir de dsR- NA/siRNA. Para a avaliação estatística de todos os dados, foi utilizada a a- nálise de modelos lineares generalizados (GLM), e após a definição do melhor modelo de ajuste para cada grupo de dados (por exemplo: número de ovos, número de J2 eclodidos, etc.) foi realizada uma análise de variância dos desvios (ANODEV) (Pacote "car", programa R.). Nos casos em que a ANODEV foi significativa, os tratamentos foram submetidos à análise de contrastes (Pacote "contrast", programa R) para determinar se existem diferenças significativas entre os tratamentos. 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. These variations are inherent in both the nematode experiment, as it is unknown how many actually penetrate the root, as well as the RNAi technique, as it is unknown how much the nematode will ingest from dsR-NA / siRNA. For statistical evaluation of all data, generalized linear model (GLM) analysis was used, and after defining the best fit model for each data group (eg number of eggs, number of hatched J2, etc.) a variance analysis was performed of deviations (ANODEV) (car package, program R.). In cases where ANODEV was significant, the treatments were subjected to contrast analysis (Contrast package, program R) to determine if significant differences exist between treatments.
Ao avaliar os dados referentes aos eventos de soja com a construção UceS8.3::GmFSMiHg, o número ovos por grama de raiz se ajustou melhor ao modelo de distribuição normal com função de ligação identidade. Ao comparar-se o número de ovos g-l de raiz entre o tratamento controle e os eventos transgênicos expressando dsRNA para FS, foi verificado que es- te número foi de 71 % a 91 % menor nos eventos transgênicos (Figura 3A), sendo essa diferença significativa peia análise de contrastes (p<0,05). Os eventos transgênicos não foram estatisticamente diferentes quando comparados entre si, exceto o evento GmFSMiHg- 4IT3 (Figura 6B). Então, plantas transformadas com o cassete de expressão da presente invenção apresen- tam, portanto, elevada resistência contra nematoides.  When evaluating data for soybean events with the UceS8.3 :: GmFSMiHg construct, the number of eggs per gram of root best fitted the normal distribution model with identity binding function. When comparing the number of gl-rooted eggs between the control treatment and the transgenic events expressing dsRNA for FS, it was found that this number was 71% to 91% lower in transgenic events (Figure 3A). significant by contrast analysis (p <0.05). Transgenic events were not statistically different when compared to each other except the GmFSMiHg-4IT3 event (Figure 6B). Thus, plants transformed with the expression cassette of the present invention therefore have high resistance against nematodes.

