| Culex pipiens pallens is a kind of sanitary insect with wide distribution, which canspread some fatal diseases. People have find some methods for preventing and curing, and themain method is using all sorts of chemical pesticides, insecticide resistance was arisen andhas been developping. The discussion and research about the machanism of the resistance inmosquito become popular. We did three parts of experiments in Culex pipiens pallens forstudying the resistance gene:1. Method of Guanidinium isothiocyanate, Trizol, Trizol plus CTAB, advanced Trizoland CTAB were compared to obtain an optimal way for extracting total RNA from Culexpipiens pallens. We compared their purity, concentration, integrity, time-using andquantitation, the results suggested that advanced CTAB provided the best quality among thefive methods, it would be a good technology for subsequent molecular experiment.2. Designed gene special primer according to the EST sequence(EC093830.1) ofresistance gene in Culex pipiens pallens, got a1637bp-long full length cDNA sequence byRACE. Finded its Open Reading Frame by the software ORF finder, we get a1521bp-longORF; analysed the bioinformation of the sequence by software PredictProtein, POLYVIEW2,PSORT et al, we get the following results: it encoded a peptide with506residues, and itssecond structure prediction suggested that it contained53.16%α-helix,14.62%β-sheet and32.21%loop,4of the α-helix is transmembrane; the subcellular localization indicated that theprotein most probably was found in mitochondria and cytoplasm, and it was a kind ofsecretory proteins; many common enzyme cutting sites, especially phosphorylation site werefound in the peptide, all these predictions suggested a conclusion that the peptide was asteady and transmembrane protein which could participate many enzyme reaction, it wasworth mentioning that its participation of metabolism in mitochondria was closely related tometabolic detoxification of insect. Quantitation was proceeded to find the difference of theexpression of this gene between resistant and sensiteve strain, the result was that the gene wasoverexoressed in resistant strain,1.58times more than sensitive strain, it added weight to the fact that the gene was related to pesticide resistance.3. As a special case, CYP6FI was thoroughly researched in the resistance gene of Culexpipiens pallens, it contained some conserved characteristics, just like Cytochrome P450, thebiggest detoxifying enzymes genes of metabolism resistance gene. We synthetized genespecific primers, and amplified from the cDNA that reverse transcripted from total RNA, theRNA was extracted from different period mosquito, including larva, pupa, and audlt. After thequantitative reaction, we found that the exoression of CYP6F1was continuously accumulatedwith the growth time going. The result suggested the gene in resistant mosquito was morethan that in sensitive larva.All of the above, we got a optimum method for extracting total RNA in Culex pipienspallens, and then cloned a resistance gene, XN-P450, analysed its structure, amino acidsequence, predicted its function, and detected the different expression between resistant andsensitive strains to ascertain its relation to resistance. We tested the tendence of expression ofCYP6F1in different period mosquitos. Through the above research, we found a optimal wayand added something new to resistance gene, provided theoretical guidance for developpingnew pepticide and its use, laid the foundation for preventing and curing mosquito. |
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