Invertebrate microRNAs

Patent metadata

Field	Value
Publication number	US-9528121-B2
Application number	US-201313746500-A
Country	US
Kind code	B2
Filing date	Jan 22, 2013
Priority date	Feb 20, 2007
Publication date	Dec 27, 2016
Grant date	Dec 27, 2016

Abstract

This invention provides plants having resistance to invertebrate pests. More specifically, this invention discloses a non-natural transgenic plant cell expressing at least one invertebrate miRNA in planta for suppression of a target gene of an invertebrate pest or of a symbiont associated with the invertebrate pest. Also provided are recombinant DNA constructs for expression of at least one invertebrate miRNA in planta, a non-natural transgenic plant containing the non-natural transgenic plant cell of this invention, a non-natural transgenic plant grown from the non-natural transgenic plant cell of this invention, and non-natural transgenic seed produced by the non-natural transgenic plants, as well as commodity products produced from a non-natural transgenic plant cell, plant, or seed of this invention. This invention further provides a method of suppressing at least one target gene of an invertebrate pest of a plant or of a symbiont associated with the invertebrate, including providing a plant including the non-natural transgenic plant cell of this invention, wherein the invertebrate is the invertebrate pest, the recombinant DNA is transcribed in the non-natural transgenic plant cell to the recombinant miRNA precursor, and when the invertebrate pest ingests the recombinant miRNA precursor, the at least one target gene is suppressed.

First claim

What is claimed is:

1. A recombinant DNA construct comprising a promoter functional in a plant cell and operably linked to DNA encoding a recombinant miRNA precursor, wherein said recombinant miRNA precursor comprises a single strand of RNA that folds into a secondary structure of at least one miRNA precursor identified from an invertebrate genome, wherein said at least one miRNA precursor comprises at least one stem-loop capable of being processed into a mature miRNA for suppressing at least one target gene.

2. The recombinant DNA construct of claim 1 , wherein said mature miRNA suppresses expression of at least one target gene of an invertebrate or of a symbiont associated with said invertebrate.

3. The recombinant DNA construct of claim 1 , wherein said invertebrate genome is a genome of an invertebrate selected from the group consisting of insects, arachnids, nematodes, molluscs, and annelids.

4. The recombinant DNA construct of claim 1 , wherein said at least one stem-loop comprises a stem region and a loop region, and wherein said loop region comprises a native loop sequence of said miRNA precursor identified from an invertebrate genome.

5. The recombinant DNA construct of claim 1 , wherein said recombinant miRNA precursor is encoded by DNA having a sequence selected from the group consisting of SEQ ID NOs:1-10, 28-35, 44-46, and 79-88, or has an RNA sequence selected from the group consisting of SEQ ID NOs:11-18, 36-43, 57-69, 92, and 96.

6. The recombinant DNA construct of claim 1 , further comprising one or more elements selected from the group consisting of a transgene transcription unit, a gene suppression element, and a transcription regulatory/transcript stabilizing element.

7. A method of causing mortality or stunting in corn rootworm larvae comprising providing in the diet of a corn rootworm larvae at least one RNA transcribed from the recombinant DNA construct of claim 1 , wherein the at least one target gene is at least one corn rootworm essential gene, and wherein ingestion of the single strand of RNA by the corn rootworm larvae results in mortality or stunting in the corn rootworm larvae.

8. The method of claim 7 , wherein the at least one miRNA precursor comprises multiple miRNA precursors.

9. The method of claim 7 , wherein the at least one target gene is multiple target genes.

10. The method of claim 7 , wherein the mature miRNA comprises 19-26 contiguous nucleotides having the reverse complementary sequence of the target gene.

11. The method of claim 7 , wherein the at least one corn rootworm essential gene is at least one gene selected from the group consisting of major sperm protein, alpha tubulin, beta tubulin, vacuolar ATPase and other ATPases, glyceraldehyde-3-phosphate dehydrogenase, RNA polymerase II, chitin synthase, cytochromes, miRNA precursor molecules, miRNA promoters, an ecdysone receptor, peptidylglycine alpha-amidating monooxygenase, sucrase/transglucosidase, translation elongation factor, eukaryotic translation initiation factor 1A, actin, alpha-actinin, a histone, a histone deacetylase, a juvenile hormone receptor, an insect peptidic hormone receptor; cathepsin B-like protease, and Caudal.

12. The method of claim 7 , wherein the at least one corn rootworm essential gene is vacuolar ATPase.

13. A method of suppressing at least one target gene of an invertebrate pest of a plant, comprising providing in the diet of said invertebrate pest at least one RNA transcribed from the recombinant DNA construct of claim 1 ,
wherein the at least one target gene is one or more selected from the group consisting of: (a) an endogenous gene of an invertebrate miRNA natively expressed from the miRNA precursor identified from an invertebrate genome, (b) other than an endogenous gene of an invertebrate miRNA natively expressed from the miRNA precursor identified from an invertebrate genome, and (c) a target gene of a symbiont associated with the invertebrate pest, and
wherein ingestion of the at least one RNA by the invertebrate pest results in suppression of the at least one target gene.

14. The method of claim 13 , wherein the invertebrate pest is selected from the group consisting of an insect, an arachnid, a nematode, a mollusk, and an annelid.

Assignees

• Monsanto Technology Llc

Inventors

• Allen Edwards
• Donovan William P
• Heck Gregory R
• Roberts James K
• Ursin Virginia
• Zhang Yuanji

Classifications

• C12N15/113

  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 title

• C12N15/8285

  for nematode resistance · CPC title

• C12N2310/531

  Stem-loop; Hairpin · CPC title

• C12N2310/141

  MicroRNAs, miRNAs · CPC title

• C12N15/8286 Primary

  for insect resistance · CPC title