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Insect resistant transgenic turf grass

An insect resistance, turfgrass technology, applied in genetic engineering, biochemical equipment and methods, plant products, etc., can solve the problems of lack of insect resistance genes, technical difficulties, hindering development, etc.

Inactive Publication Date: 2009-08-12
PHYLLOM
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

These two points demonstrate that the process of producing a complete transgenic turfgrass plant or a complete ornamental plant presents some serious technical difficulties that hinder the process
[0006] In addition, technical difficulties are also reflected in the generation of transformable plant cells or callus. The lack of an effective insect resistance gene, especially a gene that is highly effective against scarab insects, is the difficulty of producing insect-resistant transgenic lawns. Another major obstacle for grasses and other ornamental plants

Method used

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  • Insect resistant transgenic turf grass
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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0083] Example 1: Cloning cry8Da gene

[0084] The cry8Da gene was cloned from Bacillus thuringiensis strain SDS-502 according to the method described in the article published by Asano et al. ((2003), Biological Control 28, 191-196, which is hereby incorporated by reference in its entirety). A fragment of the cry8Da gene containing the active region was PCR amplified using the following two sequences as primers and the cloned cry8Da gene as a template,

[0085] 5'-GGATCCCATGAGTCCAAATAATCAAAATG (SEQ ID NO: 1)

[0086] 5'-CCCGGGTCACACATCTAGGTCTTCTTCTGC (SEQ ID NO: 2)

[0087] PCR was performed in a 100 ul reaction mixture containing 10 pg of template DNA and appropriate concentrations of other components (known to those skilled in the art). In one example, the PCR mix contains: 10u110X buffer, 2u1d-NTP, 2.5ul primer 1 (20uM), 2.5ul primer 2 (20uM), 2ul thermostable DNA polymerase, 1ul template DNA and 80ul water. According to the given plasmid manufacturing instructions, the ...

Embodiment 2

[0088] Example 2: Cloning of beetle active genes other than cry8Da

[0089] The Cry43Aa gene (GenBank accession number: AB115422) encodes a protein toxin from scarab beetles, such as beetle beetle and bean beetle. These are serious pests of turfgrass, and their predation can cause considerable damage. The Cry43Aa gene was cloned from a strain of Bacillus japonicus that was isolated from diseased green beetles and Heterophytic beetles. The full-length cry43Aa gene was amplified by PCR using the two following sequences as primers.

[0090] 5'-GGATCCATGAATCAGTATCATAACCAAAACG (SEQ ID NO: 4)

[0091] 5'-CCCGGGTTACTTTTCCATACAAATCAATTCCAC (SEQ ID NO: 5)

[0092] In the PCR reaction mixture, 1 ul of genomic DNA prepared from B. poilliae Hime strain containing about 100 ng of DNA was used.

[0093] The cry8Ca gene (GenBank accession number: U04366) also encodes a scarab protein toxin. The cry8Ca gene was cloned in Bacillus thrungiensis buibui strain obtained from Fermentation Rese...

Embodiment 3

[0098] Example 3: Cloning of the crylCa gene

[0099] The CrylCa gene (gene bank accession number: X07518) encodes a protein toxin of armyworm complexes, such as beet armyworm, fall armyworm, and American armyworm. These are serious pests of turfgrass and can cause considerable damage. The CrylCa gene was cloned in B. thringiensis subsp. aizawai HD133 strain obtained from USDA, Agricultural Research Service, Northern Research Center, Illinois, USA. The full-length crylCa gene was amplified by PCR using the two following sequences as primers,

[0100] 5'-GGATCCCATGGAGGAAAATAATCAAAATCAATGC (SEQ ID NO: 8)

[0101] 5'-CCCGGGTTATTCTCTCATAAGGAGTAATTCC (SEQ ID NO: 9)

[0102] In the PCR reaction mixture, 1 ul of genomic DNA prepared from HD-133 strain containing 100 ng DNA was used. The PCR amplified crylCa gene was cloned in pBI221 as described in Example 1 and, after testing for anti-beetle activity, was employed in turfgrass transformation as described in Example 10, below.

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Abstract

This invention relates to transgenic turf grass having resistance to turf grass pest insects. Methods of producing such insect-resistant transgenic turf grass lines are disclosed. The invention further relates to the use of the insect-resistant transgenic turf grass to eliminate or reduce the usage of spray-on insecticides to protect the turf grass from insect damage.

Description

technical field [0001] The invention includes a transgenic lawn grass, which is resistant to certain insects attacking the lawn grass. The present invention also includes methods of producing insect-resistant transgenic turfgrasses. The invention also provides the use of the transgenic grass to protect the lawn from insect attack. Background technique [0002] Bacillus thuringiensis is a spore-shaped, rod-shaped, Gram-positive bacterium closely related to the so-called Bacillus cereus. We found a large number of Bacillus thuringiensis strains that make up the subspecies of Bacillus thuringiensis, such as Bacillus thuringiensis, Bacillus thuringiensis subsp. Kustak, Bacillus thuringiensis subsp. and de Barjac's classification scheme proposed in 1963 to classify. B. thuringiensis is distinct from other Bacillus species in the production of intracellular crystals, which are insoluble protein precipitates, many of which are insecticidally active against different species of i...

Claims

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Application Information

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IPC IPC(8): A01H5/00C12N15/82
CPCC12N15/8286Y02A40/146
Inventor 广田贡浅野真一郎
Owner PHYLLOM
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