Clone And Analysis Acetylcholinesterasel Gene Of Insecticide Resistant And Susceptible Plutella Xylostella And Its Parasitoid Oomyzus Sokolowskii

Acetylcholinesterase (AChE, EC 3.1.1.7) catalyzes the hydrolysis of the neurotransmitter acetylcholine (ATCh) at cholinergic synapses to keep the normal transmission of nerve impulses,plays an important function in nerve conduction. It is the target of the organophosphate and carbamate compounds. The intensive use of insecticide had led to the pollution of environment and many insects have developed resistance. So studying the interaction of the target enzyme and insecticide, screening out the insecticides through mutant protein had become a very important project.Diamondback moth is a major pest,and also is an important insect model to study the AChE gene , not only help to explore the mechanism of insect resistance to insecticides, but also to the development of new insecticides with less harm to the environment and with high effectivity to resistant insect pests.In this study we had researched the diamondback moth acetylcholinesterase gene frequency which was impacted by temperature and the resistant and sensitive kurdjumov ace gene sequences and the relationship between structural change and resistance. According to the mutation of the diamondback moth acetylcholinesterase gene reproted,we designed bi-PASA primers to detect the mutation of resistant and sensitive diamondback moth acetylcholinesterase gene mutation which was treated at high and room temperature(25℃). We calculated the rate of the acetylcholinesterase genotypes, proved sensitivity impacted by the temperature with the acetylcholinesterase of diamondback moth at molecular level.The Oomyzus sokolowskii is a parasitic natural enemy for the diamondback moth from larvae to the pupal stage, it was reported that Oomyzus sokolowskii is one of the main parasitic natural enemies of diamondback moth. In this study, we amplified fragments of ace 1gene by the degenerate and specific primers using resistance and sensitivity Oomyzus sokolowskii. Compared the resistance AChE fragment to sensitivity AChE fragment, we found two mutations. Compared the mutations with other insect's reported mutational site, we ensured the one mutation was associated with the resistance of Oomyzus sokolowskii.The major results are summarized as following:1. Compared the amplified ace1 sequences of the diamondback moth with other insects, we found the sequences have high homology with those reported, also have the same mutation site (G to C). It can prove the diamondback moth is the resistance we used in this experiment.2. The bi-PASA experiment needed to design four primers: two outer primers, two inside primers. The inside primer allele-specific-R corresponded the wild-type, and the allele-specific-F corresponded the mutation-type. Outer primer allele-nonspecific-F and the allele-nonspecific-R amplified the fragment whose length is 348bp; allele-nonspecific-F and allele-specific-R amplified the fragment whose length is144bp; allele-specific-F and the allele-nonspecific-R amplified the fragment whose length is 254bp.3.Used bi-PASA technique detected G227A mutation from the F1 generation of field adult diamondback moth, using the F0 generation of normal-temperatured field adult diamondback moth as control, the homozygous mutation was 31.8%, the heterozygotes was 63.6%, and the wild-type was 4.6%. Compared of the treatment by normal-temperature and the high-temperature, the homozygous mutation reduced from 35.5% to 8.8%, the heterozygotes increased from 61.3% to 76.9 %, most obviously the wild-type increased from 3.2% to 14.3%.4. Compared the F1 generation of the indoor sensitive adult diamondback moth treated by normal- temperature and the high-temperature, the heterozygotes reduced from 32.3% to 30.0%, the wild-type increased from 67.7% to 70.0%, did not detect homozygous mutations in the two treatments. Using the F0 generation of normal-temperatured field adult diamondback moth as control, the heterozygotes was 33.3%, the wild-type was 66.7%, and no homozygous mutations were detected. 5. Compared the F1 generation of the indoor resistance adult diamondback moth treated by normal- temperature and the high-temperature, the homozygous mutations reduced from 62.5% to 22.6 %, the heterozygotes increased from 37.5% to 61.3%, did not detected the wild-type at normal- temperature, but at the high-temperature detected 16.1%wild type. Using the F0 generation of normal-temperatured field adult diamondback moth as control, the homozygous mutations was 68.4%, the hetero- zygotes was 32.6%, and no wild-type were detected.6. Calculated the ace1 gene frequency of susceptible and resistant adult diamondback moth F1 treated by temperature, compared the result between normal temperature (25.0℃) and high temperature(33.5℃), the gene frequency of filed resistance genes (R) reduced from 66.1% to 47.1%, the gene frequency of sensitive gene (S) increased from 33.9 % to 52.9 %. The gene frequency of resistance genes (R) of the sensitive screening for indoor (in the same treatment) reduced from 16.1% to 15.0%, and the gene frequency of sensitive gene (S) increased from 83.9% to 85.0 %, and the gene frequency of resistance genes (R) of the filtered indoor resistant reduced from 81.3% to 53.2%, the gene frequency of sensitive gene (S) increased from18.7 % to 46.8 %. Using F0 generation of normal-tempretured susceptible and resistant adult diamondback moth as control, the gene frequencises of high tempretured susceptible and resistant adult diamondback moths were reduced.7. Compared and analysed the results of the ace sequence gene between O. sokolowskii and other species, the analysis has proved that the cloned gene sequence was ace gene. And also found tow mutation sites (Phe272Leu,Lys303Arg). The Phe272Leu mutation had relation with the resistance of O. sokolowskii.