斑蝥素对草地贪夜蛾卵巢细胞凋亡基因PP2A表达的影响

用实时荧光定量PCR技术对草地贪夜蛾Spodoptera frugiperda卵巢组织细胞蛋白磷酸酶2A(protein phosphatase 2A,PP2A)基因表达进行分析以研究斑蝥素对其表达的影响。结果表明,PP2A相对表达量与斑蝥素浓度存在不显著的负相关性,斑蝥素各浓度下PP2A相对表达量差异都不显著(P<0.05),处理24h后对照的极显著高于用斑蝥素处理的(P<0.01)。在相同斑蝥素浓度下,除25μg/mL外其他浓度处理24 h后PP2A相对定量显著高于6 h的(P<0.05)。研究结果说明,斑蝥素处理对卵巢细胞内的PP2A基因相对表达量有一定的影响,且与斑蝥素的浓度和处理时间有一定关系。

Effects of heme precursors on CYP1A2 and POR expression in the baculovirus/Spodoptera frugiperda system

Objective：CYP1A2 and NADPH-CYP450 oxidoreductase（POR）were expressed in the baculovirus/ Spodoptera frugiperda（sf9）system.The aim of this study was to investigate the effects of heme precursors on the expression of CYP1A2 and POR.Methods：The heme precursors[δ-Aminolaevulinic Acid（5-ALA）,Fe^3＋and hemin]were introduced into the system to evaluate their effects on the expression of CYP1A2,POR and their co-expression.All the proteins were identified using immunoblotting,CO-difference spectroscopy,or cytochrome c assay.Results：In the present study,functional CYP1A2 and POR were successfully expressed in the baculovirus/sf9 system,and both of them showed high activities.Co-addition of 5-ALA and Fe^3＋significantly improved expression of CYP1A2 by about 50%compared with the addition of 5-ALA,Fe^3＋or hemin alone.Either co-addition of 5-ALA and Fe^3＋or addition of 5-ALA or Fe^3＋alone improved the POR expression level 2 fold and its activity 7-10 fold compared with control（no addition）.However,unlike CYP1A2,there was no difference between the co-addition and addition of these heme precursors alone.Different ratios of BvCYP1A2 to BvPOR also affected the co-expression of CYP1A2 and POR,with a 3：1 ratio of BvCYP1A2/BvPOR significantly increasing their co-expression.Surprisingly,the addition of 0.1 mM 5-ALA or Fe^3＋alone,but not their co- addition,could significantly improve the CYP1A2 and POR co-expression（P〈0.05）.Conclusion：5-ALA and Fe^3＋increased the expression of CYP1A2 and POR in a baculovirus/sf9 system,but the pattern of their expression was different between their expression alone and co-expression.

高效氯氰菊酯对草地贪夜蛾Sf9细胞自噬的诱导作用

采用噻唑蓝(MTT)染色、显微镜及透射电子显微镜观察、单丹磺酰戊二胺(MDC)荧光染色及蛋白质免疫印迹(WB)技术等方法,分别针对高效氯氰菊酯(beta-cypermethrin)作用后草地贪夜蛾Spodoptera frugiperda卵巢细胞系Sf9细胞的细胞活力、自噬发生的典型特征及自噬相关蛋白变化情况进行了测试及分析。结果表明:经不同浓度高效氯氰菊酯处理后,对草地贪夜蛾Sf9细胞活性的抑制效应与作用剂量及时间呈现依赖关系,并表现出典型的细胞自噬现象;LC3-Ⅱ/LC3-Ⅰ蛋白表达水平比和Beclin 1蛋白表达量呈现上调趋势,分别为对照组的201.32%和136.00%,p62蛋白表达量下调至对照组的68.13%。研究表明,高效氯氰菊酯对草地贪夜蛾Sf9细胞具有显著的细胞毒性,并且其对细胞活力的抑制作用与自噬诱导途径相关。

利用细菌／杆状病毒系统在昆虫细胞中表达人CYP2E1

目的:获得人CYP2E1重组酶,并用该重组酶的特征性探针底物对其进行代谢活性研究。方法:以人肝组织RNA为模板,通过RT-PCR得到CYP2E1 cDNA片断,然后与pFastBac质粒连接,得到pFastBac-CYP2E1重组质粒,将其转化E.coli DH 10Bac大肠杆菌,通过转座作用,获得重组Bacmid-CYP2E1,将其转染草地夜蛾细胞(Sf9)后,产生重组杆状病毒。将该病毒以及分别含有人CYPOR和人CYPb5的病毒共同感染Sf9细胞,收集共表达蛋白,以氯唑沙宗为底物鉴定重组酶的活性。结果:利用细菌/杆状病毒系统得到重组人CYP2E1的表达,其对氯唑沙宗的Km值为(72.4±8.7)μmol.L-1,Vmax值为(2.41±0.10)μmol.min-1.g-1蛋白。结论:利用杆状病毒系统成功表达了有催化活性的人CYP2E1重组酶,其活性与文献报道值相似。

