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Molecular Cloning, Mrna Expression and Rnai of Nadph-Cytochrome P450 Reductase Gene in Helicoverpa Armigera (Hǘbner)

Author: WangDan
Tutor: WuYiDong
School: Nanjing Agricultural College
Course: Agricultural Entomology and Pest Control
Keywords: Bollworm NDPH - cytochrome P450 reductase Gene Cloning Spatial and temporal expression RNA interference
CLC: S435.622.3
Type: Master's thesis
Year: 2011
Downloads: 25
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Abstract


The cotton bollworm, Helicoverpa armigera (Hiibner) is one of the worldwide serious pests. In 1980s, pyrethroid insecticides were widely used to control this insect in China for its high effective control to insect and low toxicity to the environment. However, due to the over-use of pesticides, the resistance to pyrethroids became a big problem, which was one of the main causes for the outbreak of cotton bollworm in northern China in the early 1990s. Afterwards the transgenic cotton was introduced to control this pest.The research about resistance mechanisms on cotton bollworm indicated that the increased activity of cytochrome P450 oxidase played a major role in the metabolic resistance of cotton bollworm to pyrethroid insecticides.NADPH-cytochrome P450 reductase (CPR) is an important component of cytochrome P450 oxidoreductase system, and a rate-limiting role in the cytochrome P450-mediated redox reactions. In this study, the full-length cDNA of CPR gene (Ha_CPR) was cloned from H. armigera by using RT-PCR and RACE techniques. Then, Ha_CPR dsRNA was imported into the 3rd instar or 5th instar larvae of cotton bollworm by feeding or injecting methods respectively, to study the effects of RNAi-mediated gene silening on the insecticidal toxicity, enzyme activity and growth development of cotton bollworm. The results of the study provide essential basis for further research on the functions of cytochrome P450 oxidoreductase system in the formation of metabolic resistance and growth development of H. armigera.1. Cloning and sequence analysis of Ha CPR gene from H. armigeraBy using RT-PCR and RACE techniques, the full-length cDNA of CPR gene (Ha_CPR) was cloned from H. armigera (GenBank accession number:HQ116527). Sequence analysis showed that the full-length cDNA was 2232bp and contained an open reading frame (ORF) of 2061bp which encoded 687 amino acid residues.The deduced amino acid sequence of Ha_CPR has the typical structural domains (the FMN-binding domain, the connecting domain, and the FAD- and NADPH-binding domains) and conserved amino acid residues (Y149, Y187, Y467, Y489, and W686) of CPR, and shows the highest amino acid similarity of 96% with Mamestra brassicae, and 70%~89% amino acid similarity with other species of insecta.2. Spatio-temporal expression of Ha_CPR geneThe relative expression level of Ha_CPR gene in different developmental stages and different tissues of H. armigera was detected by using real-time fluorescence quantitative PCR. The results showed that Ha CPR gene in H. armigera expressed throughout the whole developmental stages and in different tissues. The mRNA relative expression level of Ha_CPR gene increased gradually from egg to larval stage, and reached the maximum at 3rd-4th instar larval stage, then decreased to the minimum at pupal stage, and then increased again after emergence. The mRNA relative expression levels in each stage were respectively 0.59 (eggs),1.46 (1st~2nd),1.54 (3rd~4th),0.26/0.19 (female pupae/male pupae) and 1.21/1.29 (female/male) times than that in 5th instar larval stage. The mRNA relative expression level of Ha_CPR gene was highest in the midgut, lower in the fat body and lowest in the head; the mRNA relative expression levels in the three tissues were respectively 3.61,1.29, and 1.08 times than that in the whole body.3. Effects of silencing of Ha CPR on the insecticidal toxicity, enzyme activityThe effects of RNAi-mediated gene silening on the insecticidal toxicity and enzyme activity was investigated after Ha_CPR dsRNA was imported into the 3rd instar or 5th instar larvae of cotton bollworm by feeding or injecting respectively.After feeding Ha_CPR dsRNA (0.8μg/μlx2.5μl), the mRNA relative expression level of Ha_CPR on the 3rd instar larvae in the strains of SCD, XJ and AY-R decreased by 49%(57%),26% and 23% compared with control group. Bioassay results showed that the toxicity of fenvalerate, deltamethrin, and monocrotophos declined on the treatment groups of the three strains, and the LC50 ratio was 0.34 to 0.80 between treatment group and control group. But in XJ and AY-R, the 95% confidence interval of LC50 of fenvalerate to the treatment group was overlapped between the treatment group and the control group. T-test analysis of the mortality under a single concentration of three insecticides showed that:(1) in the strian of SCD, the difference of mortality between the two groups was highly significant at 0.01mg/ml and 0.02mg/ml of fenvalerate and 0.0025mg/ml of deltamethrin, (2) in the strian of XJ, the difference of mortality between the two groups was highly significant at 0.08mg/ml of fenvalerate, (3) in the strian of AY-R, the difference of mortality between the two groups was highly significant at 25.6mg/ml of fenvalerate.After injecting Ha_CPR dsRNA (14μg, 10μl) into 1st day 5th instar larvae of AY-R strain, the mRNA relative expression level of Ha_CPR gene in midgut and fat body decreased respectively by 12% and 25% compared with the control group. The results of detoxification enzymes assay showed that the activities of MFO (PNOD, MROD, and ECOD), GST (CDNB, DCNB) and EST were not significantly different between the two groups.4. Effects of silensing of Ha_CPR on growth developmentThe effect of RNAi-mediated gene silening on the growth development was investigated after feeding Ha_CPR dsRNA to the 3rd instar larvae of cotton bollworm. After feeding Ha_CPR dsRNA (0.8μg/μl×2.5μl) to the 3rd instar larvae, the mRNA relative expression level of Ha_CPR in the strain of SCD decreased by 49%. The duration of 3rd and 4th instar between the two groups was no significant difference, but at the 5th instar and pre-pupal stage, the duration of the treatment group was significantly longer than that of the control group. The larval weight of the treatment group was significantly heavier than that of the control group in the 1st,3rd and 7th day after gene silencing. During the pupation stage, the pupal weight and pupation rate between the two groups were not significantly different. During the emergence stage, the treatment group emerged later than the control group by one day, but the emergence rate and disable wing rate between the twol groups were not significantly different. Throughout the whole developmental stage, the mortality of the two groups showed an upward trend, but there was no significant difference.

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CLC: > Agricultural Sciences > Plant Protection > Pest and Disease Control > Crop pests and diseases and their prevention > Economic crop pests and diseases > Fiber crop pests and diseases > Cotton pests and diseases > Insect pest > Pink bollworm
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