氟化物对家蚕耐氟和氟化物敏感品种幼虫中肠羧酸酯酶及全酯酶活性的影响

为了探讨氟化物在家蚕Bombyx mori体内的代谢途径, 以家蚕耐氟品种T6和氟化物敏感品种734为研究材料, 在5龄幼虫1-7 d内分别添食经50, 100, 200和400 mg/kg NaF溶液浸泡后的新鲜桑叶, 检测家蚕中肠羧酸酯酶（CarE）和全酯酶活性的变化。结果表明： 734添氟组的CarE活性是对照组的1.21~1.98倍, 而T6添氟组约是对照组的0.72~1.10倍。734和T6添氟组的全酯酶活性数值变化规律与其各自对照组相似, 且2品种之间的酶活性数值很相近。2品种在相同浓度下, 不同天数之间的全酯酶活性差异均显著（P＜0.05）。推测氟化物对敏感家蚕中肠CarE有促进作用, 对耐氟家蚕中肠CarE有抑制作用, 但是对全酯酶活性影响不大。     

家蚕羧酸酯酶基因克隆及差异表达

家蚕浓核病毒 (Bombyx mori densonucleosis virus,BmDNV)是蚕业生产上危害比较严重的一类病毒。用完全抗浓核病中国镇江株(BmDNV-Z)的家蚕品系秋丰、感性品系华八及以华八为轮回亲本回交8代和自交8代构建的近等基因系BC₈为材料,采用mRNA荧光差显技术首次分离克隆了家蚕羧酸酯酶（B. mori carboxylesterase,BmCarE）基因全长cDNA,并用实时荧光定量PCR检测了添毒后12 h、36 h、72 h BmCarE在感、抗BmDNV-Z家蚕品系中肠内的表达差异。结果表明： （1）添毒后12 h不同品系家蚕中肠BmCarE表达差异最大,抗性品系BC₈和秋丰分别是感性品系华八的17.714倍和3.602倍,三者彼此间的差异达到极显著水平;（2）同一品系添毒后12 h与添清水后12 h BmCarE表达也有较大差异, BC₈添毒是BC₈添清水的15.08倍, 秋丰添毒是秋丰添清水的3.39倍, 差异达到极显著水平,而华八添毒和添清水的BmCarE表达量均低,二者差异不显著;（3）同一品系添毒后不同时间BmCarE表达也有较大差异, BC₈和秋丰添毒后12 h BmCarE表达量最高,显著高于各自添毒后36 h和72 h表达水平,而添毒后36 h与72 h表达无显著差异;华八添毒后12 h、36 h和72 h,BmCarE表达无显著差异。上述结果提示羧酸酯酶基因可能与家蚕抗浓核病毒有一定关系。     

寄主植物对棉蚜羧酸酯酶活性的影响

不同棉花品种对棉蚜Aphis gossypii Glov.羧酸酯酶活性具有明显的影响,在试验的7个棉花品种中,取食中棉12叶片的棉蚜α-乙酸萘酯(α-NA)羧酸酯酶活性最高(2.47微摩尔/毫克蛋白质分钟),是取食泾阳鸡脚棉种群的5.74倍.β-乙酸萘酯(β-NA)羧酸酯酶的活性以取食方渑双无的棉蚜最高(3.41微摩尔/毫克蛋白质分钟),是取食BR-S-10棉蚜的4.49倍.在取食方渑双无叶片的棉蚜中,羧酸酯酶活性大于2.5(微摩尔/毫克蛋白质分钟)的个体占28.3%(α-NA)和67.1%(β-NA),而在取食BR-S-10的种群中仅占2.6%(α-NA)和1.3%(β-NA).取食中棉12的棉蚜对β-NA与α-NA活性的比值为0.79,而取食其它棉花品种的种群β-NA与α-NA活性比值均大于1.用中棉12饲养的棉蚜对α-NA和β-NA活性均低于种群平均值的个体(CE1型)占26%;均高于平均值的个体(CE2型)占19%;对α-NA活性高于平均值,对β-NA活性低于平均值的个体(CE3型)占31%;对α-NA活性低于平均值,对β-NA活性高于平均值的个体(CE4型)占24%.取食其它6个棉花品种的棉蚜中,CE1和CE2型的比例分别为49.4—64.0%和23.4—47.2%,明显高于取食中棉12的种群,而CE3和CE4型个体仅占0—7.6%和3.4—10.4%,明显低于取食中棉12的种群.说明寄主植物对棉蚜羧酸酯酶的量和质均有影响.     

