5-氨基乙酰丙酸对中华稻蝗（Oxya chinensis）的杀虫活性及对3种酶活性的影响

 【目的】研究5-氨基乙酰丙酸（5-aminolevulinic acid,ALA）对中华稻蝗（Oxya chinensis）的杀虫活性及对3种酶活性的影响。【方法】以中华稻蝗4龄若虫为试验材料,用不同剂量的ALA（A1：250 mmol•L-1;A2：450 mmol•L-1;A3：750 mmol•L-1;A4：1 000 mmol•L-1）处理中华稻蝗,观察其对中华稻蝗的毒性效应和对其体内乙酰胆碱酯酶（AChE）、谷胱甘肽S-转移酶（GSTs）和谷胱甘肽过氧化物酶（GPx）活性的影响。【结果】不同剂量ALA处理组中华稻蝗死亡率依处理剂量呈现上升趋势,高浓度处理组A3、A4的死亡率分别达到66.19%和80.21%;LD50值为3.61（3.29～3.93）mg•g-1虫重（95%置信范围）。生化研究结果显示,最高浓度A4处理组雌、雄虫体内AChE活性分别较对照组下降了51.53%和42.65%,差异显著（P＜0.05）;GPx活性分别较对照组下降了42.82%和43.85%,差异显著（P＜0.05）。同时,中华稻蝗GSTs活性随ALA处理剂量升高而增高,A4处理组雌、雄虫体内GSTs活性分别较对照组升高了171.05%及97.42%,差异显著（P＜0.05）。【结论】ALA对雌、雄中华稻蝗均有明显的毒性效应;ALA可引起AChE和GPx光失活,从而导致中华稻蝗神经传导受阻同时抵御氧化损伤的能力下降;高剂量ALA激活GSTs,可引发昆虫对光毒性物质的自身反馈抵御反应。     

Comparative Studies of Substrate and Inhibitor Specificity of Glutathione S-Transferases in Six Tissues of Oxya chinensis （Thunberg） （Orthoptera： Acrididae）

Specific activity, substrate specificity, and kinetic parameters （Km and Vmax） of glutathione S-transferases （GSTs） towards three substrates, 1-chloro-2,4-dinitrobenzene （CDNB）, 1,2-dichloro-4-nitrobenzene （DCNB）, and p-nitrobenzene chloride （pNBC） were investigated in six tissues （foregut, midgut, hindgut, fat body, hemolymph, and muscle） of Oxya chinensis. In addition, the inhibition in vitro （ethacrynic acid, and Cibacron Blue 3GA） of Oxya chinensis in the six tissues was also investigated. Glutathione S-transferase activity was detected in all the six tissues examined. The rank order of GST activities towards CDNB was fat body 〉 midgut 〉 hindgut 〉 muscle 〉 foregut 〉 hemolymph both in females and males. Glutathione S-transferase activities in the fat body in females and males were 1.3- to 10.4-fold and 1.1- to 10.0- fold higher than those in the other tissues. The rank order of GST activities towards the other substrates changed slightly. From these results, it was inferred that GSTs in the fat body and midgut played important roles in detoxifying xenobiotics including insecticides and plant allelochemicals in O. chinensis. In the three substrates examined, CDNB seemed to be the best substrate, followed by pNBC and DCNB. The kinetic parameters of GSTs were different among the six tissues. This suggested that GSTs in different tissues have various affinities and catalytic efficiency to substrates. In vitro inhibition study showed that the median inhibition concentration （IC50） values of the two inhibitors to GSTs from the six tissues were different. The results suggested that the two inhibitors have different inhibition potency to GSTs from the different tissues. The observed changes in kinetic parameters and inhibition in vitro among the six tissues of the insect might suggest that the number and structure of isoenzymes and their rate of expression varied for the different tissues.     

