Dissertation
Dissertation > 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

Promoter Activity Analysis of Cytochrome P450 Gene CYP9A17v2 from Helicoverpa Armigera (H(?)bner)

Author YanYuCheng
Tutor WuYiDong
School Nanjing Agricultural College
Course Agricultural Entomology and Pest Control
Keywords Bollworm P450 5'- regulatory region Promoter Dual luciferase reporter gene system Polymorphism
CLC S435.622
Type Master's thesis
Year 2009
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The cotton bollworm, Helicoverpa armigera (Hiibner) (Lepidoptera:Noctuidae) is one of the most serious pests on cotton and seriously restrict the production and quality of cotton. As the control of H. armigera is mainly dependent on chemical pesticides for a long time, it has developed serious resistance to most insecticides especially to pyrethroids. The enhanced oxidative metabolism by cytochrome P450 is a major factor responsible for pyrethroid resistance in H.armigera. Overexpression of several P450 genes from CYP9 and CYP6 families were reported to be associated with xenobiotic detoxification in H. armigera. In this study, the 5’-untraslated region (UTR) of P450 genes CYP9A12 and CYP9A17v2 were obtained by PCR in a pyrethroids-resistant stain YGF and a reference stain YG, then we took a functional analysis of the promoter of CYP9A17v2 to study mechanisms underlying its regulation. Further, the amino acid polymorphism of CYP9A12, CYP9A14, CYP9A17v2, and CYP6B7 were analyzed in different strains of H. armigera.1. Molecular cloning and sequence analysis of the 5’-UTR region of CYP9A12 and CYP9A17v2 of H.armigeraOver-expression of cytochrome P450 gene CYP9A12 and CYP9A17v2 gene is associated with pyrethroid resistance in Helicoverpa armigera (Hiibner). The 5’-UTR fragments of CYP9A12 and CYP9A17v2 were cloned using specific primers based on obtained 5’-UTR sequences of the two genes in the susceptible SCD strains. The obtained fragment of 5’-UTR of CYP9A12 is about 2.6kb and of CYP9A17v2 is about 3.4kb.The transcriptional start site was predicted by the BDGP software and transcriptional factor binding sites were predicted by EEGG sequence data bank. It will provide an important basis for investigating expression regulation of cytochrome P450 genes in pyrethroid-resistant H.armigera.2. Deletion analysis of the core promoter region of CYP9A17v2 from Helicoverpa armigeraPrevious studies showed that over-expression of cytochrome P450 CYP9A17v2 gene is involved in pyrethroid resistance in Helicoverpa armigera (Hiibner). In order to study mechanisms underlying regulation of CYP9A17v2, functional analysis of the promoter of CYP9A17v2 from H. armigera (Hiibner) was performed in the present study. Luciferase reporter plasmids containing serially truncated promoter fragments (-1095~+43) of CYP9A17v2 were transiently transfected into sf9 cells, and the promoter activity was measured with dual-luciferase reporter assay system. Functional analysis showed that all seven deleted fragments had promoter activity, and a region between -197 and +43 exhibited the highest level of promoter activity. Transcription enhancer elements may exist in the region between -197 and -113 of CYP9A17v2 5’-regulation area, and transcription repressor elements may situate in the region between -1095 and -197. This study provided an important foundation for investigating transcriptional regulation mechanisms of CYP9A17v2 overexpression in H. armigera.3. Amino acid polymorphism of cytochrome P450 genes associated with pyrethroid resistance in H. armigeraThree CYP9 genes (CYP9A12, CYP9A14 and CYP9A17v2) and a CYP6 gene CYP6B7 are associated with pyrethoid resistance in H. armigera. Full length cDNAs of the four CYP genes were cloned and sequenced in four different strains including the susceptible strain SCD, field-collected strain GY, pyrethoid-resistant strain YGF, and the YG strain (the reference strain of YGF). There are many single nucleotide polymorphism (SNP) sites in the open reading frame (ORF) of the four genes, some of which resulted in amino acid substitution. Several amino acid polymorphisms were found in the P450 conserved regions such as heme-binding motif. Several amino acid substitutions were specific to the resistant YGF strain. Through the analysis of the amino acid polymorphism in the four CYP genes between different strains, some P450 alleles with meaningful polymorphisms could be selected as candidates for functional expression study.

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