【作者】 李璐；

【导师】 马玲；

【作者基本信息】 东北林业大学 ， 森林保护学， 2025， 博士

【摘要】 舞毒蛾(Lymantria dispar)作为一种世界性森林害虫,具有食性杂和分布范围广等特点,对我国林业经济带来了巨大的损失。甲维盐是一种对环境友好、高效、低残留的半合成抗生素杀虫剂,对鳞翅目害虫具有极强的毒性作用,被广泛用于林业害虫防治。G6PD酶是磷酸戊糖途径关键限速酶,调控机体的氧化还原平衡、细胞凋亡和多种生理活动。因此,探究G6PD酶在昆虫细胞中生理功能及作用机制不仅能丰富鳞翅目害虫G6PD酶的功能,还有助于研发以G6PD酶和磷酸戊糖途径为靶标的新型环境友好型杀虫剂。为探索G6PD酶对昆虫细胞氧化应激作用以及阐明甲维盐介导G6PD酶诱导昆虫细胞发生氧化还原失衡和细胞凋亡的潜在机制,本试验利用生物信息学分析对其基因和蛋白质结构,利用q RT-PCR技术分析G6PD基因进行时空表达模式,利用抗氧化酶试剂盒、G6PD酶活性检测试剂盒和q RT-PCR技术对甲维盐处理后舞毒蛾中肠氧化应激水平、G6PD酶活性和细胞凋亡进行检测,并在此基础上,使用RNAi干扰技术、添加外援物质以及过表达目的基因的方式对舞毒蛾中肠以及Sf9细胞进行处理,利用ROS染色、AO/EB染色、透射电子显微镜、多种试剂盒以及q RT-PCR技术对昆虫细胞氧化应激和细胞凋亡相关指标与基因进行检测,主要结果如下:1、Ld G6PD蛋白包含494个氨基酸,16个丝氨酸修饰位点、12个酪氨酸修饰位点、9个苏氨酸修饰位点。系统发育树表明,Ld G6PD与美国白蛾(Hyphantria cunea)、斜纹夜蛾(Spodoptera litura)、草地贪夜蛾(Spodoptera frugiperda)和粉纹夜蛾(Trichoplusia ni)的亲缘关系最近。Ld G6PD在5龄幼虫中表达量最高,是1龄幼虫(对照组)的4.530倍,4龄次之,Ld G6PD表达量为对照组的2.184倍,其余龄期与对照组无显著差异,在雄成虫期(MA)Ld G6PD基因表达量最低。Ld G6PD基因在丝腺、前肠和脂肪体中表达量最高,在表皮中表达量最低。2、甲维盐处理后舞毒蛾中肠MDA含量显著上升,抗氧化酶活性显著下降,磷酸戊糖途径关键酶活性、m RNA表达量和NADPH含量显著减低,中肠凋亡相关基因及caspase3活性显著升高。RNAi沉默后舞毒蛾中肠MDA含量显著上升,抗氧化酶活性显著下降,中肠凋亡相关基因m RNA表达量和活性显著升高。上述结果表明,甲维盐能通过诱导抑制磷酸戊糖途径关键限速酶G6PD活性,抑制抗氧化酶活性而导致舞毒蛾中肠发生氧化应激。3、不同浓度甲维盐处理后Sf9细胞活性随时间增加呈依赖性降低,甲维盐半数抑制浓度处理后Sf9细胞ROS大量生成、MDA含量显著上升,抗氧化酶活性显著下降,Sf9细胞凋亡相关基因m RNA表达量显著升高。上述结果表明,甲维盐抑制抗氧化酶活性诱导ROS生成,激活凋亡相关通路而诱导Sf9细胞发生细胞凋亡。4、不同浓度NADPH处理后Sf9细胞活性随时间增加呈依赖性增加,不同浓度NAC处理后Sf9细胞活性没有显著变化。NADPH和NAC预处理后加入甲维盐,与甲维盐组相比Sf9细胞中ROS显著降低、MDA含量恢复至正常水平,抗氧化酶活性没有显著变化,凋亡细胞含量、凋亡相关基因m RNA表达量及caspase3活性水平均恢复正常,并观察到甲维盐处理后Sf9细胞出现细胞外围轮廓模糊、染色质凝集和细胞核皱缩等凋亡典型特征,但NADPH和NAC预处理后细胞恢复正常形态。表明NADPH能够与NAC行使相似功能,维持细胞中抗氧化酶系统稳定缓解细胞凋亡发生。5、甲维盐处理后Sf9细胞磷酸戊糖途径关键酶活性、m RNA表达量和NADPH含量显著减低。Sf9细胞过表达Ld G6PD基因后加入甲维盐,细胞中MDA含量降低,抗氧化酶活性得到恢复,凋亡相关基因m RNA表达量显著下降,此外,Sf9细胞过表达Ld G6PD基因后能显著缓解甲维盐诱导的caspase3活性异常。沉默G6PD基因后Sf9细胞MDA含量显著升高,抗氧化酶活性受到显著抑制,凋亡相关基因m RNA表达量和caspase3活性显著增加。上述结果表明,甲维盐通过调控G6PD表达,降低NADPH含量而导致细胞发生氧化应激和细胞凋亡。综上所述,本研究证实了甲维盐抑制磷酸戊糖途径关键限速酶的活性,揭示了G6PD调控细胞氧化应激和凋亡的分子机制。研究结果不仅丰富昆虫G6PD基因的功能,也为创造靶向该基因的杀虫剂提供理论依据与研究基础。更多还原

