GENETIC CONTROL METHOD AND COMPOSITIONS AGAINST INSECT PESTS IN COTTON PLANTS BY THE SILENCING OF GENES OF THE LACCASE FAMILY

• US 20150368649A1
• Filed: 12/27/2013
• Published: 12/24/2015
• Est. Priority Date: 12/28/2012
• Status: Active Grant

• Current Assignee: Empresa Brasileira de Pesquisa Agropecuária, Universidade Federal Do Rio Grande Do Sul.
• Original Assignee: Universidade Federal Do Rio Grande Do Sul.
• Inventors: Mattar da Silva, Maria Cristina, Grossi de S, Maria Ftima, Pereira Firmino, Alexandre Augusto, Martins de S, Diogo, Ramos Coelho, Roberta, Lima Pepino de Macedo, Leonardo, Tristan Lourenco, Isabela
• Granted Patent: US 10,822,609 B2
• Time in Patent Office: Days
• US Class Current: 1/1
• CPC Class Codes: A01N 43/54 1,3-Diazines; Hydrogenated 1,3-diazines
• CPC Class Codes: A23V 2002/00 Food compositions, function of food ingredients or processes for food or foodstuffs
• CPC Class Codes: C12N 15/113 Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing when used in plants C12N15/8218
• CPC Class Codes: C12N 15/1137 against enzymes viral enzymes C12N15/1131; receptors C12N15/1138
• CPC Class Codes: C12N 15/8218 Antisense, co-suppression, viral induced gene silencing [VIGS], post-transcriptional induced gene silencing [PTGS]
• CPC Class Codes: C12N 15/8286 for insect resistance
• CPC Class Codes: C12N 2310/14 interfering N.A.
• CPC Class Codes: C12N 2310/141 MicroRNAs, miRNAs
• CPC Class Codes: C12N 2310/531 Stem-loop; Hairpin
• CPC Class Codes: Y02A 40/146 Genetically Modified [GMO] plants, e.g. transgenic plants

Abstract

The present invention relates to the control of infestation of pests by inhibiting or reducing the expression of genes of the family of chitin synthase. The invention further provides methods and compositions for controlling pests by feeding them with one or more double-strand RNA molecules provides by the present invention. The invention further describes a method of obtaining transgenic plants that express double-strand RNA molecules. The present invention is preferably used for cotton-plants.

Claims

1. An isolated nucleic acid molecule, characterized by being selected from the group consisting of:

a. an isolated nucleic acid molecule comprising a nucleic acid sequence substantially similar to the sequences selected from the group of;

SEQ ID No 1, SEQ ID No 2;

b. an isolated nucleic acid that hybridizes a nucleic acid sequence indicated by the sequences selected from the group of SEQ ID No 1, SEQ ID No 2, under 5×

SSC wash conditions, formamide at 50% and 42°

C.;

for 10 min;

c. a fragment of at least 19 continuous nucleotides of a nucleic acid sequence selected from the group of SEQ ID No 1, SEQ ID No 2, wherein the ingestion, by coleopterous pests, of the double-strand ribonucleotide sequence, comprising at least one complementary strand of said fragment, inhibits or reduces the proliferation of said pest; and

d. a complement of the sequence of (a), (b) or (c).

2. The isolated nucleic acid molecule according to claim 1, characterized by being selected from the group of SEQ ID No 1, SEQ ID No 2.

3. A chimeric gene, characterized by comprising:

a) a polynucleotide according to claim 1; and

b) an active promoter, operatively linked to the polynucleotide defined in (a).

4. A gene construct characterized by comprising one or more chimeric genes according to claim 3.

8. A vector characterized by comprising the isolated nucleic acid molecule according to claim 1.

9. The vector according to claim 8, characterized in that said vector is capable of promoting the expression of the molecule of interest or a fragment thereof.

10. A double-strand ribonucleotide sequence, characterized in that by being produced from the expression of a nucleic acid molecule according to claim 1.

11. The double-strand ribonucleotide sequence according to claim 10, characterized in that the ingestion or assimilation, by coleopterous pest of plants, of the double-stand ribonucleotide sequence, comprising at least one complementary strand of said fragment, inhibits or reduces the proliferation of said pest.

12. A transformed cell, characterized by comprising a nucleic acid according to claim 1.

13. The cell according to claim 12, characterized by being a prokaryotic cell.

14. The cell according to claim 12, characterized by being a eukaryotic cell.

15. The cell according to claim 12, characterized by being a plant or bacterial cell.

16. A transformed plant, characterized by comprising a nucleic acid molecule according to claim 1.

17. The plant according to claim 16, characterized in that the nucleic acid molecule is expressed in a vegetable cell, in the form of a double-strand ribonucleotide sequence, and the ingestion of diet containing an inhibiting amount, to insect pest, of said double-strand ribonucleotide sequence inhibits or reduces the proliferation of said pest.

