青花菜谷胱甘肽-S-转移酶基因克隆及其表达分析

为探讨青花菜在模拟酸雨胁迫下谷胱甘肽-S-转移酶的表达变化,克隆了青花菜谷胱甘肽-S-转移酶基因(glutathione-S-transferase,GST)的cDNA序列全长,并进行了生物信息学和表达分析。结果表明:青花菜GST基因cDNA全长为915bp,开放阅读框为642bp,编码213个氨基酸,推测分子式为C1091H1719N289O306S5,分子量为23 940.7,没有跨膜螺旋区域和信号肽。系统进化树分析表明,该青花菜基因GST与芥菜的GST聚类关系最近。实时荧光定量PCR结果显示,在模拟酸雨胁迫下,GST基因的表达量在胁迫初期显著增大,随时间延长开始下降,表明其参与了青花菜抗酸雨的应答反应。     

菜粉蝶热激蛋白70(HSP70)基因的分子特性及其对杀虫剂胁迫的响应

[目的]明确菜粉蝶Pieris rapae热激蛋白70(HSP70)基因的分子特性及其对杀虫剂胁迫的响应,为探索菜粉蝶HSP70基因在抵御杀虫剂胁迫中的功能提供前期基础.[方法]利用同源检索方法,从菜粉蝶转录组数据中鉴定HSP70基因;使用生物信息学方法分析HSP70基因的分子特性;采用实时荧光定量PCR技术分析HSP...     

甘薯谷胱甘肽-S-转移酶基因在胁迫条件下的表达分析

成功地构建了甘薯谷胱甘肽-S-转移酶基因IBGSTU1 的原核表达质粒pET-IbGST, 并在大肠杆菌 BL21(DE3)中进行IPTG 诱导表达。重组蛋白部分以包涵体形式存在, 部分以可溶性蛋白形式存在。酶活性测定表明可溶的重组蛋白具有GST的活性。纯化的重组蛋白质用于多克隆抗体的制备。半定量RT-PCR 和 Western blotting 分析结果显示, 在正常的生长条件下, 甘薯组织不启动IBGSTU1 基因的转录和翻译, 但是在冷胁迫或重金属离子等的作用下, 可以检测到该基因的mRNA和编码的蛋白质, 表明该基因在甘薯的胁迫耐受中行使重要功能, 并发现该基因的表达具有组织特异性。     

家蚕谷胱甘肽-S-转移酶基因BmGSTz1的克隆、序列分析及组织表达特征

谷胱甘肽-S-转移酶(GSTs)是一个功能广泛的超基因家族, 其中Zeta家族在动物、植物和细菌中均有分布。在哺乳动物中, Zeta GSTs具有马来酰乙酰乙酸异构酶（MAAI）活性, 参与苯丙氨酸/酪氨酸的代谢过程。本研究对家蚕Bombyx mori基因组中预测的GST基因（BmGSTz1）进行了表达序列标签的搜索, 经拼接后获得一条含有3′和5′非翻译区在内的长度为1 239 bp 的cDNA序列, 其3′端含有AATAAA加尾信号。BmGSTz1基因含有4个内含子, 外显子/内含子边界均符合GT-AG 规则。经TA克隆证实, BmGSTz1基因编码区序列全长648 bp, 共编码215个氨基酸。BmGSTz1推定的分子量为24.8 kD, 等电点pI为8.06。BmGSTz1与其他昆虫和哺乳动物GSTz1的氨基酸序列高度保守, 进化分析表明家蚕BmGSTz1与黑腹果蝇Drosophila melanogaster、冈比亚按蚊Anopheles gambiae、意大利蜜蜂Apis mellifera和赤拟谷盗Tribolium castaneum的GSTz1形成1∶1∶1∶1∶1的直系同源关系。RT-PCR和基因芯片数据表明BmGSTz1在家蚕5龄第3天幼虫各组织中都有表达。序列和组织表达特征分析结果提示家蚕BmGSTz1可能具有MAAI活性, 这将为进一步深入研究BmGSTz1基因的功能提供参考。     

