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Studies on the mechanism of resistance against pyrethroids in Helicoverpa armigera: Molecular and proteomic approach Skip to content Tez Künye Durumu Studies on the mechanism of resistance against pyrethroids in Helicoverpa armigera: Molecular and proteomic approach / Helicoverpa armigeranın piretroidlere karşı geliştirdiği dayanıklılığın mekanizması üzerine çalışmalar: Moleküler ve proteomik yaklaşım Yazar:METİN KONUŞ Danışman: PROF. MESUDE İŞCAN Yer Bilgisi: Orta Doğu Teknik Üniversitesi / Fen Bilimleri Enstitüsü / Biyokimya Ana Bilim Dalı Konu:Biyokimya = Biochemistry ; Biyoloji = Biology ; Ziraat = Agriculture Dizin:Biyokimyasal analiz = Biochemical analysis ; Böcekler = Insects ; Helicoverpa armigera = Helicoverpa armigera ; Moleküler analiz = Moleküler analysis ; Pamuk = Cotton ; Polimeraz zincirleme reaksiyonu = Polymerase chain reaction Onaylandı Doktora İngilizce 2012 201 s. Pamukta yeşil kurt (Helicoverpa armigera) tarımsal ürünlerde önemli ekonomik zaralara neden olan bir böcektir. Dünyadaki tarım alanlarında bu zararı azaltmak için H. armigera’ya karşı piretroid gibi kimyasal böcek ilaçları kullanılmaktadır. Bununla birlikte, sürekli ve yoğun ilaç kullanımı H. armigera’da direnç gelişimine neden olmaktadır. Böcekler kendilerine karşı kullanılan ilaçlara karşı direnç gelişimini aşağıda belirtlien üç ana mekanizma ile sağlarlar; böcek vücuduna giren ilacın miktarını azaltarak, ilacın etkilediği bölgede ilaca karşı duyarsızlık oluşturarak yada örneğin ilacın mide dokusundaki detoksifikasyon metabolizmasının hızını artırarak gerçekleştirirler. Bu yüzden, mide dokusundaki proteinlerin ekspresyonlarındaki değişimler protein seviyesinde iki boyutlu jel elektroforezi (2D-PAGE) ve MALDI-TOF kütle spektrofotometresi (MALDI-TOF-MS) araştırılırken ayrıca estraz (EST) ve glutatyon S-transferaz (GST) gibi detoksifikasyon enzim sistemlerinin biyokimyasal aktivitelerindeki değişimlerle birlikte incelendi. Ayrıca EST ve GST sistemlerinden seçilen bazı genlerin transkripsiyon seviyesindeki değişimleri sitokrom P450 monooksijenazlar (CYP450) enzim sisteminden seçilen genlerle birlikte gerçek zamanlı polimeraz zincir reaksiyonu metodu ile analiz edildi. Karşılaştırmalı proteom analizine gore, H. armigera tarla örnekleri piretroid stresinin üstesinden gelebilmek için ekspresyonunu artırdıkları başlıca proteinler ATP sentaz, Vakuolar ATPase A or B ve arjinin kinaz gibi enerji metabolizması ilgili proteinlerdir. Ayrıca, tiyoredoksin peroksidaz and NADPH sitokrom P450 redüktaz gibi bazı detoksifikasyon enzimlerinin ekspresyonu Mardin populasyonunda artması bu proteinlerin piretroid stresine karşı aktif olarak yer aldıklarını göstermektedir. NADPH sitokrom P450 redüktaz 3-fenoksibenzaldehit gibi toksik piretroid metabolitlerinin detoksifikasyonunda rol oynayabilir. Bununla birlikte, H. armigera’nın Adana ve Mardin populasyonlarının hassas populasyonla karşılaştırmalı proteome analizine gore GST’lerinin ekspresyonunda artış olmazken, GST (GST-CDNB, GST-DCNB ve GST-PNBC) aktivetelerinde anlamlı artışlar gösterdi. Ayrıca, H. armigera Çanakkale populasyonu da GST-DCNB and GST-PNBC aktivitelerinde anlamlı artışlar gösterdi. Enerji krizinin üstesinden gelmeye çalışılırken oksidatif stresi artmaktadır, detoksifikasyon enzimleri (GST ve tiyoredoksin peroksidaz) oksidatif stres sırasında oluşan H2O2 gibi toksik reaktif oksijen türlerinin eleminasyon yollarında yer almaktadır. Benzer şekilde, esterazlar ekspresyonu artan EST proteini belirlenmediği halde, biyokimysal EST aktiviteleri Adana ve Mardin populasyonlarında anlamlı artışlar gösterdi. Bu nedenle, EST’lerin Türkiyedeki H. armigera’daki dayanıklılık oluşumunda piretroid metabolizmasının başlatıcısı olarak rol oynadığı ileri sürüldü. Gerçek zamanlı polimeraz zincir reaksiyonu sonuçları CYP9A14 geninin ekspresiyonun analiz edilen tarla populasyonlarının tümünde arttığını gösterirken, CYP9A12 geninin Çanakkale ve Mardin populasyonlarında arttığını gösterdi. CYP4S1 geninin ekspresyonuda sadece Mardin populasyonunda arttı. Bununla birlikte, CYP6B7 geninin ekspresyonu CYP9A12 ve CYP4S1 genlerinin ekspresyonları ile birlikte Adana populasyonunda azalırken, CYP6B7 geninin ekspresyonu Çanakkale ve Mardin populasyonlarında değişiklik göstermedi. Ayrıca, tarla populasyonlarının tamamında GST, GSTX01 ve ESTX018 genlerinin ekspresyonlarında hassas populasyona göre anlamlı bir değişim göstermedi. Bu nedenle, CYP9A14, CYP9A12 