Study on Insecticidal Activities and Effect on Three Kinds of Enzymes by 5- Aminolevulinic Acid on Oxya chinensis

Insecticidal activities and effects on three enzymic activities caused by 5-aminolevulinic acid (ALA) on Oxya chinensis were studied. Fourth-instar nymphs of O. chinensis were treated with different doses of ALA (A 1,250 mM; A2, 450 mM; A3,750 mM; A4, 1 000 mM). Mortality and the activities of acetylcholinesterase (ACHE), glutathione S-transferase (GSTs), and glutathione peroxidase (GPx) were determinated. The mortality of O. chinensis rose with an increasing dose of ALA. The mortality of high-dose treatments A3 and A4 reached 66.19 and 80.21%, respectively. The value of LD50 was 3.61 (3.29-3.93) mg g-1 body weight (95% confidence interval). Biochemical studies showed that the activities of AChE and GPx in the A4 treatment declined by 51.53 and 42.82% in the female, and 42.65 and 43.85% in the male compared to the control, respectively, and the degree of decline reached a significant level at P < 0.05. Meanwhile, the GSTs activities of O. chinensis enhanced with increasing dose of ALA. The GSTs activities of female and male O. chinensis in the A4 treatment remarkably increased by 171.05 and 97.42% compared to the control (P < 0.05). ALA had an obviously toxic effect on O. chinensis. Moreover, ALA caused the photoinactivation of AChE and GPx, which induced nerve transmission blocking and the capability to defend oxidation damage declining. Meanwhile, a high dose of ALA could activate GSTs, which caused a feedback inhibition of the insect to the phototoxic substance.     

Study on Insecticidal Activities and Effect on Three Kinds of Enzymes by 5-Aminolevulinic Acid on Oxya chinensis

Insecticidal activities and effects on three enzymic activities caused by 5-aminolevulinic acid （ALA） on Oxya chinensis were studied. Fourth-instar nymphs of O. chinensis were treated with different doses ofALA （A1,250 mM; A2, 450 mM; A3,750 mM; A4, 1 000 mM）. Mortality and the activities of acetylcholinesterase （ACHE）, glutathione S-transferase （GSTs）, and glutathione peroxidase （GPx） were determinated. The mortality of O. chinensis rose with an increasing dose of ALA. The mortality of high-dose treatments A3 and A4 reached 66.19 and 80.21%, respectively. The value of LD50 was 3.61 （3.29-3.93） mg·g^-1 body weight （95% confidence interval）. Biochemical studies showed that the activities of AChE and GPx in the A4 treatment declined by 51.53 and 42.82% in the female, and 42.65 and 43.85% in the male compared to the control, respectively, and the degree of decline reached a significant level at P 〈 0.05. Meanwhile, the GSTs activities of O. chinensis enhanced with increasing dose of ALA. The GSTs activities of female and male O. chinensis in the A4 treatment remarkably increased by 171.05 and 97.42% compared to the control （P〈 0.05）. ALA had an obviously toxic effect on O. chinensis. Moreover, ALA caused the photoinactivation of AChE and GPx, which induced nerve transmission blocking and the capability to defend oxidation damage declining. Meanwhile, a high dose of ALA could activate GSTs, which caused a feedback inhibition of the insect to the phototoxic substance.     

Comparisons of Properties of Acetylcholinesterase from Two Field-Collected Populations of Oxya chinensis Thunberg (Orthoptera: Acrididae) and the Role of Acetylcholinesterase in the Susceptibility to Malathion

In this study, acetylcholinesterase (AChE) was extracted from two field-collected populations of Oxya chinensis (Xinxiang City, Henan Province and Changzhi City, Shanxi Province). AChE activities were decreased when concentrations of ATC increased, showing a characteristic phenomenon of substrate inhibition at high concentration in both populations. Such inhibition occurred at relatively low concentration for AChE from Xinxiang population but relatively high for AChE from Changzhi population. The kinetic study showed that there were no significant differences between the two populations in the Km values. The Km value in Changzhi population was only 1.09-fold higher than that in Xinxiang population. However,significant differences were observed between the two populations in Vmax values. The Vmaxvalue in Changzhi population was 1.32-fold higher than that in Xinxiang population. The inhibition study in vitro showed that the AChE from both populations exhibited similar rank order in sensitivity to inhibition by three OPs, as determined by comparison of their bimolecular rate constants (ki), from the most potent inhibition to the least was chlopyrifos-oxon ＞ paraoxon ＞demeton-s-methyl for AChE from the two populations and that the ki values in Xinxiang population were lower than those in Changzhi population. The I50 values of AChE from Xinxiang population were 4.84-, 2.66-, and 1.92-fold less sensitive to inhibition by paraoxon, chlopyrifos-oxon, and demeton-s-methyl. These results were consistent with the results in bioassay. It is inferred that AChE insensitivity to OP insecticides plays an important role in the differences of insusceptibility of Oxya chinensis to malathion between the two populations.     

