本文關(guān)鍵詞： 紫外線 低氧 電子束 物理脅迫 響應(yīng) 差異表達基因　出處：《華中農(nóng)業(yè)大學(xué)》2016年博士論文　論文類型：學(xué)位論文

【摘要】：紫外線(UV)、低氧和電子束輻射是造成昆蟲生存力降低的重要物理脅迫因子,是用作害蟲防治的有效手段。昆蟲對這些脅迫因子的響應(yīng)涉及基因表達、蛋白質(zhì)翻譯和生理變化等多種復(fù)雜的生物學(xué)過程,最終表現(xiàn)對脅迫的抗性。因此,研究昆蟲響應(yīng)物理因子脅迫的生理生化對策,有利于認(rèn)識昆蟲應(yīng)對各種物理防治方法的適應(yīng)機制。本文以赤擬谷盜(Tribolium castaneum(Herbst))和四紋豆象(Callosobruchus maculatus(Fabricius))為研究對象,分別對赤擬谷盜響應(yīng)UV脅迫和四紋豆象響應(yīng)低氧、電子束脅迫的生理及分子機制進行了系統(tǒng)的研究。主要結(jié)果如下:1.UVA脅迫誘導(dǎo)赤擬谷盜成蟲熱激蛋白基因(Hsp)和細胞色素P450基因表達。本研究發(fā)現(xiàn)1000μw/cm2強度的UVA照射赤擬谷盜成蟲后,能顯著誘導(dǎo)熱激蛋白基因(Hsp27、Hsp68、Hsp83)和細胞色素P450基因(CYP6BQ4、CYP6BQ8)的表達,3個Hsp基因表達量升高4.1~8.9倍(P0.05),2個P450基因表達量各升高2.2和2.9倍(P0.05)。上述基因的表達模式類似:在UVA照射2 h內(nèi)表達量持續(xù)上升,2 h時達到最高,隨后表達量逐漸降低。通過分別對UVA誘導(dǎo)表達的Hsp基因和P450基因的轉(zhuǎn)錄調(diào)控區(qū)域分析發(fā)現(xiàn),它們都含有HSF、KROX、NF-kappa B、AFB2和dl等多種響應(yīng)外界環(huán)境刺激轉(zhuǎn)錄因子的結(jié)合位點。以上轉(zhuǎn)錄因子可能共同調(diào)控Hsp基因和P450基因的轉(zhuǎn)錄,參與赤擬谷盜成蟲對UVA脅迫的響應(yīng)。2.UVB脅迫抑制赤擬谷盜末齡幼蟲蛻皮激素合成,推遲赤擬谷盜幼蟲變態(tài)。本研究發(fā)現(xiàn)20 k J/m2劑量的UVB照射能導(dǎo)致赤擬谷盜末齡幼蟲100%死亡;10k J/m2劑量的UVB照射后幼蟲羽化率顯著降低為61.1%(P0.001),蛹的長度和重量分別減小了0.36 mm和0.48 mg(P0.001)。研究表明UVB照射會顯著推遲赤擬谷盜幼蟲的變態(tài)。由于昆蟲的變態(tài)過程受蛻皮激素的嚴(yán)格調(diào)控,因而本研究對UVB照射后赤擬谷盜幼蟲蛻皮激素信號途徑中的關(guān)鍵基因進行表達模式分析。結(jié)果表明,UVB照射會顯著降低赤擬谷盜幼蟲蛻皮激素合成基因(Trcptth、sad、shd)和效應(yīng)基因(Ec R、Br)的表達量,表達量分別降低5.4、3.6、2.7、2.9和5.8倍(P0.05);同時,UVB脅迫能夠消除引發(fā)赤擬谷盜幼蟲蛻皮變態(tài)的蛻皮激素脈沖。3.應(yīng)激反應(yīng)、神經(jīng)調(diào)節(jié)、能量代謝和損傷修復(fù)等多種途徑參與赤擬谷盜末齡幼蟲對UVB脅迫的響應(yīng)。本研究利用數(shù)字基因表達譜技術(shù)分別分析了10 k J/m2劑量的UVB照射后4 h和24 h赤擬谷盜幼蟲的差異表達基因,鑒定出遭受UVB脅迫后,赤擬谷盜在不同時間點響應(yīng)脅迫的相關(guān)基因。UVB照射后4 h,赤擬谷盜幼蟲中被誘導(dǎo)表達的基因為222個,抑制表達的基因為88個;照射后24 h,被誘導(dǎo)表達的基因為544個,抑制表達的基因為452個。通過對差異表達基因GO富集分析發(fā)現(xiàn),UVB照射后4 h,赤擬谷盜幼蟲體內(nèi)肽聚糖的代謝過程、糖胺聚糖分解過程、肽聚糖的分解代謝過程、應(yīng)激反應(yīng)過程、氨基聚糖分解過程等相關(guān)基因被顯著富集(P0.001);照射后24 h,有機物代謝過程、蛋白酶解過程、分解代謝過程、糖類衍生物分解過程和大分子分解過程相關(guān)的基因被極顯著富集(P0.0001)。通過對差異表達基因Pathway分析發(fā)現(xiàn),UVB照射后4 h,赤擬谷盜幼蟲體內(nèi)酪氨酸代謝和Wnt信號傳導(dǎo)等途徑被激活;照射后24 h,蛋白質(zhì)代謝過程、糖酵解/糖異生作用、細胞外基質(zhì)受體相互作用和蛋白酶體形成等途徑被激活。以上結(jié)果表明,隨著UVB照射后時間的增長,參與赤擬谷盜幼蟲對UVB脅迫響應(yīng)的基因數(shù)量增加;UVB照射后早期(4 h),赤擬谷盜幼蟲主要以應(yīng)激反應(yīng)、神經(jīng)系統(tǒng)調(diào)控響應(yīng)UVB脅迫;UVB照射后晚期(24 h),赤擬谷盜幼蟲主要依靠調(diào)整能量代謝、控制器官發(fā)育、修復(fù)DNA損傷、促使無法修復(fù)的蛋白質(zhì)水解來響應(yīng)UVB脅迫。4.低氧脅迫在四紋豆象中的跨代傳遞效應(yīng)參與子代對低氧脅迫的響應(yīng)。本研究發(fā)現(xiàn)低氧處理(2%O2+18%CO2+80%N2混合氣體)四紋豆象親代4齡幼蟲、蛹和成蟲能顯著降低存活個體產(chǎn)卵量和卵的孵化率,單雌產(chǎn)卵量在低氧處理成蟲后降低最顯著(多達21粒)(P0.001),孵化率在低氧處理蛹后降低最顯著(為9.9%)(P0.001)。低氧處理親代4齡幼蟲時,其F1代對低氧會產(chǎn)生一定抗性,表現(xiàn)為:F1代4齡幼蟲再次遭遇親代經(jīng)歷的相同低氧環(huán)境時,存活個體所產(chǎn)卵的孵化率不受影響(P0.05);與親代不經(jīng)歷低氧脅迫所產(chǎn)生的F1代相比,經(jīng)歷低氧脅迫所產(chǎn)生的F1代的Cat L基因的基礎(chǔ)表達量顯著性降低2.8倍(P0.05),Hsp27基因的基礎(chǔ)表達量顯著性升高1.6倍(P0.05)。以上結(jié)果表明,低氧脅迫對四紋豆象的影響能從親代傳遞給子代,促使子代通過調(diào)整相關(guān)基因的基礎(chǔ)表達適應(yīng)親代曾經(jīng)歷的不利低氧環(huán)境。5.電子束輻照嚴(yán)重影響四紋豆象的生長發(fā)育和生殖。本研究表明四紋豆象的卵在發(fā)育過程中對電子束輻照的敏感性存在差異。0.03k Gy劑量的電子束輻照導(dǎo)致早期卵和中期卵無法孵化;0.12 k Gy劑量的電子束輻照后仍有58%的黑頭期(后期)卵可以孵化,但孵化后幼蟲的發(fā)育被抑制。雖然幼蟲中腸蛋白酶消化功能不受影響,但被0.06~0.12 k Gy劑量電子束輻照的幼蟲無法發(fā)育成正常的成蟲,死亡于蛹或非正常羽化的成蟲,表明電子束對四紋豆象的變態(tài)存在有害影響。0.06和0.09 k Gy劑量的電子束處理的蛹羽化后,成蟲壽命縮短,且無法產(chǎn)卵。0.06~2 k Gy劑量的電子束輻照成蟲,其產(chǎn)下的卵不能孵化。電子束輻照對成蟲壽命縮短的影響具有劑量依賴效應(yīng)。正反交試驗表明,四紋豆象雌性比雄性對電子束輻照更敏感。解剖雌蟲生殖系統(tǒng)發(fā)現(xiàn),電子束處理能抑制卵母細胞的形成。
