Polynucleotides encoding truncated sucrose isomerase polypeptides for control of parasitic nematodes

A technology of sucrose isomerase and polynucleotide, which is applied in the field of controlling soybean cyst nematodes and can solve problems such as yield reduction

Inactive Publication Date: 2009-12-09
BASF PLANT SCI GMBH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Actively feeding nematodes thus steal essential nutrients from the plant, resulting in lower yields

Method used

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  • Polynucleotides encoding truncated sucrose isomerase polypeptides for control of parasitic nematodes
  • Polynucleotides encoding truncated sucrose isomerase polypeptides for control of parasitic nematodes
  • Polynucleotides encoding truncated sucrose isomerase polypeptides for control of parasitic nematodes

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0081] Example 1: Cloning of a polynucleotide encoding an N-terminal truncated form of sucrose isomerase

[0082] About 0.1 μg of plasmid DNA containing the sequence of Erwinia sucrose isomerase AF279281 was used as DNA template in the PCR reaction. Primers used for PCR amplification of the truncated sucrose isomerase sequence are shown in Table 1 and were designed based on the AF279281 sequence. The primer sequence described by SEQ ID NO: 12 contained an Ascl restriction site for easy cloning. The primer sequence described by SEQ ID NO: 13 contained an XhoI restriction site for easy cloning. The primer sequences described by SEQ ID NO: 12 and SEQ ID NO: 13 were used to amplify the truncated sucrose isomerase sequence.

[0083] The size of the amplified DNA fragment was verified by standard agarose gel electrophoresis and the DNA was extracted from the gel. The purified fragment was TOPO cloned into pCR2.1 using the TOPO TA cloning kit following the manufacturer's instructi...

Embodiment 2

[0086] Example 2: Vector Construction for Transformation

[0087] In order to assess the function of the cloned Erwinia polynucleotide encoding the N-terminal truncated form of the sucrose isomerase-encoding gene, nucleosides 1-1464 corresponding to SEQ ID NO: 1 were synthesized using the restriction enzymes AscI and XhoI. Acidic gene fragments were cloned downstream of the promoter to generate expression vectors described in Table 2 operably linked to the promoter sequence. Syncytium preferred promoters include soybean MTN3 promoter SEQ ID NO: 7 (p-47116125) (USSN 60 / 899,714), Arabidopsis thaliana peroxidase POX promoter SEQ ID NO: 8 (p-At5g05340) (USSN 60 / 876,416), Arabidopsis TPP 6-phosphate trehalose phosphatase promoter SEQ ID NO: 9 (p-At1g35910) (USSN 60 / 874,375), MTN21 promoter SEQ ID NO: 10 (p-At1g21890) (USSN60 / 743,341) and At5g12170-like promoter SEQ ID NO: 11 (USSN 60 / 899,693). The plant selectable marker in the binary vector described in Table 2 is the herbicide...

Embodiment 3

[0090] Example 3: Generation of Transgenic Soybean Hairy Roots and Nematode Bioassays

[0091] The binary vectors RJT21, RJT22, RJT23, RJT51, RJT52 and RJT53 were transformed into Agrobacterium rhizogenes K599 strain by electroporation. The transformed Agrobacterium strains were used to induce soybean hairy root formation using known methods. Non-transgenic hairy roots were also generated from soybean cultivars Williams 82 (SCN susceptible) and Jack (SCN resistant) by using untransformed Agrobacterium rhizogenes to serve as controls for nematode growth in the assay.

[0092] Bioassays were performed to assess nematode resistance in transgenic hairy roots transformed with vector and non-transgenic hairy roots from Williams 82 and Jack as controls. Several independent hairy root lines were generated from each binary vector transformation for bioassays. For each transformed line, several replicate wells were inoculated with SCN according to the method outlined above. Four week...

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Abstract

The invention provides polynucleotides encoding N-terminal truncated forms of sucrose isomerase polypeptides which are capable of conferring increased nematode resistance in a plant. The invention also provides methods of producing transgenic plants with increased nematode resistance, seeds of such transgenic plants, and expression vectors, all of which comprise the polynucleotides of the invention.

Description

[0001] Cross References to Related Applications [0002] This application claims priority to US Provisional Application Serial No. 60 / 900,228, filed February 8,2007. 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 the 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 types of vegetables, fruits and ornamentals, causing $1 trillion in crop losses worldwide. A common type of nematode is the root-knot nematode (RKN), which feeds on roots and produces characteristic galls. Other root-feeding nematodes are the cyst type and the lesion type, which are more host specific. [0005] Nematodes are ub...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/82C12N9/90
CPCC12N9/90A01N63/02C12N15/8285Y02A40/146
Inventor K·赫伯斯B·茨尔施R·桑切斯-费尔南德斯R·阿申齐J·托斯伯格A·威格黄翔S·乔杜里
Owner BASF PLANT SCI GMBH
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