金属离子对红白锦鲤肠及肝胰脏蛋白酶活性的影响（摘要）（英文）

[目的]研究金属离子对红白锦鲤消化组织蛋白酶活性的影响。[方法]以酶学分析方法研究4种金属离子（Na＋、K＋、Ca2 ＋、Mg2 ＋分别来源于NaCl、KCl、CaCl2.2H2O、MgCl2.6H2O,浓度梯度均设置为25、50、75、100、125、150 mmol/L）对红白锦鲤肝胰脏、前肠、中肠、后肠蛋白酶活性的影响。蛋白酶活性的测定采用福林-酚显色法,用Thermo紫外分光光度计,根据OD680计算蛋白酶活性,以1 min水解酪蛋白产生1μg酪氨酸为一个酶活力单位,记为μg/（min.g）。[结果]在设定的浓度范围内（25 ～150 mmol/L）,4种金属离子对蛋白酶活性影响不同。K＋对肝胰脏和后肠的蛋白酶活性有不同程度的促进作用,其中K＋对中肠表现为低浓度促进,高浓度抑制的作用,对前肠蛋白酶表现为抑制作用;Na＋对肝胰脏、前肠和后肠的蛋白酶表现为不同程度促进作用,对中肠表现为抑制作用。Mg2 ＋、Ca2 ＋对肠及肝胰脏的蛋白酶均表现为不同程度的抑制作用。试验所选用的K＋、Na＋、Mg2 ＋、Ca2 ＋均属矿物元素,其中添加浓度25 mmol/LK＋对锦鲤肠蛋白酶活性促进作用最大;浓度150 mmol/L Na＋显著提高后肠和肝胰脏蛋白酶活性。这些浓度可作为饲料添加剂中的参考用量。Mg2 ＋、Ca2 ＋对锦鲤各消化器官（中肠除外）的抑制作用随浓度的增加蛋白酶活性降低,但考虑该元素系水产动物生长发育所必需,因此可选用低浓度或螯合物进行添加,以提高酶的生物学效价,提高饲料中蛋白的利用率,降低饵料系数,节约蛋白。[结论]该研究可为红白锦鲤消化生理的研究以及配合饲料的开发、优化提供基础数据和理论依据。     

中华稻蝗羧酸酯酶家族基因生物信息学及组织表达特异性分析

【目的】对重要农业害虫中华稻蝗（Oxya chinensis）羧酸酯酶（carboxylesterases,Ces）家族基因进行生物信息学分析,并对部分基因的组织表达特性进行研究,为揭示中华稻蝗Ces基因功能及研发新的分子靶标提供依据。【方法】基于中华稻蝗转录组数据库,通过关键词搜索获得Ces基因序列,采用生物信息学方法进行比对拼接、氨基酸序列推导和开放阅读框分析,选择全长序列构建系统发育树,采用在线软件分析中华稻蝗Ces氨基酸序列结构、理化性质、信号肽和N糖基化位点等特征。采用实时定量PCR技术,通过geNorm 与Normfinder 软件分析4个候选内参基因β-肌动蛋白（β-actin）、延长因子（EF1α）、3-磷酸甘油醛脱氢酶（GAPDH）和核糖体蛋白49（RP49）表达稳定性,筛选中华稻蝗5龄若虫不同组织部位最适内参基因并检测10个羧酸酯酶基因在不同组织部位相对表达量。【结果】从中华稻蝗转录组数据库搜索获得Ces基因cDNA片段180个,经筛选获得28个Ces基因全长序列进行生物信息学分析。推导的氨基酸序列与其他昆虫物种的聚类分析结果显示,中华稻蝗28个Ces分布于4个功能簇中,其中A簇直翅目和部分双翅目昆虫α酯酶（20个）、D簇表皮特异酯酶（2个）、E簇β酯酶（4个）和F簇非鳞翅目保幼激素酯酶（2个）。氨基酸序列分析结果显示,中华稻蝗大部分Ces的等电点（pI）均为偏酸性或弱酸性,pI为4.38-6.84,只有3个Ces的pI值偏碱性。中华稻蝗所有的Ces基因都具有N糖基化位点、N端保守的半胱氨酸残基及氧离子洞,信号肽预测结果表明19个Ces具有完整的信号肽;21个Ces具有典型的催化三联体S-E-H,其余7个Ces催化三联体发生了不同的替换。GeNorm与Normfinder分析结果显示,4个候选内参基因稳定性EF1α=RP49＞β-actin＞GAPDH。选择EF1α作为内参基因,RT-qPCR结果显示10个羧酸酯酶基因在7个不同组织部位均有表达,其中OcCesA1、OcCesA6、OcCesA12、OcCesA14、OcCesA18和OcCesA19在中肠和胃盲囊高表达,此外OcCesA6、OcCesA18和OcCesA19还在马氏管高表达,OcCesA2在后肠高表达。OcCesD1在马氏管表达量最高,脂肪体和表皮次之;OcCesE1在胃盲囊表达量最高;OcCesF2在中肠表达量最高。【结论】基于中华稻蝗转录组数据库获得28个全长Ces基因,聚类分析结果显示,这些基因分布于4个功能簇中,其中分布于A簇的基因数目相对较多,且多在中肠、胃盲囊和马氏管等代谢解毒器官中高表达。研究结果为Ces基因功能的深入探索和中华稻蝗Ces基因分子靶标研发提供了依据。     