18. The plant according to claim 17, characterized in that the insect pest is selected from the group consisting of Anthonomus grandis, Diabrotica virgifera, Tenebrio molitor, Tribolium castaneum and Hypothenemus hampei, Phoracantha semipunctata, Lixus angustatus, Acanthoscelides obtectus and other coleoptera that cause damages to woods and agronomically important plants of the families Scolytidae, Cerambycidae, Curculionidae and Bostrichida.

19. A commercial product, characterized by being produced from a plant according to claim 1, wherein the commercial product comprises a detectable amount of said nucleic acid molecule, or of a ribonucleotide expressed therefrom.

21. A method for controlling infestation of coleoptera, characterized in that it comprises supplying, in the diet of a coleopterous pest an agent comprising a nucleic acid molecule according to claim 1.

27. A method for improving the yield of cultivated plants subject to infestation by insect pests, characterized by comprising the steps of:

e. introducing a nucleic acid molecule according to claim 1 into said plant;

f. growing the plant obtained in “

a”

so as to enable expression of said nucleic acid molecule, wherein this expression inhibits or reduces the proliferation of said pest.

28. The method according to claim 27, characterized in that the expression of the nucleic acid molecule produces an RNA molecule that suppresses at least one first target gene in an insect pest that has ingested a portion of said plant where said target gene performs at least one function that is essential to survival of the insect to be selected from the group of differentiation and development of cuticula, egg formation, larval maturation, larval stage transition, pupation, cell division, energetic metabolism, respiration and formation and hardening of the cuticula.

29. The method according to claim 28, characterized in that the insect pest is selected from the group consisting of Anthonomus grandis, Diabrotica virgifera, Tenebrio molitor, Tribolium castaneum, Phoracantha semipunctata, Lixus angustatus, Acanthoscelides obtectus and other coleoptera that cause damages to woods and agronomically important plants of the families Scolytidae, Cerambycidae, Curculionidae and Bostrichida.

30. A method for producing a commercial product, characterized by comprising obtaining a plant as defined in claim 16, or a part thereof, and the preparing a commercial product from the plant or a part thereof.

31. A method of producing a food or animal feed, characterized by comprising obtaining a plant as defined in claim 16, or a part thereof, and preparing a food or animal feed from said plant or a part thereof.

32. A method for controlling infestation of coleoptera, characterized in that it comprises supplying, in the diet of a coleopterous pest an agent comprising a double-strand ribonucleotide sequence according to claim 10.

5. A gene construct characterized by comprising:

(a) a first region comprising a nucleic acid sequence of at least 19 consecutive nucleotides having substantial sequence similarity with at least about 19 consecutive nucleotides of the sequence of sense nucleotides as described in SEQ ID No 1, SEQ ID No 2;

(b) a second region comprising a nucleotide sequence of about 19 consecutive nucleotides having a substantial sequence similarity with the complement of about at least 19 consecutive nucleotides of the sequence of sense nucleotide as described in SEQ ID No 1, SEQ ID No 2.

6. The gene construct according to claim 5, characterized in that the first and the second region are capable of forming a double-strand region, which may have, in addition to the total length of the first and of the second region, a spacing region between them containing at least about 3 nucleotides.

7. The gene construct according to claim 6, characterized in that the spacing sequence is an intron.

20. A method for producing transgenic eukaryotic organisms, in which the expression of a target gene in the cells of the organism is reduced, characterized by comprising the steps of:

I) promoting a nucleic acid molecule according to claim;

II) inserting the molecule obtained in “

I”

into a cell or cells of the organism to produce a transgenic cell or cells; and

III) growing or regenerating a transgenic eukaryotic organism of the transgenic cell or cells.

22. The method according to claim 20, characterized in that the eukaryotic organism cell further comprises a polynucleotide sequence encoding a pesticidal agent.

23. The method according to claim 22, characterized in that the pesticidal agent is selected from the group consisting of patatin, a in insecticidal protein of Bacillus thuringiensis, an insecticidal Xenorhabdus protein, an insecticidal Photorhabdus protein, an insecticidal Bacillus laterosporous protein, an insecticidal Bacillus sphaericus protein, and a lignin.

24. The method according to claim 20, characterized in that the doleopterous pest is selected from the group consisting of Anthonomus grandis, Diabrotica virgifera, Tenebrio molitor, Tribolium castaneum e Hypothenemus hampei, Phoracantha semipunctata, Lixus angustatus, Acanthoscelides obtectus and other coleoptera that cause damages to woods and agronomically important plants of the families Scolytidae, Cerambycidae, Curculionidae and Bostrichida.

25. The method according to claim 20, characterized in that the actuation mode of the nucleic acid molecule or of the double-strand ribonucleotide sequence, upon being ingested or assimilated by the pest, is that of suppression or reduction of the expression of a gene that performs a function that is essential to the survival of the insect.

26. The method according to claim 25, characterized in that the function essential to the survival of the insect is selected from the group of differentiation and development of the cuticula, egg formation, larval maturation, transition of larval stage, pupation, digestion and assimilation of nutrients, protection against pathogens.