[Abstract]:Ultraviolet (UV), hypoxia and electron beam radiation is an important physical force reducing insect survival stress factor, is used as an effective means of pest control. These insects to stress factors in response to gene expression, biological process of protein translation and physiological changes in a variety of complex, the final performance of stress resistance. Therefore, the research of insect in response to physiological and biochemical measures of physical factors stress, is conducive to the understanding of insect adaptation mechanism to deal with all kinds of physical control methods. In this paper, Tribolium CASTANEUM (Tribolium CASTANEUM (Herbst)) and four g.maculatus (Callosobruchus maculatus (Fabricius)) as the research object, respectively, in response to UV stress and the four on Triboliumcastaneum g.maculatus in response to hypoxia, physiological and molecular mechanism electron beam stress was studied. The main results are as follows: 1.UVA stress induced by adults of Triboliumcastaneum heat shock protein (Hsp) and color Bai Jiyin cell In the expression of P450 gene. This study found that adults of Triboliumcastaneum UVA irradiation 1000 w/cm2 strength, can significantly induce heat shock protein genes (Hsp27, Hsp68, Hsp83) and cytochrome P450 gene (CYP6BQ4, CYP6BQ8) the expression of Hsp 3 gene expression was increased by 4.1~8.9 times (P0.05), 2 P450 gene expression the amount of each increase 2.2 and 2.9 times (P0.05). The similar expression pattern of genes: UVA irradiation in 2 h expression volume continued to rise at 2 h reached the highest, then the expression decreased gradually. The transcription regulatory region of Hsp gene and P450 gene were induced by UVA expression analysis found that they all contain HSF KROX, NF-kappa, B, AFB2 and DL and other transcription factor binding sites response to external environmental stimuli. These transcription factors may co transcriptional regulation of Hsp gene and P450 gene, in adults of Triboliumcastaneum response to.2.UVB UVA stress inhibited the late Triboliumcastaneum Larvae of ecdysone synthesis, delayed metamorphosis. This study found against larvae of 20 K J/m2 dose of UVB irradiation can lead to the death of 100% last instar larvae of t. castaneum; 10K J/m2 dose of UVB irradiation after larvae emergence rate were significantly reduced to 61.1% (P0.001), the