Claims

REIVINDICAÇÕES
1. Um cassete de expressão compreendendo:  1. An expression cassette comprising:
(i) um promotor funcional em planta;  (i) a plant functional promoter;
(ii) um fragmento sense substancialmente similar à SEQ ID NO: 2;  (ii) a sense fragment substantially similar to SEQ ID NO: 2;
(iii) um fragmento sense substancialmente similar à SEQ ID (iii) a sense fragment substantially similar to SEQ ID
NO: 3; NO: 3;
(iii) uma sequência separadora;  (iii) a separator sequence;
(iv) um fragmento anti-sense substancialmente similar à SEQ ID NO: 5;  (iv) an antisense fragment substantially similar to SEQ ID NO: 5;
(v) um fragmento anti-sense substancialmente similar à SEQ (v) an antisense fragment substantially similar to SEQ
ID NO: 6; ID NO: 6;
(vi) um terminador funcional em planta.  (vi) a plant functional terminator.
2. Cassete de expressão de acordo com a reivindicação 1 , ca- racterizado pelo fato do promotor funcional em plantas ser induzido por ne- matoides  Expression cassette according to claim 1, characterized in that the functional promoter in plants is induced by nematodes.
3. Cassete de expressão de acordo com qualquer uma das reivindicações 1 e 2, caracterizado pelo fato do promotor funcional em plantas ser substancialmente similar à SEQ ID NO 1.  Expression cassette according to any one of claims 1 and 2, characterized in that the plant functional promoter is substantially similar to SEQ ID NO 1.
4. Cassete de expressão de acordo com a reivindicação 1 , caracterizado pelo fato da sequência separadora ser um íntron.  Expression cassette according to claim 1, characterized in that the separator sequence is an intron.
5. Cassete de expressão de acordo com a reivindicação 4, caracterizado pelo fato do íntron ser selecionado do grupo consistindo de íntron pdk, íntron catalase da mamona, íntron Delta 12 desnaturase de algo- dão, Delta 12 desnaturase de Arabidopsis, íntron ubiquitina de milho, íntron de SV40, íntrons do gene da malato sintase.  Expression cassette according to Claim 4, characterized in that the intron is selected from the group consisting of the pdk intron, castor bean intron catalase, the Delta 12 denaturase intradage, the Arabidopsis Delta 12 denaturase, the corn ubiquitin intron. , SV40 intron, Malate synthase gene introns.
6. Cassete de expressão de acordo com a reivindicação 5, caracterizado pelo fato do íntron ser substancialmente similar à SEQ ID NO 4.  Expression cassette according to claim 5, characterized in that the intron is substantially similar to SEQ ID NO 4.
7. Cassete de expressão de acordo com qualquer uma das rei- vindicações 1 a 6, em que as sequências sense e anti-sense de (ii, iii) e (iv, v) pertencem a um gene que codifica um fator de splicing.  An expression cassette according to any one of claims 1 to 6, wherein the sense and antisense sequences of (ii, iii) and (iv, v) belong to a gene encoding a splicing factor.
8. Cassete de expressão de acordo com qualquer uma das rei- vindicações 1 a 7, em que as sequência de (ii, iv) e (iii, v) são de Heterodera glycines e Meloidogyne incógnita, respectivamente. 8. Expression cassette according to any of the claims 1 to 7, wherein the sequences of (ii, iv) and (iii, v) are from Heterodera glycines and Meloidogyne incognita, respectively.
9. Cassete de expressão caracterizado por possuir uma sequência substancialmente similar à SEQ ID NO 8.  9. Expression cassette having a sequence substantially similar to SEQ ID NO 8.
10. Vetor, compreendendo o cassete como definido em qualquer uma das reivindicações 1 a 9.  A vector comprising the cassette as defined in any one of claims 1 to 9.
11. Método para produção de uma planta resistente a múltiplas espécies de nematoides, compreendendo inserir em uma célula vegetal um cassete como definido em qualquer uma das reivindicações 1 a 9 e regene- rar uma planta a partir da referida célula vegetal.  A method for producing a plant resistant to multiple nematode species, comprising inserting into a plant cell a cassette as defined in any one of claims 1 to 9 and regenerating a plant from said plant cell.
12. Planta resistente a múltiplas espécies de nematoides, possuindo um cassete de expressão como definido em qualquer uma das reivindicações 1 a 9 integrado ao seu genoma.  Plant resistant to multiple species of nematodes, having an expression cassette as defined in any one of claims 1 to 9 integrated into its genome.
13. Planta de acordo com a reivindicação 12, em que a planta é uma planta de soja.  Plant according to claim 12, wherein the plant is a soybean plant.
14. Semente de uma planta como definida na reivindicação 13. Plant seed as defined in claim 13.
15. Produto, produzido a partir de material extraído de uma planta como definida na reivindicação 12. Product, produced from material extracted from a plant as defined in claim 12.
16. Método para o controle de nematoides em uma plantação, compreendendo cultivar planta como definida em qualquer uma das reivindicações 12 e 13.  A method of controlling nematodes in a plantation, comprising cultivating plant as defined in any one of claims 12 and 13.
PCT/BR2013/000203 2013-06-11 2013-06-11 Expression cartridge for inducing resistance to multiple nematoid species in plants, method and plants using said expression cartridge WO2014197951A2 (en)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106577796A (en) * 2016-11-22 2017-04-26 山东省农业科学院蔬菜花卉研究所 Method of prevention and treatment of tomato root knot nematode disease by castor bean meal

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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 (en) * 2007-02-05 2019-11-26 Empresa Brasileira De Pesquisa Agropecuaria Embrapa compositions and methods for modifying gene expression using the ubiquitin conjugation protein gene promoter from soybean plants

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106577796A (en) * 2016-11-22 2017-04-26 山东省农业科学院蔬菜花卉研究所 Method of prevention and treatment of tomato root knot nematode disease by castor bean meal

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BR112015024341A2 (en) 2018-10-23
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WO2014197951A3 (en) 2015-04-09

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