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Clone and Analysis Acetylcholinesterasel Gene of Insecticide Resistant and Susceptible Plutella Xylostella and Its Parasitoid Oomyzus Sokolowskii

Author: LiChangWei
Tutor: WuGang
School: Fujian Agriculture and Forestry University
Course: Biochemistry and Molecular Biology
Keywords: Resistance Sensitive Diamondback moth Oomyzus Bee ace1 Clone
CLC: S433
Type: Master's thesis
Year: 2009
Downloads: 66
Quote: 1
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Abstract


Acetylcholinesterase (acetylcholinesterase, AChE, EC3.1.1.7.) Is a key enzyme in the insect nerve conduction in nerve conduction plays an important function in the cholinergic synapses by catalytic neurotransmitter acetylcholine (acetylcholine, ATCh) to maintain the normal transmission of nerve impulses. AChE is the target of organophosphate and carbamate insecticides role. The diamondback moth is a major pest, is also an important mode of insects, AChE gene study of diamondback moth, not only helps to explore insect for insecticide resistance mechanisms also contribute to novel agents provide no pollution to the environment for the development of the pest efficient scientific basis and guidance for insecticide resistance management decisions. The study to study the effects of temperature on the impact resistance of the diamondback moth acetylcholinesterase gene (ace1) of frequency sensitive dish the moth Tetrastichus ace1 partial gene sequence of its amino acid structure change resistant. The detection primers designed bi-PASA reported that diamondback moth ace1 of mutation sites were detected by high temperature and room temperature handling confrontational, sensitive to the diamondback moth future generations ace1. Detection sample of ace1 of different genotypes than the column and ace1 gene frequency calculated from the molecular level that the temperature of the diamondback moth resistant and sensitive diamondback moth offspring ace1 genotype and ace1 frequency. Oomyzus bee (Oomyzus sokolowskii) larvae to pupae of the diamondback moth (Plutella xylostella) intertemporal parasitic wasps, reported diamondback moth parasitoids. This study as a research object to Oomyzus bee ace1 the designed degenerate and specific primers amplified resistance for the first time, the sensitivity Oomyzus bee ace1 gene fragments by comparing resistant and sensitive sexual Oomyzus bee acetylcholinesterase fragment found two mutations, and comparative analysis with other insects, electric rays and other organisms have been reported acetylcholinesterase sequence obtained a mutation may diamondback moth Tetrastichus bee resistance. The main results of this study are as follows: 1. Obtained through the the diamondback moth ace1 amplified fragment sequences the sequence reported anti-propionate parathion diamondback moth ace1 sequence homology mutant sites with reported consistent (G to C). Therefore, it can be drawn in this experiment, the materials used for the resistance Plutella. 2.bi-PASA detection experiments, designed four primers: two outer primers (allele-nonspecific-F and allele-nonspecific-R), the two inner primers (allele-specific-F and allele-specific-R) . The allele-specific-F 3 'end corresponding to the mutant allele-specific-R 3' end of the corresponding sensitive. Outer primer allele-nonspecific-F and allele-nonspecific-R amplified fragment length 348bp; allele-nonspecific-F and the allele-specific-R amplified fragment length 144bp; allele-specific-F and allele-nonspecific-R The amplified fragment length of 254bp. 3. Using the bi-PASA detected Plutella xylostella F1 generation adults G227A mutation, room temperature processing of F0 generation control: 31.8% homozygous, 63.6% heterozygous and 4.6% of the wild-type. At room temperature homozygote processing than the high-temperature processing decreased by 35.5% to 8.8%, heterozygous rose to 76.9% from 61.3%, but the most obvious is the wild-type ratio increased from 3.2% to 14.3%. 4 indoor room temperature handling sensitive diamondback moth F1 generation adults than the high temperature treatment of heterozygous decreased by 32.3% to 30.0%, the wild-type ratio increased from 67.7% to 70.0%, the two treatments were not detected in the homozygote. The normal temperature processing F0 generation is not detected as a control: 33.3% of heterozygous and 66.7% of wild-type, homozygous. 5. Room temperature processing indoor resistant diamondback moth F1 generation adult homozygous mutations than the high-temperature processing decreased from 62.5% to 22.6% the heterozygous ratio increased from 37.5% to 61.3%, room temperature treatment was not detected in the wild-type The F1 generation high temperature treatment accounted for 16.1% of the wild-type. Room temperature processing control: 68.4% homozygous, 32.6% heterozygous F0 generation does not detect the wild-type. Calculated temperature treatment resistant and susceptible diamondback moth F1 generation adult ace1 of gene frequency, Plutella xylostella room temperature (25.0 ℃) treatment with high-temperature processing compared gene frequency of the resistance gene (R) decreased from 66.1% to 47.1% sensitive gene (S) gene frequency, from 33.9% to 52.9%. For the indoor screening sensitive diamondback moth (after the same treatment) resistance gene (R) gene frequency decreased from 16.1% to 15.0% sensitive gene (S) gene frequency from 83.9% to 85.0%, while the indoor screening resistance diamondback moth resistance gene (R) gene frequency sensitive gene (S) gene frequency decreased from 81.3% to 53.2%, from 18.7% to 46.8%. The contrast normal temperature processing resistance, sensitive Plutella F0 generation, the gene frequency of the resistance gene (R) after heat treatment are reduced. Get resistance, sensitive the diamondback moth Tetrastichus ace1 sequence and other insects, electric rays and other biological the acetylcholinesterase amino acid sequence alignment analysis, the results proved cloned into ace1 gene sequences. And found two mutations that: Phe272Leu (TTT to CTT), Lys303Arg (AAG to AGG). Through comparative analysis with other insects, known Phe272Leu (TTT to CTT), and Oomyzus bee resistance-related, according to the AChE structure analysis shows, Phe272Leu (TTT to CTT) is located near the mouth of the acyl pocket.

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CLC: > Agricultural Sciences > Plant Protection > Pest and Disease Control > Plant pest and its control
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