Nucleic acid sequence of wild emmer alpha-amylase inhibiting factor and its application

An amylase inhibition, emmer technology, applied in application, genetic engineering, plant genetic improvement, etc., can solve problems such as differences in inhibitory activity and inability to select inhibitor genes.

Inactive Publication Date: 2007-06-06
SICHUAN AGRI UNIV
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  • Claims
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AI Technical Summary

Problems solved by technology

The 24kDa wheat dimeric α-amylase inhibitor (WDAI) is one of the natural insect-resistant proteins, which can inhibit the α-amylase of certain pests. The inhibitors in this protein family all have 124 amino acid residues The two factors of WDAI-0.19 and WDAI-0.53 have only 7 substitutions in the amino acid sequence, and they have 94% homology. Insect α

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0075] 1. Genomic DNA extraction:

[0076] Genomic DNA was extracted by CTAB method. Take 2 g of fresh young leaves, grind them into fine powder with liquid nitrogen, add 2×CTAB extract (2% CTAB; 1.4M NaCl, 0.1M Tris-HCl, pH8.0, 0.1MEDTA, pH8.0) preheated to 65°C , add 2% β-mercaptoethanol) 15ml before use, and mix well.

[0077] 65°C water bath for 30-45min, during which shake gently to mix

[0078] After cooling to room temperature, add an equal volume of chloroform:isoamyl alcohol (24:1), mix gently until the supernatant becomes milky, and centrifuge at 4000rpm for 10min

[0079] Take the supernatant, add an equal volume of isopropanol, and place in an ice bath to precipitate DNA

[0080] Hook up the DNA, wash twice with 70% alcohol, wash once with absolute ethanol, air-dry the DNA, and dissolve in an appropriate amount of TE solution with pH 8.0. Add RNase to a final concentration of 100 μg / μl

[0081] Detection of DNA concentration and quality by agarose gel electrop...

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PUM

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Abstract

The present invention discloses coding gene sequence and amino acid sequence of wild emmer alpha-amylase inhibiting factor, and belongs to the field of biological gene engineering technology. The sequences are shown in SEQ ID Nos. 1-6 of the sequence list. The gene sequence may be introduced via different methods to wheat to culture excellent wheat breed with high pest resistance. In the same time, specific convenient molecular marker system may be established based on the cloned gene sequence for directive breeding. Furthermore, yeast expression vector containing the alpha-amylase inhibiting factor coding gene may be established and expressed in yeast to obtain alpha-amylase inhibiting factor, which is used as botanical pesticide added into common pesticide product.

Description

technical field [0001] The invention belongs to the technical field of biogenetic engineering, and in particular relates to a nucleic acid sequence of a gene encoding a wheat α-amylase inhibitor and the application of the sequence to improve insect resistance of wheat through biotechnological methods. Background technique [0002] Due to the attack of various pests, the world's food production will be reduced by 37% every year. The in-depth research and wide application of chemical insecticides have achieved remarkable insect control effects in production, but at the same time, the cost of chemical insecticides to control insect pests is high, the environment is polluted, and it is easy to cause insecticide resistance and ecological balance imbalance. Therefore, in recent years, the focus of research has been on how to improve the insect resistance of plants, and the breeding for the introduction of heterologous (plant, animal, microorganism) insect resistance genes has been...

Claims

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Application Information

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IPC IPC(8): C12N15/29C07K14/415C12N15/63
Inventor 郑有良王际睿魏育明颜泽洪李伟
Owner SICHUAN AGRI UNIV
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