家蚕羧酸酯酶基因Bmae35的克隆、 序列分析及表达

昆虫羧酸酯酶是一类能对外源化合物解毒和气味分子降解的重要酶系。本研究选取在家蚕Bombyx mori幼虫嗅觉感器中有表达, 并与蛀茎夜蛾Sesamia nonagrioides触角酯酶基因Snon-EST可能为直系同源基因的Bmae35进行克隆和外源表达研究。结果表明： 该基因编码区长1 581 bp, 共编码526个氨基酸。与其他昆虫触角酯酶的多序列比对分析发现, Bmae35编码的蛋白具有酯酶活性必须的催化残基Ser191, Glu313和His429, 也保持着α-酯酶家族特征基序Gly-x-Ser-x-Gly。RT-PCR分析显示, Bmae35在家蚕5龄第3天各组织中均有表达, 其中在头、 脂肪体、 马氏管、 体壁和丝腺中的表达量较高。Bmae35在雌蛾性信息腺中表达, 并与性信息素合成呈正相关, 暗示其在信息素合成中起重要作用。构建Bmae35与pET28(a) 重组载体, 经异丙基-β-D-硫代半乳糖苷诱导,电泳检测发现该基因以包涵体形式表达, 以镍亲和层析柱纯化, Western blotting鉴定证实Bmae35在大肠杆菌Escherichia coli中正确表达并得以纯化。本实验通过对家蚕Bmae35基因的克隆、 原核表达与纯化, 为进一步深入研究其表达定位和气味降解等功能奠定基础。     

繁殖寄主对赤眼蜂羧酸酯酶和乙酰胆碱酯酶的影响

通过测定赤眼蜂Trichogramma羧酸酯酶和乙酰胆碱酯酶的活性、对底物的亲和力以及对抑制剂的敏感度研究了繁殖寄主对松毛虫赤眼蜂T.dendrolimi和螟黄赤眼蜂T.chilonis的影响。柞蚕卵和米蛾卵繁殖的赤眼蜂羧酸酯酶对底物的亲和力有不同程度的影响,柞蚕卵繁殖的赤眼蜂羧酸酯酶对α-乙酸萘酯或β-乙酸萘酯的亲和力最高是米蛾卵的2倍以上。繁殖寄主对乙酰胆碱酯酶对底物亲和力没有明显的影响。米蛾卵繁殖的松毛虫赤眼蜂羧酸酯酶活性明显高于柞蚕卵繁殖的种群,而米蛾卵繁殖的螟黄赤眼蜂种群羧酸酯酶的活性明显低于柞蚕卵繁殖的种群。用柞蚕卵繁殖的松毛虫赤眼蜂种群对对氧磷的敏感度明显低于米蛾卵繁殖的种群,而增效磷则正好相反。繁殖寄主对松毛虫赤眼蜂吉林种群乙酰胆碱酯酶对DDVP和毒扁豆碱的敏感度没有明显的影响,而在松毛虫赤眼蜂广东种群和螟黄赤眼蜂中,柞蚕卵繁殖的种群乙酰胆碱酯酶对DDVP和毒扁豆碱的敏感度明显低于米蛾卵繁殖的种群。     

柑橘全爪螨两个田间种群抗性监测及羧酸酯酶生化特性研究

采用叶碟浸渍法测定了重庆北碚和万州地区柑橘全爪螨Panonychus citri(McGregor)田间种群对阿维菌素、毒死蜱、甲氰菊酯和哒螨灵的抗性水平。结果表明,同室内敏感品系相比,北碚种群对毒死蜱、甲氰菊酯和哒螨灵的相对抗性水平分别达到3倍、3倍和22倍;万州种群对阿维菌素、毒死蜱、甲氰菊酯和哒螨灵的相对抗性水平分别达到2倍、35倍、10倍和2倍。柑橘全爪螨2个地理种群的羧酸酯酶CarE的生化特性研究发现,CarE酶活的增高和毒死蜱的抗性存在一定的相关性。毒死蜱对不同地理种群柑橘全爪螨CarE的抑制效果不同,对抗性倍数较高的万州种群抑制效果最差。     

