Trypsin inhibitor gene with insecticide resistance in populus nigra and application thereof

A trypsin inhibition and gene technology, applied in the fields of molecular biology and biology, can solve the problems of deterioration of the ecological environment, high production costs, harm to humans and animals, etc., to reduce the survival rate, reduce malignant pollution, maintain balance and sustainable development Effect

Inactive Publication Date: 2012-03-28
TIANJIN UNIV
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  • Application Information

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Problems solved by technology

At present, the direct economic loss caused by forest diseases and insect pests in my country alone has reached 100 billion yuan each year, and the use of chemical pesticides to control forest diseases and insect pests not only has high pro

Method used

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  • Trypsin inhibitor gene with insecticide resistance in populus nigra and application thereof
  • Trypsin inhibitor gene with insecticide resistance in populus nigra and application thereof
  • Trypsin inhibitor gene with insecticide resistance in populus nigra and application thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0013] 1. Isolate the trypsin inhibitor gene from Populus nigra, a model plant of the genus Populus.

[0014] 1.1 Search the KTI gene in the gene database by BLAST, and perform multiple sequence alignment.

[0015] 1.2 Design gene-specific primers, and use reverse transcription-polymerase chain reaction to isolate the member genes of the Kunitz-type trypsin inhibitor gene family from Populus nigra.

[0016] 2. Analyze the response characteristics of the above-mentioned Kunitz-type trypsin inhibitor genes by real-time quantitative PCR and semi-quantitative methods.

[0017] 3. Select the highly inducible Kunitz type trypsin inhibitor gene, and clone its full-length coding sequence: PnKTI A6 DNA sequence (shown in SEQ ID NO.1) and PnKTI D2 DNA sequence (shown in SEQ ID NO.2) :

[0018] 24 hours after being bitten by the black poplar worm, the leaves were collected and the results of the RT-PCR test were performed. Using the 18S rRNA gene as a reference, the relative expression...

Embodiment 2

[0020] Response characteristics of PnKTI A6 DNA sequence (shown in SEQ ID NO.1) and PnKTI D2 DNA sequence (shown in SEQ ID NO.2) induced by artificial mechanical damage and spring inchworm bite.

[0021] Overexpression of the poplar protease inhibitor gene in Arabidopsis enhances resistance to diamondback moth

[0022] 1. Use the known sequences shown in the isolated SEQ ID NO.1 and SEQ ID NO.2 to design primers

[0023] Upstream primer 5'-ATGAAGATCACTAAAT-3' (shown in SEQ ID NO.3);

[0024] Downstream primer 5'-AATGGTGTCAGAATGA-3' (shown in SEQ ID NO.4);

[0025] The amplified fragment was digested and ligated into pCGN1548 binary vector, and then transformed into Agrobacterium C58 by electric shock for Arabidopsis transformation.

[0026] 2. Transformation of Arabidopsis thaliana with Agrobacterium

[0027] Agrobacterium was inoculated in 10ml of YEB medium, cultured overnight at 28°C, and then transferred to 200ml of YEP medium at a ratio of 1:50 for expansion until the ...

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Abstract

The invention discloses a trypsin inhibitor gene with insecticide resistance in populus nigra and application thereof. The trypsin inhibitor gene with insecticide resistance in populus nigra is shown as SEQ ID NO.1 or SEQ ID NO.2 in a sequence table; populus nigra Kunitz type trypsin inhibitor gene (PnKTIs) separated in the invention has high inductivity to two external inducing stimulations, namely mechanical damage and insect bite. Experiments prove that: the trypsin inhibitor gene has insecticide resistance, and the survival rate of lepidopteran insects is remarkably reduced. According to the trypsin inhibitor gene with insecticide resistance in populus nigra and the application thereof, a novel insecticide-resistance gene is provided to related searchers working on insect-resistant transgenic engineering and biological control, and a novel insecticide resistance means is developed on the basis of the application, pesticide use and vicious pollution to the environment are reduced, and balance of an ecological system and sustainable development are maintained.

Description

technical field [0001] The invention belongs to the fields of molecular biology and biotechnology. It relates to a black poplar gene and its application. Background technique [0002] Tree plants are the main life forms in most ecosystems and have the largest biomass and biological productivity in all terrestrial ecosystems, so they have important economic and ecological value. At present, the direct economic loss caused by forest diseases and insect pests in my country alone has reached 100 billion yuan each year, and the use of chemical pesticides to control forest diseases and insect pests not only has high production costs, but also causes the deterioration of the ecological environment, and also causes serious harm to humans and animals. Using genetic engineering Breeding pest-resistant plants has become an important means of plant pest control. In the work of using genetic engineering to improve the ability of forest species to resist diseases and insect pests, findin...

Claims

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Application Information

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IPC IPC(8): C12N15/15A01N65/08A01P7/04
Inventor 王洁华杨少辉宋英今陈姗
Owner TIANJIN UNIV
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