【作者】 杨丽；

【导师】 鲁玉杰； 卢少华；

【作者基本信息】 河南工业大学 ， 农业昆虫与害虫防治， 2024， 硕士

【摘要】 氮气气调是一种安全且环保的绿色储粮技术,其杀虫机理主要是缺氧而窒息死亡,但很多昆虫在长期的低氧胁迫下产生了低氧耐受性。低氧胁迫会破坏脂质、抑制蛋白质合成并提升活性氧（reactive oxygen species,ROS）的水平,从而引起氧化损伤。为了中和ROS的毒性,昆虫进化出了抗氧化酶系统来抵御氧化损伤,抗氧化酶系统可以清除体内过多的ROS,维持体内ROS代谢的平衡。超氧化物歧化酶、过氧化氢酶和过氧化物酶（peroxidase,POD）是昆虫体内ROS防御系统的主要抗氧化酶系。POD中的过氧化物还原酶（peroxiredoxins,Prxs）可以通过清除ROS从而维持细胞稳态。Prxs是一种半胱氨酸依赖性过氧化物酶超家族,Prx6属于1-Cys Prxs。丝裂原活化蛋白激酶（mitogen activated protein kinase,MAPK）能够通过激活14-3-3ε作为伴侣蛋白,调控Prx6的亚细胞定位,导致其向酸性细胞器转运。然而,赤拟谷盗TcPrx6基因对低氧胁迫的响应机制及其蛋白和TcMAPK蛋白的互作关系尚不清楚。本研究以赤拟谷盗为研究对象,在研究低氧对赤拟谷盗不同虫态的胁迫作用的基础上,运用分子生物学和生物信息学等手段,对TcPrx6基因进行鉴定和功能研究,为揭示TcPrx6调控赤拟谷盗低氧耐受性的机制提供理论基础,为更科学有效地开展储藏物害虫的防治提供新方向。研究结果如下:（1）低氧对赤拟谷盗的胁迫作用和过氧化物酶活性的影响采用低氧胁迫赤拟谷盗卵、幼虫、蛹和成虫,统计死亡率,并测定低氧胁迫赤拟谷盗幼虫后体内H₂O₂含量、MDA含量、POD酶活性和Prx6酶活性。结果表明,随着氮气浓度的升高和低氧处理时间的延长,赤拟谷盗各虫态的死亡率逐渐升高,赤拟谷盗成虫对低氧的耐受性最强,其次是蛹,卵和幼虫对低氧的耐受性最弱。即使在98%浓度氮气处理赤拟谷盗各虫态7 d,仍然没有观察到100%死亡,说明赤拟谷盗具有一定的低氧耐受性。在98%浓度氮气下,随着低氧处理时间的延长,赤拟谷盗幼虫体内H₂O₂含量没有显著变化（P>0.05）,而MDA含量呈现先逐渐升高后降低的趋势。随着低氧处理时间的延长,POD和Prx6酶活性均呈现先升高后降低的趋势,不同的是,POD酶活性在处理24 h后开始下降,而Prx6酶活性在处理72 h后开始下降。说明低氧胁迫赤拟谷盗引发了脂质过氧化损伤,而Prx6酶活性的升高维持了赤拟谷盗体内H₂O₂含量的稳定。（2）赤拟谷盗TcPrx6的鉴定及生物信息学分析运用RT-PCR技术克隆获得赤拟谷盗TcPrx6基因的CDS,TcPrx6的CDS序列长度为672 bp,编码223个氨基酸。TcPrx6蛋白属于Thioredoxin＿like超家族,具有亲水性,无信号肽和跨膜区,属于非分泌蛋白。TcPrx6蛋白具有N端半胱氨酸残基（Cys45）,一个组氨酸（His 37）和一个精氨酸（Arg 129）,表明TcPrx6属于1-Cys Prxs。系统发育分析显示,TcPrx6与其他鞘翅目昆虫的Prx6属同一亚群,且TcPrx6氨基酸序列与黑拟谷盗（Tribolium madens）的Prx6同源性为96.86%,表明Prx6高度保守。采用RT-q PCR技术分析TcPrx6的时空表达模式,发现TcPrx6在晚期幼虫和中期幼虫的表达量相对较高,而在晚期成虫、早期成虫、早期幼虫、早期卵和晚期卵的表达量较低。此外,正常生理条件下,TcPrx6在赤拟谷盗幼虫所有被测组织中普遍表达,且TcPrx6在脂肪体和血淋巴中高表达,说明TcPrx6基因表达具有阶段特异性和组织特异性。（3）TcPrx6基因对低氧胁迫的响应机制采用RT-q PCR技术分析TcPrx6对低氧的响应模式,98%浓度氮气处理赤拟谷盗幼虫不同时间后,TcPrx6表达水平呈现两次先升高后降低的趋势,表明低氧可显著诱导TcPrx6的表达。沉默TcPrx6的赤拟谷盗幼虫在98%浓度氮气处理24 h后死亡率显著升高（P<0.05）,但是在处理48 h后差异不显著（P>0.05）。表明赤拟谷盗TcPrx6基因在短期低氧胁迫下发挥了重要作用。此外,沉默TcPrx6的赤拟谷盗幼虫在低氧胁迫下体内H₂O₂含量显著升高,表明TcPrx6基因在赤拟谷盗应对低氧胁迫的过程中发挥了重要作用,特别是通过清除H₂O₂发挥作用。（4）Prx6与MAPK和14-3-3ε蛋白的互作在赤拟谷盗低氧耐受性中的作用初探干扰TcPrx6后,TcMAPK基因表达量显著下调（P<0.01）,下调了0.45倍,同样Tc14-3-3ε基因表达量显著下调（P<0.05）,下调了0.23倍,表明沉默TcPrx6基因能够影响TcMAPK和Tc14-3-3ε基因的表达。采用酵母双杂交和GST-Pull down技术验证了TcPrx6和TcMAPK、Tc14-3-3ε间存在相互作用。沉默TcMAPK和Tc14-3-3ε基因后赤拟谷盗的低虫氧耐受性均显著降低,且沉默TcMAPK后,Tc14-3-3ε和TcPrx6表达量均显著降低,表明TcPrx6和TcMAPK、Tc14-3-3ε可能以某种相互作用和调控模式参与调控赤拟谷盗低氧耐受性。综上所述,TcPrx6在保护赤拟谷盗免受低氧胁迫以及氧化损伤的过程中发挥了重要作用。通过初步探讨TcPrx6和TcMAPK、Tc14-3-3ε蛋白的互作在低氧耐受性中的作用,以期为深入了解TcPrx6调控赤拟谷盗低氧耐受性的机制,为开发有效的储粮害虫防治新靶标提供理论基础,同时为绿色储粮技术的发展提供理论依据。更多还原

【Abstract】 Nitrogen controlled atmosphere is a safe and environmentally friendly green grain storage technology.Its insecticide mechanism is mainly that asphyxia leads to death due to hypoxia.However,many insects have developed hypoxic tolerance under long-term hypoxia stress.Hypoxia stress could damage lipids,inhibit protein synthesis,and increase the level of reactive oxygen species（ROS）,thus causing oxidative damage.In order to neutralize the toxicity of ROS,insects have evolved an antioxidant enzyme system to resist oxidative damage,which could remove excessive ROS and maintain the balance of ROS metabolism in the body.Superoxide