length and weight of pupae were reduced by 0.36 mm and 0.48 Mg (P0.001). Research shows that UVB irradiation can significantly delay the Tribolium larval metamorphosis. Due to the strict regulation of insect metamorphosis by ecdysone, so the research on the key gene of Tribolium larval ecdysone signal pathway of UVB after irradiation in the expression pattern analysis. The results showed that UVB irradiation significantly reduced the ecdysone synthesis genes against larvae (Trcptth, sad, SHD) and the effect of gene (Ec R Br) expression, expression levels were reduced by 5.4,3.6,2.7,2.9 and 5.8 times (P0.05); at the same time, UVB can eliminate the stress caused by Triboliumcastaneum Adjust the ecdysonepulses.3. stress response, larval molt and metamorphosis of the nerve, energy metabolism and injury repair of multiple pathways involved in Tribolium castaneum last instar larvae in response to UVB stress. This study using digital gene differential gene expression spectrum technology are analyzed respectively. 10 K J/m2 UVB dose after 4 h and 24 h against larvae of expression, identification the UVB suffered stress, Tribolium castaneum at different time points of genes related to stress response after.UVB irradiation was 4 h, t.castaneum larvae induced the expression of 222 genes, inhibiting the expression of the genes for 88; 24 h after irradiation, induced the expression of the genes for 544, inhibited the expression of 452 genes. Based on the differential gene expression GO enrichment analysis showed that UVB 4 h after irradiation, the metabolic process of Tribolium larvae of peptidoglycan, decomposition of glycosaminoglycans, metabolism of peptidoglycan, stress response Cheng, aminoglycan decomposition process and other related genes were significantly enriched (P0.001); 24 h after irradiation, organic metabolism process, solution of protease, metabolism, carbohydrate derivatives decomposition process and molecular decomposition process related genes was extremely significantly enriched (P0.0001). The expression of Pathway gene analysis showed that the difference of UVB. 4 h after irradiation, activated against larvae in vivo tyrosine metabolism and Wnt signal transduction pathway; 24 h after irradiation, protein metabolism, glycolysis / gluconeogenesis, ECM receptor interaction and protein enzyme formation pathways were activated. These results show that with the time after exposure to UVB growth in response, the number of t.castaneum larvae to UVB stress was increased; the early stage after exposure to UVB (4 h), mainly against larvae of stress, nervous system regulation in response to UVB stress; late after UVB irradiation (24 h), The larvae mainly rely on the adjustment of energy metabolism, control organ development, DNA damage repair, the hydrolysis