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Method and special primer of method for fast identifying plutella xylostella ryanodine receptor gene mutation

A technology of ryanodine receptor and diamondback moth, applied in biochemical equipment and methods, measurement/testing of microorganisms, DNA/RNA fragments, etc., to achieve high sensitivity and delay the development of resistance

Inactive Publication Date: 2014-06-18
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the control of Plutella xylostella is still dominated by chemical pesticides. Due to the irrational use of insecticides and the biological characteristics of Plutella xylostella, it has developed resistance to almost all commonly used insecticides.

Method used

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  • Method and special primer of method for fast identifying plutella xylostella ryanodine receptor gene mutation
  • Method and special primer of method for fast identifying plutella xylostella ryanodine receptor gene mutation
  • Method and special primer of method for fast identifying plutella xylostella ryanodine receptor gene mutation

Examples

Experimental program
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Effect test

Embodiment 1

[0024] Example 1. Analysis of the ryanodine receptor gene of Plutella xylostella xylostella individuals with different susceptibility to diamide insecticides, thereby discovering and verifying the relationship between the G4899E mutation of the ryanodine receptor gene and the susceptibility to resistance

[0025] The ryanodine receptor gene was amplified by PCR and verified by sequencing on 100 individuals of Plutella xylostella xylostella collected from the field in Guangzhou City, Guangdong Province in 2011. Sequencing results show that there is a SNP site in the ryanodine receptor gene of Plutella xylostella, which is the 14696th position of the open reading frame of the ryanodine receptor gene of Plutella xylostella resistant to bisamide insecticides from the 5' end The nucleotide is mutated from G to A, resulting in the 4899th amino acid residue at the N-terminal of the ryanodine receptor being mutated from glycine (G) to glutamic acid (E), that is, the G4899E mutation. T...

Embodiment 2

[0029] Embodiment 2, primer design

[0030] Based on the findings of Example 1, the allele-specific PCR primers were designed as follows:

[0031] Primer 1 (sequence 1 of the sequence listing): 5'-TGTTGGACGTGGCTGT G G -3';

[0032]Primer 2 (sequence 2 of the sequence listing): 5'-TGTTGGACGTGGCTGT G A -3';

[0033] Primer 3 (Sequence 3 of the Sequence Listing): 5'-TGTTTCCCGTTATGCGTGAC-3'.

[0034] Primer 1 is a dedicated sensitive primer, primer 2 is a dedicated resistant primer, and primer 3 is a downstream primer used in conjunction with a dedicated sensitive primer or a dedicated resistant primer during PCR detection. The first nucleotide at the 3' end of primer 1 (see the underline) is consistent with the nucleotide before the mutation, and the first nucleotide at the 3' end of primer 2 (see the underline) is the same as the nucleotide after the mutation unanimous. In addition, in order to increase the specificity of the allele-specific PCR method, a mismatched ba...

Embodiment 3

[0035] Embodiment 3, the application of sensitive primer pair and resistant primer pair

[0036] 1. Detection of the resistance of Plutella xylostella to chlorantraniliprole

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Abstract

The invention discloses a method and a special primer of the method for fast identifying the plutella xylostella ryanodine receptor gene mutation. The invention provides a specific primer combination, which consists of a single chain DNA (deoxyribonucleic acid) molecule shown as a sequence 1 of a sequence table, a single chain DNA molecule shown as a sequence 2 of the sequence table and a single-chain DNA molecule shown as a sequence 3 of the sequence table. The specific primer combination can be used for the following (a) or (b): (a) the specific primer combination can be used for identifying whether plutella xylostella generates the G4899E mutation of the ryanodine receptor gene or not, and the G4899E mutation of the ryanodine receptor gene refers to that an open reading frame of the ryanodine receptor gene is mutated into A from G in the 14696 locus nucleotide of the 5' tail end; and (b) the specific primer combination is used for the auxiliary identification of the resistance of plutella xylostella on bisamide pesticides. The specific primer combination and the application of the specific primer combination have the important significance in the aspects of monitoring the resistance gene frequency of plutella xylostella field populations on the bisamide pesticides and the bisamide pesticide resistance generation and development trend of the plutella xylostella field populations, timely regulating the plutella xylostella preventing and control strategy, guiding the reasonable pesticide use and delaying the resistance development.

Description

technical field [0001] The invention relates to a method for quickly identifying mutations in the ryanodine receptor gene of diamondback moth and special primers thereof. Background technique [0002] The diamondback moth (Plutella xylostella) belongs to the Lepidoptera Plutellaidae, and its English name is "Diamondback moth". It is a pest that is extremely harmful to cruciferous plants. It is distributed all over the country and causes serious damage in southern my country all the year round. . At present, the control of Plutella xylostella is still dominated by chemical pesticides. Due to the irrational use of insecticides and the biological characteristics of Plutella xylostella, it has developed resistance to almost all commonly used insecticides. [0003] The bisamide insecticides represented by chlorantraniliprole and flubendiamide have good control effects on Lepidoptera pests. Its mechanism of action is unique. By activating insect ryanodine receptors, a large amoun...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68C12N15/11
Inventor 梁沛郭磊高希武史雪岩宋敦伦
Owner CHINA AGRI UNIV
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