胡椒碱对SF-21细胞株的杀伤作用及对其形态影响的研究

目的检测胡椒碱对草地贪夜蛾SF-21细胞的毒性作用和生长抑制作用,以便进一步研究其对昆虫细胞离子通道的影响。方法胡椒碱处理SF-21细胞后,用MTT微量酶反应比色法、改良微波Giemsa染色法检测细胞死亡率及形态学改变,用冲击式测试法检测细胞生长率及受损细胞的恢复。结果胡椒碱处理24h后,对SF-21细胞的半数毒性浓度(IC50)为95.37μg/ml,且毒性作用强度随着药物浓度增加而增强。胡椒碱浓度在10μg/ml以上时可诱发SF-21细胞形态学改变,表现为细胞呈多形性、细胞间有间隙、胞质内充满颗粒,以后随药物浓度的升高,胞质出现空泡、染色质凝成粗大颗粒或无结构大块、大片细胞脱落、崩解、死亡。低浓度胡椒碱(5μg/ml)对SF-21细胞增殖没有影响,较高浓度胡椒碱(10、20、40μg/ml)则对SF-21细胞增殖有明显的抑制作用,抑制作用强度随药物浓度的增加而增加,去除药物作用后,受抑制的细胞又重新恢复生长、分裂。结论胡椒碱对昆虫细胞有较强的毒性作用和生长抑制作用,可作为新型杀虫剂的功能物质。

Transcriptome-wide analysis of mRNA N^(6)-methyladenosine modification in the embryonic development of Spodoptera frugiperda

N^(6)-methyladenosine(m^(6)A)RNA is the most abundant modification of mRNA,and has been demonstrated in regulating various post-transcriptional processes.Many studies have shown that m^(6)A methylation plays key roles in sex determination,neuronal functions,and embryonic development in Drosophila and mammals.Here,we analyzed transcriptome-wide profile of m^(6)A modification in the embryonic development of the destructive agricultural pest Spodoptera frugiperda.We found that the 2 key mRNA m^(6)A methyltransferases SfrMETTL3 and SfrMETTL14 have high homologies with other insects and mammals,suggesting that SfrMETTL3 and SfrMETTL14 may have conserved function among different species.From methylated RNA immunoprecipitation sequencing analysis,we obtained 46869 m^(6)A peaks representing 8587 transcripts in the 2-h embryos after oviposition,and 41389 m^(6)A peaks representing 9230 transcripts in the 24-h embryos.In addition,5995 m^(6)A peaks were differentially expressed including 3752 upregulated and 2243 downregulated peaks.Functional analysis with Gene Ontology and Kyoto Encyclopedia of Genes and Genomes suggested that differentially expressed m^(6)A peak-modified genes were enriched in cell and organ development between the 2-and 24-h embryos.By conjoint analysis of methylated RNA immunoprecipitation-seq and RNA-seq data,we found that RNA m^(6)A methylation may regulate the transcriptional levels of genes related to tissue and organ development from 2-to 24-h embryos.Our study reveals the role of RNA m^(6)A epigenetic regulation in the embryonic development of S.frugiperda,and provides new insights for the embryonic development of insects.

日本血吸虫胞蚴的体外培养和操作初探

目的探究一种体外培养日本血吸虫胞蚴的新方法，并通过体外注射，感染阴性钉螺，培养阳性钉螺。方法将日本血吸虫虫卵孵化的毛蚴与作为滋养层的草地夜蛾（Spodoptera frugiperda）卵巢细胞（庐细胞）共培养，观察毛蚴的生长情况，并拍摄生长照片。用显微注射针将3～5只母胞蚴注射到钉螺体内，计算钉螺的存活率及尾蚴的阳性率，用钉螺逸出的尾蚴人工感染昆明鼠，检测尾蚴的活力，用组织化学染色法和PCR方法鉴定尾蚴的相关基因特性。结果毛蚴培养至第12小时观察到纤毛板已脱落，培养2～3d的虫体呈不断的伸缩运动，此后母胞蚴维持该状态并不转化成子胞蚴。3次实验的钉螺存活率依次为24．72％、28．23％和57．89％，活螺阳性率依次为4．55％、8．57％和14．29％。阳性钉螺释放的尾蚴具有感染性，且具有与日本血吸虫相同的基因特性。结论体外培养毛蚴通过显微注射可以人工感染钉螺并逸出有感染活力的尾蚴，为日本血吸虫幼虫的体外操作提供一种新的方法。

α-三联噻吩致草地贪夜蛾Sf9细胞损伤的可视化探索

[目的]用可视化的方法研究光活化物质α-三联噻吩（α-T）对离体培养的草地贪夜蛾卵巢细胞系Sf9细胞（Sf9）的损伤机理。[方法]用Cell Counting Kit-8法测定α-T对Sf9的细胞毒力,通过流式细胞仪、激光扫描共聚焦显微镜观察α-T在Sf9细胞内的吸收、分布和光照损伤的影响。[结果]在黑暗和光照条件下,α-T处理Sf9 24 h的IC50值分别为66.359、0.003 mg/L;α-T能够跨越细胞膜在细胞质内随机分布但不能跨越核膜进入细胞核,经光照之后,细胞大小没有明显变化,形态异常,细胞膜出现褶皱,细胞核体积明显胀大。[结论]光活化物质α-T对Sf9的增殖抑制效果显著,主要损伤细胞核和细胞膜。