中华稻蝗几丁质酶基因10（OcCht10）的分子特性及功能

【目的】获得中华稻蝗（Oxya chinensis）几丁质酶基因10（OcCht10）的cDNA序列,预测其功能域,并做聚类分析明确该基因的系统发育关系。绘制OcCht10在5龄若虫不同组织部位和发育时间的表达图谱,研究其在中华稻蝗蜕皮过程中的生物学功能,为害虫防治提供高效安全的候选基因。【方法】从中华稻蝗转录组数据库搜索OcCht10 cDNA片段,经过blast分析、比对、拼接后,翻译为氨基酸序列,预测其功能域,并与其他昆虫几丁质酶家族基因进行聚类分析,进一步确认该基因的系统发育关系并进行准确命名;选取中华稻蝗5龄第6天不同组织部位及5龄若虫不同天数表皮样本,总RNA提取后反转录为cDNA模板,采用Real-time quantitative PCR（qPCR）方法获得OcCht10在5龄稻蝗若虫不同组织部位和不同发育阶段的表达谱;设计OcCht10 dsRNA引物,用试剂盒体外合成dsOcCht10,采用注射法进行RNA干扰;取注射24 h稻蝗表皮样本,用qPCR方法检测OcCht10基因沉默效率;并仔细观察dsRNA注射后稻蝗表型,统计其死亡率。【结果】经对中华稻蝗转录组数据库的搜索,获得OcCht10 cDNA片段长度为9 318 bp,开放阅读框为8 613 bp,编码2 870个氨基酸;其3′非编码区为705 bp,推测OcCht10 5'端有500多碱基尚未获得;预测其氨基酸序列具有多个结构域,包含有5个几丁质酶催化域和6个几丁质结合域;与其他昆虫几丁质酶基因聚类分析结果显示OcCht10属于昆虫几丁质酶家族基因Ⅱ组,该组基因与昆虫蜕皮相关;5龄若虫组织特异性分析表明该基因主要在表皮、前肠和后肠等外胚层发育而成的组织器官中表达,提示OcCht10可能参与表皮几丁质代谢;发育时间表达图谱表明该基因在5龄第6-7天,即蜕皮前的表皮中高表达,表明OcCht10可能负责蜕皮时表皮几丁质降解;5龄若虫第2天注射该基因的dsRNA,24 h后取表皮检测其干扰效率,结果显示该基因被沉默70%;与注射dsGFP的对照组相比,注射dsOcCht10 5龄若虫表现为龄期延长,旧表皮无法开裂,蜕皮受阻,昆虫无法正常活动导致死亡,死亡率达到100%。【结论】获得OcCht10的部分cDNA序列,该基因在昆虫蜕皮前表皮中高表达;OcCht10参与中华稻蝗的蜕皮发育,注射dsOcCht10可有效沉默靶基因,并导致试虫无法完成正常蜕皮而死亡。     

