赤拟谷盗与四纹豆象对物理因子胁迫的响应机制研究

发布时间：2018-02-26 02:17

本文关键词： 紫外线低氧电子束物理胁迫响应差异表达基因　出处：《华中农业大学》2016年博士论文　论文类型：学位论文

【摘要】：紫外线(UV)、低氧和电子束辐射是造成昆虫生存力降低的重要物理胁迫因子,是用作害虫防治的有效手段。昆虫对这些胁迫因子的响应涉及基因表达、蛋白质翻译和生理变化等多种复杂的生物学过程,最终表现对胁迫的抗性。因此,研究昆虫响应物理因子胁迫的生理生化对策,有利于认识昆虫应对各种物理防治方法的适应机制。本文以赤拟谷盗(Tribolium castaneum(Herbst))和四纹豆象(Callosobruchus maculatus(Fabricius))为研究对象,分别对赤拟谷盗响应UV胁迫和四纹豆象响应低氧、电子束胁迫的生理及分子机制进行了系统的研究。主要结果如下:1.UVA胁迫诱导赤拟谷盗成虫热激蛋白基因(Hsp)和细胞色素P450基因表达。本研究发现1000μw/cm2强度的UVA照射赤拟谷盗成虫后,能显著诱导热激蛋白基因(Hsp27、Hsp68、Hsp83)和细胞色素P450基因(CYP6BQ4、CYP6BQ8)的表达,3个Hsp基因表达量升高4.1~8.9倍(P0.05),2个P450基因表达量各升高2.2和2.9倍(P0.05)。上述基因的表达模式类似:在UVA照射2 h内表达量持续上升,2 h时达到最高,随后表达量逐渐降低。通过分别对UVA诱导表达的Hsp基因和P450基因的转录调控区域分析发现,它们都含有HSF、KROX、NF-kappa B、AFB2和dl等多种响应外界环境刺激转录因子的结合位点。以上转录因子可能共同调控Hsp基因和P450基因的转录,参与赤拟谷盗成虫对UVA胁迫的响应。2.UVB胁迫抑制赤拟谷盗末龄幼虫蜕皮激素合成,推迟赤拟谷盗幼虫变态。本研究发现20 k J/m2剂量的UVB照射能导致赤拟谷盗末龄幼虫100%死亡;10k J/m2剂量的UVB照射后幼虫羽化率显著降低为61.1%(P0.001),蛹的长度和重量分别减小了0.36 mm和0.48 mg(P0.001)。研究表明UVB照射会显著推迟赤拟谷盗幼虫的变态。由于昆虫的变态过程受蜕皮激素的严格调控,因而本研究对UVB照射后赤拟谷盗幼虫蜕皮激素信号途径中的关键基因进行表达模式分析。结果表明,UVB照射会显著降低赤拟谷盗幼虫蜕皮激素合成基因(Trcptth、sad、shd)和效应基因(Ec R、Br)的表达量,表达量分别降低5.4、3.6、2.7、2.9和5.8倍(P0.05);同时,UVB胁迫能够消除引发赤拟谷盗幼虫蜕皮变态的蜕皮激素脉冲。3.应激反应、神经调节、能量代谢和损伤修复等多种途径参与赤拟谷盗末龄幼虫对UVB胁迫的响应。本研究利用数字基因表达谱技术分别分析了10 k J/m2剂量的UVB照射后4 h和24 h赤拟谷盗幼虫的差异表达基因,鉴定出遭受UVB胁迫后,赤拟谷盗在不同时间点响应胁迫的相关基因。UVB照射后4 h,赤拟谷盗幼虫中被诱导表达的基因为222个,抑制表达的基因为88个;照射后24 h,被诱导表达的基因为544个,抑制表达的基因为452个。通过对差异表达基因GO富集分析发现,UVB照射后4 h,赤拟谷盗幼虫体内肽聚糖的代谢过程、糖胺聚糖分解过程、肽聚糖的分解代谢过程、应激反应过程、氨基聚糖分解过程等相关基因被显著富集(P0.001);照射后24 h,有机物代谢过程、蛋白酶解过程、分解代谢过程、糖类衍生物分解过程和大分子分解过程相关的基因被极显著富集(P0.0001)。通过对差异表达基因Pathway分析发现,UVB照射后4 h,赤拟谷盗幼虫体内酪氨酸代谢和Wnt信号传导等途径被激活;照射后24 h,蛋白质代谢过程、糖酵解/糖异生作用、细胞外基质受体相互作用和蛋白酶体形成等途径被激活。以上结果表明,随着UVB照射后时间的增长,参与赤拟谷盗幼虫对UVB胁迫响应的基因数量增加;UVB照射后早期(4 h),赤拟谷盗幼虫主要以应激反应、神经系统调控响应UVB胁迫;UVB照射后晚期(24 h),赤拟谷盗幼虫主要依靠调整能量代谢、控制器官发育、修复DNA损伤、促使无法修复的蛋白质水解来响应UVB胁迫。4.低氧胁迫在四纹豆象中的跨代传递效应参与子代对低氧胁迫的响应。本研究发现低氧处理(2%O2+18%CO2+80%N2混合气体)四纹豆象亲代4龄幼虫、蛹和成虫能显著降低存活个体产卵量和卵的孵化率,单雌产卵量在低氧处理成虫后降低最显著(多达21粒)(P0.001),孵化率在低氧处理蛹后降低最显著(为9.9%)(P0.001)。低氧处理亲代4龄幼虫时,其F1代对低氧会产生一定抗性,表现为:F1代4龄幼虫再次遭遇亲代经历的相同低氧环境时,存活个体所产卵的孵化率不受影响(P0.05);与亲代不经历低氧胁迫所产生的F1代相比,经历低氧胁迫所产生的F1代的Cat L基因的基础表达量显著性降低2.8倍(P0.05),Hsp27基因的基础表达量显著性升高1.6倍(P0.05)。以上结果表明,低氧胁迫对四纹豆象的影响能从亲代传递给子代,促使子代通过调整相关基因的基础表达适应亲代曾经历的不利低氧环境。5.电子束辐照严重影响四纹豆象的生长发育和生殖。本研究表明四纹豆象的卵在发育过程中对电子束辐照的敏感性存在差异。0.03k Gy剂量的电子束辐照导致早期卵和中期卵无法孵化;0.12 k Gy剂量的电子束辐照后仍有58%的黑头期(后期)卵可以孵化,但孵化后幼虫的发育被抑制。虽然幼虫中肠蛋白酶消化功能不受影响,但被0.06~0.12 k Gy剂量电子束辐照的幼虫无法发育成正常的成虫,死亡于蛹或非正常羽化的成虫,表明电子束对四纹豆象的变态存在有害影响。0.06和0.09 k Gy剂量的电子束处理的蛹羽化后,成虫寿命缩短,且无法产卵。0.06~2 k Gy剂量的电子束辐照成虫,其产下的卵不能孵化。电子束辐照对成虫寿命缩短的影响具有剂量依赖效应。正反交试验表明,四纹豆象雌性比雄性对电子束辐照更敏感。解剖雌虫生殖系统发现,电子束处理能抑制卵母细胞的形成。
[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.

【学位授予单位】：华中农业大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S379.5

【相似文献】