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Nematode-resistant transgenic plants

A technology of transgenic plants and plants, applied in the directions of genetic engineering, biochemical equipment and methods, introduction of foreign genetic material using vectors, etc., can solve the problems of plant regulation without removing the resistance of transgenic nematodes, etc.

Inactive Publication Date: 2012-02-22
BASF PLANT SCI GMBH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0014] Although there have been many efforts to control parasitic nematodes using RNA interference, so far, transgenic nematode-resistant plants have not been deregulated in any country

Method used

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Examples

Experimental program
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Effect test

Embodiment 1

[0082] Example 1: Target gene cloning and vector construction

[0083] Using the available soybean target gene cDNA clone sequences, DNA fragments approximately 200-500 bp in length were isolated by PCR, which were used to construct the binary vectors described in Table 1 and discussed in Example 2. This PCR product was cloned into TOPO Pcr2.1 vector (Invitrogen, Carlsbad, CA) and the insert was confirmed by sequencing. This method was used to isolate target gene GmTPP-like, GmGLABRA-like, and GmMIOX-like gene fragments. Alternatively, available cDNA clone sequences of soybean target genes were used to identify DNA fragments approximately 200-300 bp in length that were used to construct the binary vectors described in Table 1 and discussed in Example 2. The DNA sequences of the identified soybean target genes were synthesized, cloned into pUC19 (Invitrogen) vector, and verified by sequencing. DNA synthesis was used to isolate target gene GmHD-like, GmRingH2 finger-like, GmUN...

Embodiment 2

[0090] Example 2 Bioassay of Double-stranded RNA Targeting Soybean Target Gene

[0091] The binary vectors described in Table 1 were used in the rooting plant assay system disclosed in commonly-owned co-pending US Patent Publication 2008 / 0153102. Transgenic roots were generated after transformation with the binary vector described in Example 1. Multiple transgenic root lines were subcultured and inoculated with surface decontaminated race 3SCN second stage larvae (J2) at a level of approximately 500 J2 / well. Four weeks after nematode inoculation, the number of cysts in each well was counted. For each transformation construct, the number of cysts for each line was calculated to determine the average cyst number and standard error for the construct. The cyst counts for each transformation construct were compared to the cyst counts for the empty vector control tested in parallel to determine whether the tested construct resulted in a reduction in cyst number. Bioassay results ...

Embodiment 3

[0092] Example 3: Identification of additional soybean sequences targeted by binary constructs

[0093] As disclosed in Example 2, construct RAW484, when operably linked to an SCN-inducible promoter and expressed in soybean roots, resulted in the expression of a double-stranded RNA molecule targeting SEQ ID NO: 4 and reduced cyst counts. The sense fragment of the GmHD-like gene contained in RAW484 described by SEQ ID NO:6 corresponds to nucleotides 592-791 of the GmHD-like sequence described by SEQ ID NO:4. At least one 21-mer obtained from processing of a double-stranded RNA molecule expressed from RAW484 can target another soybean sequence described by SEQ ID NO:7. Figure 2 shows the amino acid alignment of the identified target of the double-stranded RNA molecule expressed from RAW484 described by the GmHD-like target gene SEQ ID NO:5 and GM50634465 described by SEQ ID NO:8. Figure 6 shows the identified target of the double-stranded RNA molecule expressed from RAW484 desc...

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Abstract

The present invention provides expression vectors encoding double stranded RNAs that target certain plant genes required for maintenance of parasitic nematode infection, nematode-resistant transgenic plants that express such double-stranded RNAs, and methods associated therewith. The targeted plant gene is a GLABRA-like gene, a homeodomain-like gene, a trehalose-6-phosphate phosphatase-like gene, an unknown gene having at least 80% homology to SEQ ID NO:16, a ringH2 finger-like gene, a zinc finger-like gene, or a MIOX-like gene.

Description

[0001] This application claims the benefit of priority to US Provisional Patent Application Serial No. 61 / 161,776, filed March 20, 2009, the entire contents of which are incorporated herein by reference. Background of the invention [0002] Nematodes are tiny nematodes that feed on more than 2,000 species of row crops, vegetables, fruits, and ornamentals, causing an estimated $100 billion in crop losses worldwide. A variety of parasitic nematode species infect crop plants, including root-knot nematode (RKN), cyst-forming nematode and lesion-forming nematode. Root-knot nematode, characterized by causing root gall formation at feeding sites, has a relatively broad host range and is therefore pathogenic on a variety of crop species. Cyst and lesion forming nematodes have a more limited host range but still cause considerable losses in susceptible crops. [0003] The disease-causing nematode is now widespread throughout the United States, with the highest concentrations occurring...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/82
CPCC12N15/8285Y02A40/146
Inventor A·威格B·麦凯格
Owner BASF PLANT SCI GMBH
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