A dna fragment with insecticidal effect and its application in agriculture

A fragment and sequence technology, applied in the field of biological transgenics, can solve the problems of affecting toxin activity, insecticidal effect, and lectin protein activity, etc., to achieve the effect of improving activity and ensuring stability

Active Publication Date: 2019-02-22
北京依科曼生物技术股份有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the barriers of the insect's outer skin and the peritrophic membrane, the spider toxin cannot effectively enter the insect body/ion channel, directly affecting its insecticidal effect. Therefore, chitinase and plant lectin are added to the spider toxin , chitinase can break through the barrier effect of insect outer skin, and plant lectin can bind to the surface of insect peritrophic membrane, glycoconjugates of digestive tract epithelial cells, or glycosylated digestive enzymes to induce lesions in insect digestive tract for the purpose of killing insects
For example: the prior art publication number is the document of CN103154022A, chitinase is connected on the spider poisonous sequence through the secretion signal sequence, and the spider toxin sequence is connected with the plant lectin sequence to reduce the protective effect of the insect exoskeleton, ma

Method used

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  • A dna fragment with insecticidal effect and its application in agriculture
  • A dna fragment with insecticidal effect and its application in agriculture
  • A dna fragment with insecticidal effect and its application in agriculture

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] In the present invention, the expression selection sequence fragments 1, 3, 5, and 7 are sequentially connected in the plant transformation of upland cotton to form the DNA fragment of the present invention. For cotton transgenic methods, please refer to transgenic cotton methods and protocols (edited by Baohong zhang). The CaMV35S promoter is used as the promoter of the present invention, and the target gene is constructed into the vector pCIMBIA2300, and the transgenic process is completed by the tissue culture method mediated by Agrobacterium. The method is very mature and well-known in the art, so it will not be repeated here. The hypocotyls of 23 cotton varieties of China Cotton Research Institute were selected as the recipient material to complete the transgenic process.

[0027] Positive cotton plants were obtained by Kana resistance and PCR detection, cotton needles were selected, total protein was extracted, and whether the protein was expressed in cotton was d...

Embodiment 2

[0034] The present invention is compared with the expression amount and insecticidal effect of the prior art

[0035] The selected sequence fragments 1, 10, 12, and 7 are sequentially connected to form the DNA fragment of the present invention, which is marked as fragment A.

[0036] Selected sequence fragments 9, 12, and 7 are sequentially connected to form a DNA fragment described in the prior art, marked as B. That is to say, the plant secretion leader peptide is connected with chitinase, spider toxin and GNA.

[0037] The above two fragments were used to construct vectors for transgenic comparison of Arabidopsis thaliana.

[0038] Transgenic method: Arabidopsis transgenic method is a mature method in the field of biological research, so it will not be described here. The reference method in this example comes from Simplified Arabidopsis Transformation Protocol, SteveClough and Andrew Bent, University of Illinois at Urbana-Champaign

[0039] During the transformation proc...

Embodiment 3

[0045] The corresponding transgenic Arabidopsis of the present invention is compared with the transgenic Arabidopsis without auxiliary sequence and the wild type, and the anti-aphid effects are compared.

[0046] The present invention: the selected sequence fragments 1, 14, 16, and 7 are sequentially connected to form the DNA fragment of the present invention. Transformation was carried out on Arabidopsis thaliana, and experiments were carried out on the obtained T3 generation plants, and the three strains with high, medium and low expression levels of the fusion protein were named Z-1, Z-2, and Z-3;

[0047] Control 1: The new DNA fragment 1 formed by removing the auxiliary sequence of the above DNA fragments, that is, 1, 16, and 7 in sequence, was transgenic in the same way to obtain T3 generation plants, and the fusion protein expression levels were measured as high, medium and low. The strains were named ZWF-1, ZWF-2, ZWF-3;

[0048] Control 2: wild-type Arabidopsis;

[...

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Abstract

The invention relates to a DNA fragment having an insecticidal effect and agricultural application of the DNA fragment, belonging to the technical field of biological transgenosis. The DNA fragment having the insecticidal effect consists of a signal peptide sequence, a toxin protein sequence, a phytolectin sequence and an auxiliary sequence; the DNA fragment having the insecticidal effect is formed by linking the spider toxin sequence and the phytolectin sequence which take an insecticidal effect in a special mode and by adding the auxiliary sequence to increase expression quantity, and the formed DNA fragment has a positive effect in the aspects of pest repelling and control. The agricultural application mode of the DNA fragment includes the follows: DNA is transcribed and translated into fusion protein, or a polynucleotide composition, which is constructed by the DNA fragment and a plant promoter, is transformed and expressed in a plant body so as to form a resistant plant.

Description

Technical field: [0001] The invention belongs to the technical field of biological transgenes, and relates to a DNA segment with insecticidal effect and its application in agriculture. Background technique: [0002] In the 1980s, people have begun to use biotransgenic technology to control pests and cultivate insect-resistant plants. However, until now, the insect-resistant gene is still dominated by the Bacillus thuringiensis toxin gene (Bt). After years of application in cotton, it has been found that sucking pests such as miridae have gradually risen from secondary pests to major pests, mainly because the above-mentioned insect-resistant genes have no obvious effect on sucking mouthparts pests. At the same time, the long-term use of a single insect-resistant gene can lead to an increase in pest resistance, which also limits the further application of such insect-resistant genes. Therefore, the development of a new environmentally friendly and efficient insect-resistant g...

Claims

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Application Information

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IPC IPC(8): C12N15/62C12N15/82A01H5/00A01H6/20A01H6/82A01N47/44A01P7/04
Inventor 陈育邓青李永亮王广兴梁兴慧杜进平周震
Owner 北京依科曼生物技术股份有限公司
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