Characterization of the Purified Glutathione S-transferases from Two Psocids Liposcelis bostrychophila and L.entomophila

Glutathione S-transferases（GSTs） from Liposcelis bostrychophila Badonnel and L.entomophila（Enderlein）（Psocoptera：Liposcelididae） were purified by glutathione-agarose affinity chromatography,and characterized subsequently by their Michaelis-Menten kinetics toward the artificial substrates 1-chloro-2,4-dinitrobenzene（CDNB） and reduced glutathione（GSH）,respectively.The specific activity of the purified GST toward CDNB was 2.3-fold higher in L.bostrychophila than in L.entomophila.Though the specific activities of purified enzymes varied between the two species,the purification yields were similar.SDS-PAGE revealed one band at 23 kDa for both the species.GSTs of L.entomophila exhibited higher Michaelis-Menten constants（Km） but lower maximal velocity（Vmax） values than those of L.bostrychophila.The optimum pH for CDNB conjugation of L.bostrychophila and L.entomophila GSTs was 7.0 and 7.5,and optimum temperature was 35 and 40°C,respectively.Inhibition kinetics showed that cibacron blue,curcumin,bromosulfalein,ethacrynic acid,and carbosulfan had excellent inhibitory effects on GSTs in both species,but the inhibitory effects of beta-cypermethrin,fenpropathrin,tetraethylthiuram disulfide,and diethyl maleate were not significant.     

pH对南方白甲鱼消化酶活性的影响

[目的]为南方白甲鱼饲料中各营养成分的配比提供参考。[方法]分别采用福林-酚试剂法、碘-淀粉比色法、碱滴定法及吸光度测定的方法测定了蛋白酶、淀粉酶、脂肪酶的最适pH,并比较了各消化酶的活性。[结果]蛋白酶、淀粉酶和脂肪酶的最适pH分别为:7～7.5、7.5～8、6.5～7.5。南方白甲鱼不同组织蛋白酶活性大小顺序为:前肠>后肠>肝胰腺>中肠>胆;淀粉酶活性大小顺序为:肝胰腺>胆>中肠>前肠>后肠;脂肪酶活性大小顺序为:肝胰腺>胆>中肠>前肠>后肠。[结论]南方白甲鱼是一种杂食性或偏草食性的鱼类,对糖类物质有较强的消化能力。     

Comparative Study of Malathion Toxicity and General Esterases in Larvae and Adults from a Field Population of Oxya chinensis (Thunberg)(Orthoptera:Acridoidea)

The susceptibility of Oxya chinensis to malathion was compared in larvae and adults from a field population, collected from Jinyuan outskirt, Shanxi Province. The results showed that Oxya chinensis was more susceptible to malathion in the adult stage than in the larval stage. The LD50 values for malathion susceptibility of Oxya chinensis were 4.94 and 2.44 mg g-1 body weight in the larvae and adults respectively. The results indicated that the larvae were 2.02-fold less susceptible to malathion than the adults. The general esterases and the kinetics were characterized and compared between the two life stages and between females and males. Larval preparations of Oxya chinensis were more active than adult preparations in females and males. The larvae showed 1.18-, 1.49-, and 1.17-fold higher specific activities than the adults in females with α -NA, α -NB and β -NA respectively. In males, the ratios were 1.34-, 1.70-, and 1.06-fold. Female preparations were more active than those of males in the adults. The reverse results were observed in the larvae where male preparations were more active than female preparations. Kinetic studies showed that Km values of general esterases hydrolyzing α -NA, α -NB, and β -NA in the adult stage were 1.36-, 1.32- and 1.39-fold respectively, higher than those in the larval stage in females. In males, the ratios were 1.24-, 2.14-, and 1.20-fold. The esterase from male insects had a higher affinity (lower Km value) to the substrate than those from females. The results also showed that the Vmax values of general esterase hydrolyzing α -NA, α -NB, and β -NA in the two stages were similar. From the results of bioassays and biochemical analyses, it has been inferred that a higher level of resistance to malathion in larvae than in adults would appear to result from differences in the expression of resistance mechanisms in these two life stages. Enhanced esterase activities appeared to play a major role in resistance to malathion in both larvae and adults. From the analysis of inhibition in vitro, the esterases in the two life stages were B-type,and carboxylesterases were predominant enzymes in the composition of the esterases in the two stages.     