dismutase,catalase and peroxidase（POD）are the main antioxidant enzymes in the ROS defense system of insects.Among POD,peroxiredoxins（Prxs）could maintain cell homeostasis by scavenging ROS.Prxs are a cysteine-dependent peroxidase superfamily,and Prx6 belongs to 1-Cys Prxs.Mitogen activated protein kinase（MAPK）could regulate the subcellular localization of Prx6by activating 14-3-3εas a chaperone protein,leading to its transport to acidic organelles.However,the response mechanism of TcPrx6 to hypoxia stress and the relationship of the interaction between TcPrx6 and TcMAPK protein in Tribolium castaneum remain unclear.In the study,we used T.castaneum as the research insect.On the basis of studying the effect of hypoxia stress on T.castaneum different developmental stages,TcPrx6 gene was identified and its function was studied by means of molecular biology and bioinformatics,so as to provide a theoretical basis for revealing the mechanism of TcPrx6 regulating the hypoxic tolerance of T.castaneum.It provided a new direction for more scientific and effective control of stored-product pests.The results are as follows:（1）The effects of hypoxia on stress and peroxidase activity of T.castaneum.T.castaneum eggs,larvae,pupae and adults were exposed to nitrogen for different time.Then,the mortality rate was calculated.The contents of H₂O₂and MDA and the activities of POD and Prx6 enzyme in T.castaneum larvae under hypoxia stress were determined.The results showed that with the increase of nitrogen concentration and the prolongation of exposure time,the mortality of each developmental stage of T.castaneum increased gradually.The adults of T.castaneum had the strongest tolerance to hypoxia,followed by pupae,and eggs and larvae had the weakest tolerance to hypoxia.Even though different developmental stages of T.castaneum were exposed with 98%nitrogen for 7 d,no 100%death was observed,indicating that T.castaneum had a certain hypoxic tolerance.At 98%nitrogen,the content of H₂O₂ in T.castaneum larvae did not change significantly with the prolongation of hypoxia exposure time（P>0.05）,while the content of MDA showed a trend of increasing first and then decreasing.With the prolongation of hypoxia exposure time,POD and Prx6 enzyme activities increased first and then decreased.The difference was that POD enzyme activity began to decrease after 24 h of exposure,while Prx6 enzyme activity began to decrease after 72 h of exposure.The results indicated that hypoxia stress induced lipid peroxidation damage,and the increase of Prx6 enzyme activity maintained the stability of H₂O₂content in T.castaneum.