辣椒碱对烟粉虱体内羧酸酯酶、谷胱甘肽S-转移酶和乙酰胆碱酯酶活性的影响

【背景】烟粉虱作为世界性的多食性害虫,对我国农业生产造成了严重危害。已有研究表明,许多植物的次级代谢产物能够对烟粉虱解毒酶和靶标酶活性产生影响。【方法】采用常规生化方法研究了辣椒碱对烟粉虱体内羧酸酯酶（CarE）、谷胱甘肽S-转移酶（GSTs）和乙酰胆碱酯酶（AChE）活性的影响。【结果】与对照相比,500mg·L^-1辣椒碱处理烟粉虱成虫后6h,CarE活性被激活,其他时段被抑制;1000mg·L^-1辣椒碱处理后6、12、48h,CarE活性受到抑制,其他时段被激活;2000mg·L^-1辣椒碱处理后6h,CarE活性受到抑制,其他时段被激活;d000mg·L^-1辣椒碱处理后3、12h,CarE活性受到抑制,其他时段被激活;8000mg·L^-1辣椒碱处理后1h,CarE活性被激活,其他时段受到抑制。辣椒碱对烟粉虱体内GSTs活性的诱导存在剂量和时间效应,8000mg·L^-1辣椒碱处理烟粉虱成虫后24和48h,GSTs活性受到明显抑制,其余各处理在任何时段GSTs活性均被激活而高于对照。辣椒碱各浓度处理对烟粉虱体内AChE活性的诱导存在时间效应,各处理均在处理后48h对AChE活性产生了抑制作用。【结论与意义】辣椒碱对烟粉虱体内的解毒酶CarE和GSTs、靶标酶AChE活性具有明显影响,且存在剂量或时间效应,这可为辣椒碱在生物农药领域的应用提供理论依据。     

溴氰菊酯对飞蝗羧酸酯酶基因表达的影响

【目的】研究溴氰菊酯作用下飞蝗羧酸酯酶基因的mRNA表达特性,为溴氰菊酯的代谢解毒及飞蝗Locusta migratoria防治中抗性风险的评估提供基础资料。【方法】本文采用不同剂量溴氰菊酯处理3龄飞蝗,提取总RNA,体外反转录合成cDNA模板,采用Real-time PCR技术分析飞蝗羧酸酯酶基因在溴氰菊酯不同浓度和不同时间处理后的表达模式。【结果】飞蝗经不同浓度溴氰菊酯处理12 h后,LmCesA3和LmCesE1表现为诱导效应;除LmCesA2外,其余羧酸酯酶基因经溴氰菊酯LD30剂量处理后分别在不同的时间点表现为诱导效应。【结论】5个羧酸酯酶基因LmCesA1、LmCesA3、LmCesD1、LmCesE1和LmCesI1可以被溴氰菊酯诱导,表明其可能参与飞蝗对溴氰菊酯的代谢解毒及抗性产生。     

四种杀虫剂对两种书虱羧酸酯酶和乙酰胆碱酯酶的抑制作用

选用有机磷类杀虫剂(敌敌畏、毒死蜱、对氧磷)和氨基甲酸酯类杀虫剂(丁硫克百威),通过生物测定(药膜法)和生化测定(比色法)比较了嗜卷书虱和嗜虫书虱对所选药剂的敏感差异性。根据LC50可知嗜虫书虱对所选药剂比嗜卷书虱敏感。离体酶活性分析结果显示嗜卷书虱和嗜虫书虱的羧酸酯酶只对敌敌畏敏感,且嗜卷书虱比嗜虫书虱更敏感;4种药剂对乙酰胆碱酯酶均有强烈的抑制作用,同样是嗜卷书虱比嗜虫书虱敏感。乙酰胆碱酯酶的动力学研究结果和离体酶活性测定相一致。聚丙烯酰胺凝胶电泳分析显示,4种杀虫剂离体条件下对2种书虱的酯酶同工酶的抑制能力有明显差异,其中敌敌畏的抑制力最强;但对不同同功酶的抑制趋势(对大分子的抑制似乎较强)是一致的。酶的敏感性分析结果与生测结果比较表明,2种书虱的耐药力差异与其乙酰胆碱酯酶和酯酶对药剂的敏感性无关。如要弄清耐药力机制,需做进一步研究。     

