Use of alanine racemase genes to confer nematode resistance to plants

A technology of alanine racemase and transgenic plants, applied in plant products, genetic engineering, plant genetic improvement, etc., can solve problems such as yield loss

Inactive Publication Date: 2009-12-16
BASF PLANT SCI GMBH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Actively feeding nematodes in turn steal essential nutrients from the plant, resulting in yield loss

Method used

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  • Use of alanine racemase genes to confer nematode resistance to plants
  • Use of alanine racemase genes to confer nematode resistance to plants
  • Use of alanine racemase genes to confer nematode resistance to plants

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0085] Example 1: Cloning of the gene encoding alanine racemase

[0086] Alr (SEQ ID NO: 7) and DadX (Seq ID NO: 5), two forms of alanine racemase, were cloned from E. coli genomic DNA using the PCR primers shown in Table 1 .

[0087] Table 1: Primers used to clone the gene encoding ARLNCP.

[0088] Primer

Embodiment 2

[0089] Example 2: Vector construction for transformation and generation of transgenic roots

[0090] The PCR product generated in Example 1 was sequenced and cloned into a mass expression vector containing a syncytia-preferred (nematode-induced) promoter. Syncytium preferred promoters include soybean MTN3 SEQ ID NO: 9 (p-47116125) (USSN 60 / 899,714), Arabidopsis peroxidase POX SEQ ID NO: 10 (p-At5g05340) (USSN 60 / 876,416) and Arabidopsis TPP trehalose-6-phosphate phosphatase SEQ ID NO: 11 (p-At1g35910) (USSN 60 / 874,375). Constitutive super promoters were also used. The selection marker used for transformation was a mutated acetohydroxyacid synthase (AHAS) gene from Arabidopsis that confers resistance to the herbicide ARSENAL (imazepyr, BASF Corporation, Mount Olive, NJ). Expression of the mutant AHAS is driven by the Arabidopsis actin 2 promoter.

[0091] Table 2: Expression vectors comprising SEQ ID NO: 5 or 7

[0092] carrier

Embodiment 3

[0093] Example 3: Generation of transgenic soybean hairy-roots and nematode bioassays

[0094] Vectors RSH118, RSH120, RSH122, RSH117, RSH125, RSH127, RSH129 and RSH124 were transformed into A. rhizogenes K599 strain by electroporation. The transformed Agrobacterium strains were used to induce soybean hairy root formation using known methods. Non-transgenic hairy roots from soybean cultivars Williams 82 (SCN susceptible) and Jack (SCN resistant) were also produced by using untransformed A. rhizogenes as controls for nematode growth in the assay.

[0095] Nematode resistance was evaluated by bioassays on transgenic hairy roots transformed with the vector and non-transgenic hairy roots from Williams 82 and Jack as controls. Hairy root cultures of each line occupying at least half of the wells were inoculated with surface decontaminated second stage larvae (J2) of soybean cyst nematode (SCN) race 3. The plates were then sealed and placed back into the incubator at 25°C in the d...

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PUM

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Abstract

The invention provides alanine racemase encoding polynucleotides, which are capable of conferring increased nematode resistance in a plant. Specifically, the invention relates to methods of producing transgenic plants with increased nematode resistance, expression vectors comprising polynucleotides encoding alanine racemase, and transgenic plants and seeds generated thereof.

Description

[0001] Cross reference with related applications [0002] This application claims priority to US Provisional Application Serial No. 60 / 899,746, filed February 6, 2007. field of invention [0003] The present invention relates to the control of nematodes, especially soybean cyst nematodes. Disclosed herein are methods of producing transgenic plants with increased nematode resistance, expression vectors comprising polynucleotides encoding functional proteins, and transgenic plants and seeds produced therefrom. Background of the invention [0004] Nematodes are microscopic worm-like animals that feed on the roots, leaves and stems of more than 2,000 species of vegetables, fruits and ornamentals, causing an estimated $100 billion in crop losses worldwide. One common type of nematode is the root-knot nematode (RKN), whose feeding causes characteristic galls on the roots of a variety of plant species. Other root-eating nematodes are the more host-specific cyst and lesion nematod...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/82C12N15/61C12N9/90A01H5/00
CPCC12N9/90C12N15/8285Y02A40/146
Inventor S·希尔
Owner BASF PLANT SCI GMBH
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