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COT102 insecticidal cotton

A technology of polynucleotides and contiguity, applied in the field of genetic engineering of plants, can solve problems such as impact, emergence of drug-resistant insect varieties, etc.

Active Publication Date: 2005-12-14
SYNGENTA PARTICIPATIONS AG
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Good insect pest control can thus be achieved, but these chemicals sometimes also affect other beneficial insects
Another problem caused by the widespread use of chemical insecticides is the emergence of resistant insect species

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0054] Example 1: Cloning and Transformation

[0055] 1.1 Vector cloning

[0056] Transformation vector pNOV3001 was constructed using standard gene cloning techniques of restriction digestion and fragment ligation from home-made vectors. The vector contains a selectable marker cassette comprising the ubiquitin (UBQ3) promoter, the UBQ3 intron, the gene sequence encoding a protein that confers resistance to hygromycin, and a nos polyadenylation sequence. The vector also contains the target gene expression cassette comprising the actin (Act2) promoter, the Act2 intron, the sequence encoding the VIP3A gene and the nos polyadenylation sequence that has been codon optimized for expression in maize . The selectable marker cassette and the cassette containing VIP3A were cloned into the T-DNA region of vector pNOV3001, between the left and right border sequences. For prokaryotic selection, the vector also contains a gene that confers resistance to the antibiotic, spectinomycin.

...

Embodiment 2

[0070] Embodiment 2: using TaqMan TM Perform COT102 detection

[0071] 2.1 DNA extraction

[0072] Using Wizard TM Magnetic 96 DNA Plant System (Promega, #FF3760), DNA was extracted from leaf tissue according to the manufacturer's instructions, while the following additional steps were performed before starting the method: After grinding the leaf material, 0.9 ml of cotton extract was added to each well buffer (0.2M Tris pH 8.0, 50mM EDTA, 0.25M NaCl, 0.1% v / v 2-mercaptoethanol, 2.5% w / v polyvinyl-pyrrolidone), resuspend the plant tissue and centrifuge the plate at 4,000rpm (2755g) 10 minutes. After pipetting and discarding the supernatant, 300 [mu]l Lysis Buffer A (Promega) was added and from this point on the manufacturer's protocol was used. This method yielded approximately 85 μl of purified genomic DNA at a concentration of approximately 10 ng / μl.

[0073] 2.2 TaqMan PCR reaction

[0074] Setting Up TaqMan Using Standard Reaction Mixtures TM PCR reaction, the rea...

Embodiment 3

[0107] Example 3: COT102 detection by PCR

[0108] 3.1 Genomic DNA extraction

[0109] Genomic DNA was extracted from COT102 as described in Example 2.1.

[0110] 3.2 Multiplex PCR zygosity assay

[0111]PCR primers were designed to bind the cotton genomic DNA sequence upstream of the COT102 cassette insertion site (SEQ ID NO: 18); the COT102 cassette sequence itself (SEQ ID NO: 19); and the cotton genomic DNA sequence (SEQ ID NO: 20). The primer pair SEQ ID NO: 18 and 19 amplified a PCR fragment of 962 bp size when the COT102 insert was present. For each sample to be tested, set up a 50 μl PCR reaction as follows:

[0112] 1x JumpState ReadyMix REDTaq PCR (SigmaP-1107) 25μl

[0113] 40pmole Primer 1 (SEQ ID NO: 18) 4μl

[0114] 40pmole Primer 2 (SEQ ID NO: 19) 4μl

[0115] 40pmole Primer 3 (SEQ ID NO: 20) 4μl

[0116] 40ng genomic DNA 4μl

[0117] wxya 2 O 9μl

[0118] The PCR reaction was heated at 94°C for 2 minutes in a thermal cycler, followed by 35 cycles of: ...

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PUM

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Abstract

The present invention relates to insect resistant transgenic cotton plants. In particular, the present invention relates to a specific event designated COT102. The invention also relates to polynucleotides unique to COT102 events, plants comprising said polynucleotides, and methods of detecting COT102 events. This COT102 event showed a new genotype containing 2 expression cassettes. The first cassette contains a suitable promoter for expression in plants operably linked to a gene encoding a VIP3A insecticidal toxin useful for controlling broad-spectrum leprosy and a suitable polyadenylation signal. Pteroptera insect pests. The second box contains genes that can be used as selectable markers when expressed.

Description

technical field [0001] The present invention relates to the genetic engineering of plants, particularly insect-resistant transgenic cotton plants. The invention also relates to methods of detecting material derived from the plant. Background technique [0002] Plant pests are a major factor in the loss of important agricultural crops around the world. In the United States, approximately $8 billion is lost annually due to attacks on plants by non-mammalian pests, including insects. In addition to crop loss on farmland, insect pests are a burden to vegetable and fruit growers, to producers of ornamental flowers, and to home gardeners. [0003] Insect pests are controlled primarily through the widespread use of chemical insecticides that have activities that inhibit the growth of insects, prevent insect feeding or reproduction, or cause death. Good insect pest control can thus be achieved, but these chemicals sometimes also affect other beneficial insects. Another problem p...

Claims

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Application Information

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IPC IPC(8): C12N15/82C12Q1/68C12Q1/6888
CPCC12N15/8286C12Q1/6888Y02A40/146
Inventor D·M·埃利斯D·V·奈格罗特施良F·A·施特科斯基C·R·托马斯
Owner SYNGENTA PARTICIPATIONS AG
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