氟化物对家蚕肠道微生物菌群的影响

摘要:【目的】探讨氟化物对家蚕肠道微生物菌群的影响,开发具有潜在应用价值的益生菌以提高家蚕对氟化物的抗性。【方法】PCR扩增家蚕肠道内细菌16S rRNA基因并构建克隆文库;用核糖体DNA限制性分析(Amplified ribosomal DNA restriction analysis,ARDRA)方法对克隆子进行分型。从家蚕T6和734肠道样品中共获得14种分类操作单元(Operational taxonomic unit,OTUs),以16S rRNA基因为基础构建系统发育树进行分析。再经巢式PCR-DGGE技术检测家蚕肠道内优势菌群的变化。【结果】结果表明:家蚕氟中毒后肠道内肠球菌属Enterococcus和芽孢杆菌属Bacillus细菌菌群减少,而葡萄球菌属Staphylococcus的细菌增多。【结论】氟化物能通过改变家蚕肠道内细菌的多样性和比例,从而破坏家蚕肠道微生物菌群平衡,且对家蚕734的影响作用大于T6。     

BmICE-2 is a novel pro-apoptotic caspase involved in apoptosis in the silkworm, Bombyx mori

In this study we identified a potential pro-apoptotic caspase gene, Bombyx mori(B. mori)ICE-2 (BmICE-2) which encoded a polypeptide of 284 amino acid residues, including a ¹⁶⁹QACRG¹⁷³ sequence which surrounded the catalytic site and contained a p20 and a p10 domain. BmICE-2 expressed in Escherichia coli (E. coli) exhibited high proteolytic activity for the synthetic human initiator caspase-9 substrates Ac-LEHD-pNA, but little activity towards the effector caspase-3 substrates Ac-DEVD-pNA. When BmICE-2 was transiently expressed in BmN-SWU1 silkworm B. mori cells, we found that the high proteolytic activity for Ac-LEHD-pNA triggered caspase-3-like protease activity resulting in spontaneous cleavage and apoptosis in these cells. This effect was not replicated in Spodoptera frugiperda 9 cells. In addition, spontaneous cleavage of endogenous BmICE-2 in BmN-SWU1 cells could be induced by actinomycin D. These results suggest that BmICE-2 may be a novel pro-apoptotic gene with caspase-9 activity which is involved apoptotic processes in BmN-SWU1 silkworm B. mori cells.     

Expression of heat shock protein 70a mRNA in Bombyx mori diapause eggs

In an effort to understand whether heat shock protein 70 (Hsp70) participates in the environmental 5 °C signal reception/transduction toward breaking embryonic diapause of the silkworm Bombyx mori, we isolated a cDNA for Hsp70a and examined the expression of Hsp70a mRNA in B. mori diapause and nondiapause eggs by quantitative real-time PCR. Hsp70a mRNA gradually increased in diapause eggs continuously kept at 25 °C after oviposition to maintain diapause. When diapause eggs were exposed to the diapause-terminating condition of 5 °C beginning at 2 days post-oviposition, Hsp70a mRNA increased beginning at 5 days post-cold treatment. Even in nondiapause eggs, Hsp70a mRNA increased slightly with exposure to 5 °C. These results suggest that Hsp70a is involved in reception/transduction of the diapause-terminating (5 °C) signal via gene activation. The expression patterns of Hsp70a mRNA are discussed in relation to those of the cold-response gene Samui.     

Establishment and characterization of an ovarian cell line of the silkworm, Bombyx mori

A cell line BmN-SWU1 was established from the ovarian tissues of 3-day-old fourth instar Bombyx mori larvae of the 21-872nlw variety by performing primary cultures in Grace's medium supplemented with 20% fetal bovine serum (FBS). The cell line primarily consisted of short spindle cells and round cells. The frequency of cells with chromosome number 2n = 56 was 80.5%; therefore, the cell line was considered to be a diploid cell line. The population-doubling time (PDT) at 45th passage line was 57.7 h. This cell line was susceptible to the B. mori nuclear polyhedrovirus (BmNPV), and the median tissue culture infective dose (TCID₅₀) at a cell density of 10⁵ cells/ml was 16.3 OBs/ml. The transient expression efficiency of the green fluorescent protein (GFP) gene in this cell line was 54.8%. We used the BmN-SWU1 cell line to select and establish a GFP transgenic cell line.     