中华稻蝗（Oxya chinensis）对重金属镉的累积及排泄

为了研究重金属镉(Cd)在植食性昆虫体内的累积和排泄规律,通过Cd慢性染毒的方法,用不同浓度Cd溶液培养的麦苗饲喂中华稻蝗(从4龄若虫至成虫),采用原子吸收法测定Cd在中华稻蝗(Oxyachinensis)整虫和中肠的蓄积及Cd在其粪便和蜕中的排泄量。研究结果表明,Cd在中华稻蝗整虫和中肠中的累积规律相似,即随着麦苗中Cd含量的增加,整虫和中肠中Cd的累积量明显升高,且各处理间存在显著差异(P<0.05)。部分Cd可以通过粪便的排泄和蜕皮过程而排出体外。用不同Cd浓度的麦苗饲喂中华稻蝗后,其粪便和蜕中的Cd排泄量与对照组相比显著升高(P<0.05),且各处理间差异显著(P<0.05)。中华稻蝗整虫、中肠、粪便和蜕中的Cd含量与食物(麦苗叶片)中的Cd含量存在显著的剂量-反应关系,其相关系数分别为:整虫0.977、中肠0.920、粪便0.967、蜕0.840。研究结果可为进一步研究Cd在中华稻蝗体内积累和排泄的动态变化规律提供理论和实验依据。     

锦鲤肠道不同部位菌群组成结构及多样性分析

【目的】研究锦鲤（Cyprinus carpio var.koi）肠道的菌群组成结构及多样性,为深入研究锦鲤肠道菌群的功能及肠道不同部位的生理功能的分化提供参考。【方法】收集锦鲤前肠、中肠和后肠的肠道细菌,通过传统培养技术,采用细菌微量生化鉴定管对肠道细菌进行各项生理生化指标的测定;分别提取3个肠段的肠道菌群基因组DNA,进一步采用16S rDNA高通量测序技术,分析3个肠段的菌群组成和生物多样性。【结果】前肠、中肠和后肠样本测序后分别得到48659、47013和47819条有效序列,按97%相似性水平划分OTU后,分别得到1555、1294和1423个OUTs。α多样性分析结果显示,后肠菌群样本的Shannon指数、Chao1指数最高。在门水平上,前肠、中肠和后肠丰度最高的5个菌门均是变形菌门（Proteobacteria）、梭杆菌门（Fusobacteria）、拟杆菌门（Bacteroidetes）、厚壁菌门（Firmicutes）和黏胶球形菌门（Lentisphaerae）,推测这些菌门是锦鲤肠道的核心细菌类群,但各菌门在不同部位的相对丰度有差异。构建的物种丰度热图显示相对丰度最高的前10类菌属相同,但某些菌属在肠道不同部位的相对丰度有较大差异,如代尔夫特菌属（Delft）、弓形菌属（Toxoplasma）。UniFrac分析显示中肠和后肠的肠道菌群聚为一支,表明这2个部位菌群组成结构相似度较高。【结论】锦鲤肠道不同部位具有独特的菌群结构,可能会影响肠道不同部位功能的分化。     