【Abstract】 Lymantria dispar,as a worldwide forest pest,has the characteristics of mixed feeding and wide distribution,which has brought huge losses to forestry economy in China.Emamectin benzoate is an environmentally friendly,highly effective,and low-residue semisynthetic antibiotic insecticide that has extremely toxic effects on Lepidopteran pests and is widely used in forestry pest control.G6PD enzyme is a key rate-limiting enzyme of the pentose phosphate pathway,which regulates the body’s redox homeostasis,apoptosis and various physiological activities.Therefore,exploring the physiological function and mechanism of G6PD enzyme in insect cells can not only enrich the function of G6PD enzyme in lepidopteran pests,but also help to develop new environmentally friendly insecticides targeting G6PD enzyme and pentose phosphate pathway.In order to explore the effect of G6PD enzyme on oxidative stress in insect cells and elucidate the underlying mechanism of EMB-mediated redox imbalance and apoptosis in insect cells.In this study,bioinformatics analysis was used to analyze the structure of genes and proteins,q RT-PCR technology was used to analyze the spatiotemporal expression pattern of G6PD gene,and antioxidant enzyme kit,G6PD enzyme activity detection kit and q RT-PCR technology were used to detect the level of midgut oxidative stress,G6PD enzyme activity and apoptosis of L.dispar after EMB treatment.On this basis,RNAi interference technology,addition of foreign aid substances and overexpression of target genes were used to treat the midgut and Sf9 cells of L.dispar,and the indexes and genes related to oxidative stress and apoptosis in insect cells were detected by ROS staining,AO/EB staining,transmission electron microscopy,various kits and q RT-PCR technology,and the main results were as follows:1、Ld G6PD protein contains 494 amino acids,16 serine modification sites,12 tyrosine modification sites,and 9 threonine modification sites.The phylogenetic tree showed that Ld G6PD was most closely related to Hyphantria cunea,Spodoptera litura,Spodoptera frugiperda,and Spodoptera ni.The expression level of Ld G6PD was the highest in the 5^th-instalarvae,which was 4.530 times that of the 1^th-instalarvae（control group）,followed by the4th instar,and the expression level of Ld G6PD was 2.184 times that of the control group.The expression level of Ld G6PD gene was highest in silk glands,foregut and fat body,and lowest in epidermis.2、After EMB treatment,the content of MDA in the midgut was significantly increased,the activity of antioxidant enzymes was significantly decreased,the activities of key enzymes in the pentose phosphate pathway,the expression of m RNA and the content of NADPH were significantly decreased,and the genes related to apoptosis in the midgut were significantly increased.After RNAi silencing,the content of MDA in the midgut increased significantly,the activity of antioxidant enzymes decreased significantly,and the m RNA expression of apoptosis-related genes in the midgut increased significantly.These results indicated that EMB could induce oxidative stress in the midgut of the L.dispar by inducing and inhibiting the activity of G6PD,a key rate-limiting enzyme in the pentose phosphate pathway,and inhibiting the activity of antioxidant enzymes.3、The activity of Sf9 cells decreased with time after different concentrations of EMB,and the amount of ROS was generated,the content of MDA increased significantly,the activity of antioxidant enzymes decreased significantly,and the m RNA expression of apoptosis-related genes in Sf9 cells increased significantly.These results indicated that EMB inhibited the activity of antioxidant enzymes to induce ROS production,activate apoptosis-related pathways and induce apoptosis in Sf9 cells.4、The activity of Sf9 cells increased with time after treatment with different concentrations of NADPH,and there was no significant change in the activity of Sf9 cells after treatment with different concentrations of NAC.After NADPH and NAC pretreatment,the ROS in Sf9 cells was significantly reduced,the MDA content returned to normal level,the antioxidant enzyme activity was not significantly changed,and the content of apoptotic cells and the m RNA expression of apoptosis-related genes and the activity of caspase3 returned to normal compared with the EMB group.Typical apoptosis characteristics of Sf9 cells,such as blurred peripheral contour,chromatin agglutination and nuclear shrinkage,were observed after EMB treatment,but the cells returned to normal morphology after NADPH and NAC pretreatment.These results indicated that NADPH could perform similar functions to NAC to maintain the antioxidant enzyme system in cells and alleviate the occurrence of apoptosis.5、The key enzyme activities,m RNA expression and NADPH content of pentose phosphate pathway in Sf9 cells were significantly reduced after EMB treatment.Overexpression of Ld G6PD in Sf9 cells significantly alleviated the abnormal activity of caspase3 induced by emamectin benzoate.After the overexpression of Ld G6PD in Sf9 cells,the MDA content in the cells decreased,the activities of antioxidant enzymes recovered,and the m RNA expression of apoptosis-related genes decreased significantly.After silencing G6PD gene in Sf9 cells,MDA content was significantly increased,the activities of antioxidant enzymes were significantly inhibited,and the m RNA expression of apoptosis-related genes and caspase3 activity were significantly increased.These results indicated that EMB could lead to oxidative stress and apoptosis by regulating the expression of G6PD and reducing the content of NADPH.In summary,this study confirmed the activity of EMB to inhibit the key rate-limiting enzyme of the pentose phosphate pathway,and revealed the molecular mechanism of G6PD regulating cellular oxidative stress and apoptosis.The results of this study not only enrich the function of the G6PD gene in insects,but also provide a theoretical basis and research basis for the creation of insecticides targeting this gene.更多还原