of proteins cannot be repaired in response to inter generational transmission effects in offspring of hypoxic stress response to UVB stress in.4. hypoxia stress in four maculatus. This study found that low oxygen treatment (2%O2+18%CO2+80%N2 mixture) four g.maculatus parent 4 larvae, pupae and adults can significantly reduce the survival of individual fecundity and egg hatching rate, single female fecundity in adults after hypoxia treatment decreased most significantly (up to 21) (P0.001), the hatching rate in pupae decreased most significantly after hypoxia treatment (9.9%) (P0.001). Hypoxia parental 4 instar larvae when the F1 generation will have a certain resistance to hypoxia, as follows: F1 4 instar larva encounters the parental experience the same hypoxic environment, the egg hatching rate of individual survival is not affected (P0.05); and the parent does not experience hypoxia stress Force generated by F1 generation, F1 generation Cat experience based L gene hypoxia stress generated by expression significantly decreased 2.8 times (P0.05), Hsp27 gene expression was significantly increased 1.6 times (P0.05). The results show that the effects of hypoxia stress on four g.maculatus can be passed from parents to the offspring, the offspring through the basis of adjusting the expression of genes related to hypoxia to adapt to adverse environment.5. electron beam irradiation has serious impact on parental growth and reproductive four g.maculatus. This study shows that electron beam irradiation sensitivity of four g.maculatus eggs according to the electron beam radiation in the development process there exist differences in the dose of Gy.0.03k in early and mid eggs from hatching eggs; electron beam irradiation dose of 0.12 K Gy was still 58% black period (late) eggs can be hatched, but after hatching and larval development was inhibited. Although larval midgut protease digestion Is not affected, but was unable to 0.06~0.12 K Gy dose of electron beam irradiation larvae develop into normal adults, died pupae or non normal eclosion adults showed that the electron beam of four g.maculatus metamorphosis has deleterious effects of.0.06 and K 0.09 Gy dose of the electron beam treatment of eclosion, adult longevity, adult.0.06~2 K Gy and cannot spawn dose of electron beam irradiation, the eggs did not hatch. Effect of electron beam irradiation on adult longevity shortened with dose dependence. The reciprocal cross test showed that four g.maculatus than the males are more sensitive to electron beam irradiation. The anatomy of the female reproductive system that can inhibit the formation of electron beam processing oocytes.

【學(xué)位授予單位】：華中農(nóng)業(yè)大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：S379.5

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