中华稻蝗几丁质脱乙酰基酶2基因的分子特性和生物学功能

【目的】研究中华稻蝗（Oxya chinensis）几丁质脱乙酰基酶（chitin deacetylase,CDA）基因的分子特性和生物学功能,为新型农药靶标筛选提供科学依据。【方法】采用生物信息学方法在稻蝗转录组数据库中搜索获得几丁质脱乙酰基酶基因cDNA序列,对其进行保守区域分析,选取赤拟谷盗（Tribolium castaneum）、果蝇（Drosophila melanogaster）、冈比亚按蚊（Anopheles gambiae）、家蚕（Bombyx mori）和云杉卷叶蛾（Choristoneura fumiferana）等昆虫物种的同源序列与中华稻蝗几丁质脱乙酰基酶氨基酸序列进行聚类分析,进一步构建系统发育树;运用real-time PCR方法检测几丁质脱乙酰基酶基因在中华稻蝗5龄若虫不同组织部位和不同发育天数表皮中的表达特性;进一步采用RNA干扰（RNAi）技术研究该基因对中华稻蝗蜕皮发育的影响。【结果】在中华稻蝗转录组数据库中搜索获得2条几丁质脱乙酰基酶基因的cDNA全长序列,生物学软件分析其氨基酸,发现2条序列均具有信号肽,开放阅读框包含3个几丁质脱乙酰基酶保守区域：几丁质结合区（ChBD）、低密度脂蛋白受体区（LDLa）和几丁质脱乙酰基催化结合域（CDA）。聚类分析表明2条序列分别与所选用的5种昆虫CDA2聚为一支。剪切子聚类分析发现2条序列分别与5种昆虫CDA2a和CDA2b相聚为一支,综合序列分析和聚类结果,认为所获得的序列属于几丁质脱乙酰基酶2基因的2个不同剪切子,分别命名为OcCDA2a和OcCDA2b。定量PCR分析表明OcCDA2a和OcCDA2b均在中华稻蝗表皮、前肠和后肠中高表达;在5龄第1天的表皮中表达量较高,之后逐步下降,蜕皮前又略有上升;RNAi研究发现5龄第2天的若虫注射dsOcCDA2a或dsOcCDA2b 24 h后,与注射dsGFP的对照相比,目的基因表达量显著降低,沉默效率分别为96.72%和80.43%;进一步观察试虫的表型,与对照组相比,注射dsOcCDA2和dsOcCDA2a的大多数虫体出现龄期延长,脊线开裂,旧表皮不能顺利剥离而导致虫体扭曲,最终死亡。少数若虫在蜕皮前死亡,未出现异常表型,部分若虫虽蜕至成虫,但四肢无力,行动缓慢。死亡率分别达到87.5%和94.4%;注射dsOcCDA2b的试虫无可见表型,可正常蜕皮发育至成虫。【结论】中华稻蝗几丁质脱乙酰基酶2基因具有2个剪切子,分别为OcCDA2a和OcCDA2b。OcCDA2a参与虫体蜕皮,对其生长发育起着重要的作用,该基因的沉默导致中华稻蝗因蜕皮困难而死亡,而OcCDA2b对中华稻蝗的生长发育没有影响。     

枸杞多糖对尼罗罗非鱼生长发育、消化酶活性及肠道组织形态的影响

【目的】明确枸杞多糖对罗非鱼机体生长发育及消化功能的影响,为罗非鱼健康养殖提供理论参考,同时为促进罗非鱼养殖产业的可持续健康发展提供新思路。【方法】在尼罗罗非鱼基础饲料中添加5个不同剂量（0~2000 mg/kg）的枸杞多糖,进行40 d的养殖试验后测量尼罗罗非鱼的体重及全长,计算相对增重率（WG）、相对增长率（LG）、特定生长率（SGR）和饲料系数（FCR）,采用ELISA试剂盒分别检测淀粉酶（Amylase）、蛋白酶（Protease）及脂肪酶（Lipase）活性,并采集前肠、中肠和后肠组织制作切片,通过光学显微镜观察肠道组织形态的变化特征。【结果】于基础饲料中添加不同剂量枸杞多糖对尼罗罗非鱼的生长发育均起促进作用,其中以添加1500 mg/kg处理组的促进效果最佳,至养殖试验结束时尼罗罗非鱼的终末全长、终末体重、WG、LG和SGR均达最高值,分别为13.45 cm、48.74 g、6.96%、0.94%和5.19%/d;而FCR最小,仅为1.88。尼罗罗非鱼中肠组织的蛋白酶和淀粉酶活性在1500 mg/kg处理组中显著升高（P<0.05,下同）,胃组织的蛋白酶和淀粉酶活性、肝脏组织的脂肪酶活性也是以1500 mg/kg处理组最高,但与对照组（0 mg/kg）相比无显著差异（P>0.05）。肠绒毛长度在尼罗罗非鱼前肠组织中以1500 mg/kg处理组的最短（0.351 mm）、在后肠组织中则以1500 mg/kg处理组的最长（0.320 mm）,与对照组（0 mg/kg）相比差异显著;肌层厚度在前肠和中肠组织中均以500 mg/kg处理组的最厚,分别为0.089和0.087 mm,在后肠组织中则以1000 mg/kg处理组的最薄（0.065 mm）、2000 mg/kg处理组的最厚（0.099 mm）。【结论】枸杞多糖可提高罗非鱼的消化酶活性,改善其肠道组织形态,从而影响罗非鱼的生长发育,且以添加剂量为1500 mg/kg的效果最佳。     