（2）Identification and bioinformatics analysis of TcPrx6 gene.The CDS sequence of TcPrx6 gene was cloned by RT-PCR.The CDS sequence length of TcPrx6 was 672 bp,encoding 223 amino acids.TcPrx6 protein belonged to the Thioredoxin＿like superfamily,which was a non-secreted protein with hydrophilic properties,no signal peptide and transmembrane region.The TcPrx6 protein had an N-terminal cysteine residue（Cys 45）,a histidine（His 37）and an arginine（Arg 129）,indicating that TcPrx6belonged to 1-Cys Prxs.Phylogenetic analysis showed that TcPrx6 belonged to the same subgroup as Prx6 of other Coleoptera insects,and the amino acid sequence homology of TcPrx6 and Prx6 of Tribolium madens is 96.86%,indicating that Prx6 was highly conserved.The temporal and spatial expression patterns of TcPrx6 were analyzed by RT-q PCR.It showed that the expression of TcPrx6 was relatively high in late larvae and middle larvae,but low in late adults,early adults,early larvae,early eggs and late eggs.In addition,under normal physiological conditions,TcPrx6 was widely expressed in all tested tissues of T.castaneum larvae,and TcPrx6 was highly expressed in fat body and haemolymph,indicating that the expression of TcPrx6 gene was stage-specific and tissue-specific.（3）The response mechanism of TcPrx6 gene to hypoxia stress.The response of TcPrx6 to hypoxia in T.castaneum was analyzed by RT-q PCR.After exposure with 98%nitrogen for different time,the expression of TcPrx6 increased twice and then decreased,indicating that hypoxia could significantly induce the expression of TcPrx6.The mortality of TcPrx6-silenced T.castaneum larvae increased significantly after exposure with 98%nitrogen for 24 h（P<0.05）but there was no significant difference after wxposure for 48 h（P>0.05）.These results indicated that TcPrx6 played an important role in short-term hypoxia stress of T.castaneum.In addition,the H₂O₂ content of TcPrx6-silenced T.castaneum larvae increased significantly under hypoxia stress,which indicated that TcPrx6 played an important role in the response to hypoxia stress,especially by scavenging H₂O₂.（4）The interaction between Prx6 and MAPK and 14-3-3εprotein in the hypoxic tolerance of T.castaneum.After interfering with TcPrx6,the expression of TcMAPK was significantly down-regulated by 0.45 times（P<0.01）,and the expression of Tc14-3-3εwas also significantly down-regulated by 0.23 times（P<0.05）,indicating that silencing TcPrx6 could affect the expression of TcMAPK and Tc14-3-3ε.The interaction between TcPrx6 and TcMAPK and Tc14-3-3εwas verified by yeast two-hybrid and GST-Pull down.The hypoxic tolerance of T.castaneum decreased significantly after silencing TcMAPK and Tc14-3-3ε,and the expression of Tc14-3-3εand TcPrx6 decreased significantly after silencing TcMAPK,indicating that TcPrx6,TcMAPK,and Tc14-3-3εmay be involved in the regulation of hypoxic tolerance of T.castaneum in some interaction and regulation mode.In summary,TcPrx6 played an important role in protecting T.castaneum from hypoxia stress and oxidative damage.The interaction between TcPrx6 and TcMAPK and Tc14-3-3εprotein in hypoxic tolerance was preliminarily discussed in order to further understand the mechanism of TcPrx6 regulating the hypoxic tolerance of T.castaneum.It provided a theoretical basis for developing effective new targets for the prevention and control of stored grain pests and the development of green grain storage technology.更多还原