Studies On Resistance Of Bactrocera Dorsalis To Avermectin

Bactrocera dorsalis is the world's more harmful fruit pests,but also resistant to the emergence and development of one of the more serious pests.Avermectin is one of the main agents for the control of B.dorsalis,and the B.dorsalis is more resistant to it.In order to clarify the antibiotic resistance of the B.dorsalis,the sequence of the GluCl gene of the flies was studied by RT-PCR and RACE.The sequence of the GluCl gene was analyzed.The mRNA expression of GluCl gene was analyzed by fluorescence quantitative PCR.The full-length sequence of GluCl gene was sensitive and resistant to avermectin,and the full length of GluCl gene was amplified.Sequence analysis and sequencing were performed to find the site of mutations in the gene.A prokaryotic expression and purification of the extracellular domain of GluCl,a susceptible strain,was successfully carried out and the purified protein was obtained.A yeast two-hybrid library of the susceptible strain GluCl gene.The results are as follows:(1)The full length of the GluCl gene was amplified by 1377 bp,459 amino acids were encoded and 70%of the GluCl amino acid sequence was 80%similarity.GluCl receptor amino acid sequence signal peptide cleavage site between the 19th and 20th position,the signal peptide sequence there are twenty amino acids,the specific sequence is:MGSGHYIWAIFFFASLCSAS.GluCl receptor for amino acid secondary structure prediction,found that it has 129 alpha helix,139 Extended Strand,42 Beta turn,148Random Coil.In predicting the transmembrane structure of the GluCl receptor,it was found that it had four transmembrane domains consistent with the typical characteristics of the GluCl receptor,specifically including TM1/TM2/TM3/TM4,the presence of cysteine Outer zone,transmembrane region,C-terminal extracellular domain,and the like.The first transmembrane structure is from 5 to 22,contains amino acids:HYIWAIFFFASLCSASLA;the second transmembrane structure is at 244~266,contains amino acids:FSYYLIQIYIPCCMLVIVSWVSF;the third transmembrane structure is in the range of 309~331,The amino acids contained are:VWTGVCLTFVFGALLEFALVNYA;the fourth transmembrane structure is at 427-449,and the amino acids are:VISRITFPLVFALFNLVYWSTYL.GluCl receptor protein encoding a total of 20 amino acids,which accounted for the most leu,Ser and Ile,leu up to52,accounting for 11.4%of amino acid composition;followed by Ser,33,accounting for amino acid composition 7.2%;Ile 29,accounting for 6.3%of the amino acid composition.The results showed that the N-terminal and C-terminal of the sequence formed two hydrophilic regions,which were the sites where the protein was modified and functionalized,and the hydrophobic groups were mostly distributed in the middle,That is,the area where the functional locus is located.The molecular weight and isoelectric point of GluCl receptor protein were predicted.The molecular weight was 52452.95D and the isoelectric point was 8.99?(2)The results showed that the mRNA expression of GluCl gene in adult stage was higher than that in other developmental stages.The results showed that the expression of GluCl gene in adult stage was higher than that in other developmental stages.The expression of the foot,the head and the chest is higher than that of the abdomen and the wing.(3)There were 13 mutations in the amino acid sequence relative to the susceptible population.The 240th locus is located in the intracellular region between the first and second transmembrane regions,the 262th locus is located on the second transmembrane region,the 325th locus is located on the third transmembrane region,334-337,338,339,340,341,342,343,403 and 420 to 423 are located in the intracellular domain between the third and fourth transmembrane regions and the 432th locus is located on the fourth transmembrane region.(4)The prokaryotic expression of the intracellular domain between the third and fourth transmembrane regions of the GluCl gene was successfully performed and the protein was successfully expressed and purified.The polyclonal antibody was prepared with the protein.Western blot was used to detect the GluCl receptor protein bands,which indicated that the GluCl receptor protein was present in the susceptible strain,and the GluCl receptor protein was found in the susceptible strain.(5)The eukaryotic expression vector of GluCl gene was constructed by pcDNA3.1plasmid,and the eukaryotic expression was successfully performed in 293 cells.(6)A yeast two-hybrid library of GluCl gene was constructed,and the quality of the library was 4.8×10~5.