Specific molecular marker of wild emmer alpha-amylase inhabiting factor and its utilization

An amylase inhibition and molecular marker technology, which can be used in applications, sugar derivatives, genetic engineering, etc., can solve problems such as differences in inhibitory activity, and achieve the effects of accurate results, fast and efficient detection, and good promotion prospects.

Inactive Publication Date: 2007-06-06
SICHUAN AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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-amylase, there is also a 100-fold difference in the inhibitory activity of the same amylase
[0005] However, there is no research on the differences between different inhibitors at the gene level, and it is impossible to select a more suitable inhibitor gene for transgenic work to inhibit amylases from different sources

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] 1. Genomic DNA extraction:

[0053] 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.

[0054] 65 ℃ water bath for 30-45min, during which gently shake to mix.

[0055] 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.

[0056] Take the supernatant, add an equal volume of isopropanol, and place in an ice bath to precipitate DNA.

[0057] 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.

[0058] DNA concentration and quality were detected by agarose gel ele...

Embodiment 2

[0103] 1, with the first step of embodiment 1.

[0104] 2. Primer design:

[0105] According to the sequence alignment results of DNAMAN software, the specific primers for specific amplification of the site were used at the 47 bp of the coding region according to the site specificity. The primers are as follows:

[0106] F47: R-terminal primer: 5′ACTCATTT / CGCTTGACTAGGC 3′

[0107] F-terminal primer: 5′AGTACARCGCATGGAGTGG 3′

[0108] F47: length 19bp, GC content 52.6%, molecular weight 5.94kDa, Tm 56.7 degrees

[0109] 3. Gene amplification:

[0110] The PCR reaction system was 5μL of 10×PCR buffer, 1.5U of Ex TaqTM DNA polymerase, 2mmol / LMgCl2, 0.2mmol / L dNTP, 150ng of each primer, 100ng of template DNA, and double distilled water to a total of 50μl. The PCR reaction program was pre-denaturation at 94°C for 4 min; denaturation at 94°C for 45 s, annealing at 57°C for 45 s, extension at 72°C for 1 min, a total of 35 cycles; extension at 72°C for 5 min.

[0111] 4, wit...

Embodiment 3

[0113] 1, with the first step of embodiment 1.

[0114] 2. Primer design:

[0115] According to the sequence alignment results of DNAMAN software, the specific primers for specific amplification of the site were used at the 125 bp coding region according to the site specificity. The primers are as follows:

[0116] F125: R-terminal primer: 5′ACTCATTT / CGCTTGACTAGGC 3′

[0117] F-terminal primer: 5′CTGTCGTCCATTGCTTAG 3′

[0118] F125: length 18bp, GC content 50.0%, molecular weight 5.50kDa, Tm 51.9 degrees

[0119] 3. Gene amplification:

[0120] The PCR reaction system was 5μL of 10×PCR buffer, 1.5U of Ex TaqTM DNA polymerase, 2mmo / LMgCl2, 0.2mmol / L dNTP, 150ng of each primer, 100ng of template DNA, and double distilled water to a total of 50μl. The PCR reaction program was pre-denaturation at 94°C for 4 min; denaturation at 94°C for 45 s, annealing at 62.5°C for 45 s, extension at 72°C for 1 min, a total of 35 cycles; extension at 72°C for 5 min.

[0121] 4, with t...

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Abstract

The present invention discloses four specific primers based on the specific sites in 19bp, 47bp, 125bp and 276bp of wheat alpha-amylase and with one of the specific sites specifically amplified. The four specific primers have simple clear structure and convenient use, and may be used in fast and efficiently establishing molecular marker by means of PCR technology for fast precise detection of different kinds of alpha-amylase inhibiting factor in wheat. The present invention has quick and obvious effect and excellent application foreground.

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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Patent Type & Authority Applications(China)
IPC IPC(8): C07H21/04C12N15/29C12Q1/68
Inventor 郑有良王际睿魏育明颜泽洪李伟
Owner SICHUAN AGRI UNIV
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