辛硫磷及毒死蜱对中华稻蝗的毒力研究

本文采用点滴法分别测定了辛硫磷和毒死蜱对中华稻蝗5龄若虫的毒力,做出了毒力回归曲线并得到毒力回归方程及LD50值。辛硫磷的毒力回归方程为Y=2.79X-6.07,LD50=1.96μg/g虫重,毒死蜱的毒力回归方程为Y=2.86X-7.01,LD50=3.69μg/g虫重。比较两种农药的毒力回归方程、LD50值和95%置信区间,结果表明辛硫磷对中华稻蝗5龄若虫的药效较毒死蜱强。     

中华稻蝗内黄酮组分的抗氧化活性

利用70%的乙醇提取中华稻蝗黄酮,并用乙酸乙酯和正丁醇依次进行萃取,采用DPPH自由基、超氧阴离子、羟基自由基、鳌合亚铁离子和还原力、总抗氧化的反应体系,检测了中华稻蝗组分的体外抗氧化活性,并与化学合成抗氧化剂BHT进行比较.结果表明:在所测浓度范围内,水相残留物的清除能力、总抗氧化能力和总还原能力最好;IC50值,羟基自由基(25.16±0.64) μg/ml、超氧阴离子自由基(35.06±0.86) μg/ml、DPPH为(19.56±1.24) μg/ml、脂质过氧化(43.75±0.94) μg/ml,且均强于BHT. 其它几种萃取物与BHT相当,因此,稻蝗黄酮类组分具有很好的抗氧化能力.     

Molecular cloning and expression patterns of two small heat shock proteins from Chilo suppressalis(Walker)

Small heat shock proteins(sHSPs) are a very complex protein superfamily that increase insect temperature tolerance. In order to deeply understand the function and role of sHSPs in Chilo suppressalis(Walker), this study isolated and identified two CsHSP genes lacking introns from C. suppressalis, Cshsp23.9 and Cshsp27.3. The cDNA full-length of Cshsp23.9 and Cshsp27.3 were 909 and 1 036 bp encoding 220 and 242 amino acids, respectively. Alignment with homologs and phylogenetic analysis indicated Cshsp23.9 and Cshsp27.3 were two new types of Cshsps in C. suppressalis. Real-time quantitative PCR(qPCR) revealed that Cshsp23.9 had the highest relative expression in hindgut compared with other tissues(head, epidermis, foregut, midgut, fat body, Malpighian tubules, and hemocytes), while Cshsp27.3 expressed the highest in fat body content. When exposed to thermal stress from –11 to 43℃ for 2 h, two genes showed different expression patterns. Cshsp23.9 did not respond to low temperature, but could be up-regulated by high temperature and the highest expression temperature was at 36℃. Cshsp27.3 could only be induced by mild temperature, with the highest expression at 15 and 30℃. In conclusion, Cshsp23.9 and Cshsp27.3 existed in different tissues/organs of C. suppressalis, and played different important roles in C. suppressalis to resist temperature stress and regulate physiological activities.     

二化螟幼虫抗药性相关酶系的组织及亚细胞分布特征

研究了二化螟幼虫体内抗药性相关酶系的组织及亚细胞分布特征。结果表明,二化螟幼虫体内的解毒酶均以脂肪体和消化道组织中的活性较高,而血淋巴中的活性明显较低;乙酰胆脂酶在各组织中均检测到酶活性,其中以消化道的比活力最高,而脂肪体、体壁和血淋巴中的比活力相当;从亚细胞分布来看,MFOs和AChE主要分布在细胞核和细胞碎片、线粒体和微粒体等有膜的亚细胞结构中,而胞质溶液的活性明显较低;GSTs和Care均以胞质溶液中的比活力最高,其次为线粒体、细胞核和碎片,微粒体的比活力最低。