Establishment and characterization of the Bombyx mandarina cell line

A new cell line, designated as NIAS-Boma-529b, was established from the larval fat bodies of Bombyx mandarina (B. mandarina), which is believed to be an ancestor of Bombyx mori (B. mori). This cell line has been cultured for approximately 150 passages during 2 years in an IPL-41 medium supplemented with 10% fetal bovine serum at a constant temperature of 26 °C. The morphology of this line includes adhesive round and spindle-shaped cells. Random-amplified polymorphic DNA analysis (RAPD) using 7 primers and a statistical analysis based on Nei’s genetic distance revealed that this cell line was closely related to B. mori-derived cell lines. An infection study also revealed that this cell line was susceptible to B. mori nucleopolyhedrovirus (BmNPV); however, it had no apparent susceptibility to Autographa californica NPV (AcNPV), which is closely related to BmNPV. Nevertheless, cells infected with AcNPV showed an extensive cytopathic effect (CPE), including a rough cell surface, rounding, nuclear expansion, and cell blebbing. These results suggest that this cell line can be useful to clarify the mechanism of host range determination of BmNPV and AcNPV.     

A homeotic mutation influences the wing vibration patterns during mating in males of the silkworm moth Bombyx mori

An abnormality in the wing vibration pattern in males of the E^Nc homeotic mutant of Bombyx mori was investigated. The wild-type (+/+) males show a switching of the rhythmic wing vibrations from a sequential pattern to an intermittent pattern during mating, whereas the E^Nc mutants show a sequential pattern both before and during mating. Wing motions in +/+ males became small during mating, but those in +/E^Nc males did not. Ablation of the head ganglia of +/+ and +/E^Nc males during mating caused no change in the motor patterns of wing vibrations. Ablation or cooling of the posterior abdomen in the +/+ males during mating caused sequential wing vibrations, suggesting that the change in wing vibrations is induced by signals from the posterior abdomen. The pterothoracic ganglion in the +/E^Nc males is separated into two ganglia, in contrast to the complete ganglionic fusion in the +/+ males. The neurons in the pterothoracic ganglion stained from abdominal nerve cords are homologus in +/+ and +/E^Nc males, but many of these in +/E^Nc males are elongated along the anteroposterior axis. These results suggest that the wing vibration pattern is restricted by genetic factors through reconstruction of the thoracic nervous system during metamorphosis.     

Spatial expression of the mevalonate enzymes involved in juvenile hormone biosynthesis in the corpora allata in Bombyx mori

The developmental expressions of the mRNA of JH synthetic enzymes have been studied using homogenates of the corpora cardiaca-corpora allata (CC-CA) complexes in Bombyx mori [Kinjoh, T., Kaneko, Y., Itoyama, K., Mita, K., Hiruma, K., Shinoda, T., 2007. Control of juvenile hormone biosynthesis in Bombyx mori: cloning of the enzymes in the mevalonate pathway and assessment of their developmental expression in the corpora allata. Insect Biochemistry and Molecular Biology 37, 808-818]. The in situ hybridization analyses in the CC-CA complex showed that the distribution of the mRNAs of all the mevalonate enzymes and juvenile hormone (JH) acid O-methyltransferase occurred only in the CA cells, indicating that the fluctuations of the enzyme mRNA amounts in the CC-CA complexes were derived solely from the CA. In addition, the size of the CA and their nuclei was not associated with the JH synthetic activity by the CA until the pharate adult. Only female adult CA synthesized JH in B. mori, and the CA and the nuclei were significantly larger than those of male CA which do not synthesize JH.     

Fat body catalase activity as a biochemical index for the recognition of thermotolerant breeds of mulberry silkworm, Bombyx mori L.

The fifth instar larvae of the silkworm Bombyx mori L. were exposed to selected high temperatures (35 and 40 °C) in order to understand the changes in the level of catalase activity in the three tissues of fat body, midgut and haemolymph of the five selected bivoltine breeds and their 9 quantitative traits, namely larval weight, cocoon weight, shell weight, shell ratio, filament length, filament weight, denier, renditta and effective rearing rate (ERR), also the correlation between them under high temperature conditions were examined. Catalase activity resulting in fat body revealed a positive correlation between the control (28±1 °C) and 40±1 °C. The CSR₂ breed showed the most level of thermotolerance and catalase activity, compared with the CSR₄, JROP, NB₄D₂ and KA breeds. It was found that the level of catalase activity in fat body may be a reliable biochemical index to recognize thermotolerant breeds in order to develop resistant hybrids for tropical areas.