Construction And Application Of An Insecticide Resistance-Associated DNA Microarray

With the continuous application of insecticides on control of agricultural and disease vector insect pests, insect resistance to insecticides has been developed and caused serious problems in agriculture and human health. The study on insecticide resistance, especially at molecular level, has uncovered a number of mechanisms insects used to reduce the effects of insecticides on their survivance in toxic environments. Recently microarrays were used to study insecticide resistance and most of them were used for flies and mosquitoes and focused on the differential expression of cytochrome P450 genes. In this study, 6 insect species are selected as the templates and 41 kinds of insecticide resistance-associated genes and a housekeeping gene are obtained from them with 58 pairs of primers which were designed based on 2761 insecticide resistance-associated gene sequences. An insecticide resistance-associated DNA microarray containing 2808 target spots is constructed with all these target genes and is used to detect the differential gene expression between resistant and susceptible strain of Musca domestica and Culex pipiens. These works provided a sensitive and efficient technical tool for studying resistance and molecular mechanism of insecticide resistance.Main results of this dissertation included the following three parts:1. Clone of the target genes for insecticide resistance-associated DNA microarray(1) According to the functions of insecticide resistance, 41 kinds of genes were selected for construction of this microarray, which included 28 kinds of cytochrome P450 genes, 4 kinds of ion channel genes, 2 kinds of protective enzyme genes, 4 kinds of target site genes and 3 kinds of other resistance-associated genes. These genes included most of the known insecticide resistance-associated gene kinds. Additionally, positive and negative controls were determined: a house-keeping gene (β-actin) was selected as positive control of microarray hybridization and a partial sequence of pUC-18 plasmid was used as negative control. 58 pairs of primers were designed based on 2761 insecticide resistance-associated gene sequences, which were used for amplification of the target genes. These primers included 39 pairs of ordinary primersand 19 pairs of CODEHOP primers.(2) The target genes were cloned from 6 insect species and used for construction of insecticide resistance-associated DNA microarray. These 6 insect species are Apis cerana, Blattella germanica, Spodoptera exigua, Musca domestica, Nilaparrata lugens and Culex pipiens, all of which are important economic insects. After cloning the target DNA fragments, their sequences were measured and bioinformatics analysis was performed. A total of 646 target fragments were determined as insecticide resistance-associated genes and positive control genes. In these 646 target genes, there were 512 cytochrome P450 genes, 45 ion channel genes, 17 protective enzyme genes, 37 target site genes, 29 other kinds of resistance-associated genes and 6 positive control genes. According to the origin of genes, 143 target genes came from M. domestica, 125 from C. pipiens, 79 from A. cerana, 106 from B. germanica, 88 from S. exigua and 105 from N. lugens. Moreover, the length distribution of target gene fragments was analyzed. The results showed that the length of 97 gene fragments was between 100-199 bp, 117 between 200-299 bp, 137 between 300-399 bp, 105 between 400-499 bp, 83 between 500-599 bp, 77 between 600-699 bp and 30 above 700 bp. In general, the length of most target gene fragments are between 200-600 bp (442, which is the 68.4% of them).2. Preparation and validation of insecticide resistance-asssociated DNA microarray(1) In this study, the optical microarray printing concentration was determined. The M. domestica 0-actin gene was selected as the target gene and its PCR products were diluted to 50, 100, 150, 200 and 300 ng/yLin printing buffer. Then the diluted products were spotted onto the slide and hybridized with the probes composed of M. domestica 0-actin gene labeled with different dyes. The results showed that the hybridizing signal values of target spots were positively linearly correlated with the printing concentrations within the ranges of 50-200 ngAi L and the linear coefficient was 0.9662. However, the signal value boost up rapidly and there was no linear relativity between printing concentration and hybridizing signal value when the concentration was more than 300 ng/uL. Additionally, signal ratios of two fluorescence in five different printing concentrations were analyzed. The results showed that all of them were close to 1, but the ratio of 200 ng/yLis mostly close to 1. Accordingly, the optical microarray printing concentration was determined as 250 ng/ u L because the higher concentration is propitious to detect the hybridizing signal.