ve CYP4S1 genlerinin toksik piretroid metabolitlerinin detoksifikasyonunda NADPH cytochrome P450 redüktazın regülasyonuyla yer almış olabilecekleri öne sürülmektedir.Sonuç olarak, dayanıklılık oluşumunda ana mekanizmalardan birinin H. armigera’nın mide dokusundaki enerji metabolizmasındaki artış olduğu öne sürülmektedirki bu artış enerji gerektiren detoksifikasyon işlemleri için gerekli enerjinin sağlanmasında genel bir ön koşul olabilmektedir. Helicoverpa armigera is an insect, causes important economical losses in crops. To reduce this loss, chemical insecticides such as pyrethroids have been commonly used against H. armigera in farming areas all over the world. However, excess and continuous usages of them cause resistance development in H. armigera. Insects develop resistance against applied insecticides by following three main mechanisms; by reducing the amount of insecticide entering into the insect body, developing insensitivity of the insecticide effective site and increasing detoxification metabolism of insecticides such as increased metabolism of them in midgut tissue of H. armigera. Therefore, changes in differentially expressed midgut proteins were analysed at protein level with two-dimensional gel electrophoresis (2D-PAGE) and matrix-assisted laser desorption/ionization-time-of-flight-mass spectrometry (MALDI-TOF-MS) together with examine biochemical activity changes of certain detoxification enzymes such as esterases (EST) and glutathione S-transferases (GST). Moreover, transcriptional level analysis of certain genes from EST and GST systems together with cytochrome P450 monooxygenases (CYP450) system were done with quantitative real-time PCR method, too. According to the comparative proteome analysis, it was found that H. armigera field samples overcome pyrethroid stress mainly by increasing energy metabolism related proteins expressions such as ATP synthase, Vacuolar ATPase A and B and arginine kinase proteins. Furthermore, certain detoxification enzymes such as thioredoxin peroxidase and NADPH cytochrome P450 reductase were up-regulated in Mardin population, suggesting that they were actively participating in response to pyrethroid stress. NADPH cytochrome P450 reductase could play a role in detoxification of toxic pyrethroid metabolites such as 3-phenoxybenzaldehyde. However, while glutathione S-transferases (GSTs) were not found up-regulated in the comparative proteome analysis, biochemical assays (GST-CDNB, GST-DCNB and GST-PNBC) showed significant increases in enzyme activities in the Adana and in the Mardin field population, as compared to the susceptible strain. Furthermore, GST-DCNB and GST-PNBC activities showed significant increase in Çanakkale population. As overcoming energy crisis may lead to an increase in oxidative stress, detoxification enzymes (GSTs and thioredoxin peroxidase) might be involved in pathways for eliminating toxic reactive oxygen species such as H2O2. Similarly, although esterases (EST) were not found as differentially expressed, biochemical assays for ESTs showed significant increases in enzymatic activities in the Adana and the Mardin field populations. Thus, ESTs are also proposed to be involved in developing resistance as an initiator of pyrethroid metabolism in H. armigera from Turkey. Quantitative real-time PCR results showed that while CYP9A14 gene expression was up-regulated in all analyzed field populations, CYP9A12 gene expression was up-regulated in both Çanakkale and Mardin populations. CYP4S1 gene expression was also up-regulated only in Mardin field population. However, while CYP6B7 gene expression together with CYP9A12 and CYP4S1 genes expressions were down-regulated in Adana population, CYP6B7 gene expression was not significantly changed in both Çanakkale and Mardin populations. In addition, GST, GSTX01 and ESTX018 gene expressions were not significantly changed in all field populations in comparison to susceptible population. Therefore, CYP9A14, CYP9A12 and CYP4S1 genes proposed to be involved in detoxification of toxic pyrethroid metabolites possibly through regulation of NADPH cytochrome P450 reductase. In conclusion, it is suggested that one of the main mechanisms of resistance development is increased energy metabolism in the midgut tissue of H. armigera which may be a general prerequisite for compensating the costs of energy-consuming detoxification processes.
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