(2) The insecticide resistance-associated DNA microarrays were prepared withaldehyde glass slides. The microarray includes 2 same hybridizing areas and each hybridizing area includes 8 arrays. The arrangement mode of arrays is 2 X 4 and each array includes 15 X 12 spots. Except 72 blank spots, there are 2808 sample spots on the microarrays. After printing, the prepared microarrays are detected by DNA microarray scanner. The scanning results showed that the arrangement of the arrays and the spots on the microarray was orderly, the morphology was similar and the background of fluorescence W￡s weak. The results also showed that the rate of eligible target spots was 96.0%. The mean diameter of eligible spots was 94.80um and the SD was 19.07um, which was close to the diameter of the spotting pin (100 um). All the results indicated that the quality of prepared microarrays was credible.(3) The microarray hybridization specificity was validated. A PCR probe composed of 18 target genes was prepared and hybridized with the microarray. The results showed that all the target spots on the microarray hybridized with the probe and hybrid rate was 100%. The average hybridizing signal values was 1240.15. Except the 18 pairs of target spots, there were else 6 pairs of non-target spots found to be hybridized with the probe. The sequence similarities of these 6 gene fragments to the 18 target gene fragments are analyzed and the results showed that the similarity indexes of 2 non-target genes (A. cerana CYP4 and M. domestica JHE) and 2 target genes (M. domestica CYP4 and B. germanica JHE) were very high and the signal ratios of them were both close to 1. So they are considered as repeats for each other and their hybridized results are considered as right. However, the similarity indexes of the other 4 non-target genes are low and their results are considered as false positive. In conclusion, the positive rate of this validating test was 83.3%.3. Application of constructed DNA microarray on profiling the expression of insecticide resistance-associated genes in M. domestica and C. pipiens(1) With the constructed microarray in this study, the differential gene expression in a M. domestica strain resistant to both malathion and cypermethrin and a susceptible M. domestica strain was studied. The results showed that the ratios of 29 target genes on the microarray between resistant and susceptible strain were more than 2 or less than 0.5, of which 22 target genes come from M. domestica and 7 from other insect species. Among the 22 target genes from M. domestica, there were 14 cytochrome P450 genes, 3 ion channel genes, 1 protective enzyme gene and 4 other kinds of resistance genes and all of them over-expressed in resistant strain. Among the 7 target genes from other insects, there were 4 cytochrome P450 genes, 1 ion channelgene, 1 Mucin gene and 1 JHE gene. Except the N. lugens JHE gene, the ratios of other 6 genes were all more than 2. The analysis showed that most of the 18 up-regulated cytochrome P450 genes belonged to CYP4 and CYP6 families, which implied that the cytochrome P450 genes of these families might play important roles in resistance to both malathion and cypermethrin in M. domestica. Additionally, though ion channel genes are regarded as target resistance genes, several ion channel genes were found to be up-regulated in resistant M. domestica in this study, which provided a chance to study the relationship between the differential expression of them and insecticide resistance.(2) With the constructed microarray in this study, the differential gene expression in a C. pipiens strain resistant to fenvalerate and a susceptible C. pipiens strain was also studied. The results showed that the ratios of 8 target genes on the microarray between resistant and susceptible strain were more than 2, of which 6 target genes come from C. pipiens and 2 from other insect species. Among the 6 target genes from C. pipiens, there were 4 cytochrome P450 genes, 1 target-site gene and 1 protective enzyme gene. The other 2 up-regulated genes are 2 cytochrome P450 genes which come from M. domestica. All of them over-express in resistant strain. The analysis showed that 3 of the 6 up-regulated cytochrome P450 genes belonged to CYP4 family and 2 of them belonged to CYP6 families, which implied that the cytochrome P450 genes of these families might play important roles in resistance to fenvalerate in C. pipiens and resembled the studying result in M. domestica. Additionally, GABA gene, a target resistance gene, was found to be up-regulated in resistant C. pipiens in this study, which provided a chance to study the relationship between its differential expression and insecticide resistance.(3) The results by microarray of 3 genes differentially expressed in different M. domestica strains, CYP4D2v2, CYP6A24 and CYP6Dlvl gene, were validated using real-time PCR. The results of validation showed that the expressing level of CYP4D2v2, CYP6A24 and CYP6Dlvl gene were 9.48E+06, 1.26E+07 and 6.55E+07 copies/ug total RNA in resistant strain respectively and 3.98E+06, 3.46E+06 and 7.15E+06 copies/ug total RNA in susceptible strain respectively. Accordingly, the over-expressed times of CYP4D2v2, CYP6A24 and CYP6Dlvl gene in resistant strain are 2.38, 3.64 and 9.16 respectively. Compared with the detecting results by microarray, of which the over-expressed times of these 3 genes were 2.86, 3.79 and 8.85 respectively, the detecting results by real-time PCR is very close to them, which indicated that the hybridizing results by microarray in this study was credible.