芸香苷对家蚕谷胱甘肽-S-转移酶部分基因的诱导表达

谷胱甘肽-S-转移酶(glutathione-S-transferases, GSTs) 是对一种机体的解毒代谢起重要作用并可以被诱导的酶系。为了从分子水平上探究家蚕Bombyx mori GSTs 基因与植物次生物质芸香苷代谢的关系, 本实验采用双跟踪标定定量PCR (dual-spike-in qPCR)方法, 用 5×10^-1, 5×10^-2, 5×10^-3 ng/μL 3 个浓度芸香苷溶液处理家蚕5龄幼虫, 并对各组织中 GSTs Epsilon 家族不同基因的转录水平进行检测。结果显示, 浓度为 5×10^-2 ng/μL 的芸香苷溶液能诱导GSTs 基因的转录水平发生显著变化。在中肠中, BmGSTe1, BmGSTe2 和 BmGSTe6的诱导转录水平较高, 在诱导后24 h达到最大值; 在脂肪体中BmGSTe1, BmGSTe6和BmGSTe7的转录水平相对较高且分别在诱导后 2 h, 2 h和4 h 达到最大值; 而GSTs基因在马氏管中表达量均很低或者检测不到表达。与 5×10^-2 ng/μL 浓度相比, 5×10^-1 ng/μL 的芸香苷溶液诱导后各基因的转录水平上升的幅度较小并且达到峰值的时间不同, 而 5×10^-3 ng/μL 浓度的芸香苷溶液并不能使GSTs基因的诱导转录水平发生变化。结果提示, 家蚕 GSTs Epsilon 家族基因对芸香苷的代谢具有重要作用。     

野桑蚕谷胱甘肽-S-转移酶（GSTs）基因的诱导表达定量分析

谷胱甘肽-S-转移酶（GSTs）在机体的解毒代谢和抗氧化中起重要作用。为了从分子水平上探究野桑蚕Bombyx mandarina对杀虫剂抗性的产生机理,本研究采用实时荧光定量RT-PCR方法,对GSTs基因在正常饲养野桑蚕5龄幼虫及用敌敌畏和溴氰菊酯处理5龄幼虫不同组织中的转录水平进行检测,并采用Actin3内源参照基因对检测结果进行归一化处理。结果表明：用敌敌畏和溴氰菊酯处理5龄幼虫24 h后,GSTs基因在各组织中的诱导转录水平存在差异,但GSTs基因在脂肪体中的诱导转录水平最高,其次为中肠,可能与这2种组织是野桑蚕主要的解毒器官有关。其中,GSTe2和GSTe5基因诱导转录水平相对较高,推测这2个基因可能主要参与野桑蚕对外源物质的解毒代谢。     

不同浓度阿维菌素对鲤鱼过氧化氢酶和谷胱甘肽-S-转移酶活性的影响

采用半静态水质接触染毒法,研究不同浓度(0、3.2、5.6、7.5、10.0和18.0μg.-1)阿维菌素对鲤鱼(Cyprinus carpio)肝胰脏和肌肉过氧化氢酶(CAT)、谷胱甘肽-S-转移酶(GSTs)活性的影响。结果表明:除3.2μg.L-1浓度对CAT和GSTs活性无显著影响外,其余各浓度的阿维菌素均对鲤鱼组织中CAT和GSTs产生显著影响,总体表现为低浓度诱导,高浓度先诱导后抑制;5.6和7.5μg.L-1浓度组的肌肉CAT活性与对照组相比无显著差异,肝胰脏CAT活性以及肌肉和肝胰脏GSTs活性均显著升高;10.0和18.0μg.L-1浓度组的肌肉和肝胰脏中CAT、GSTs活性先显著升高,随后又显著下降;解除污染胁迫10d,18.0μg.L-1浓度组的肝胰脏中酶活性不能恢复到正常水平,其余各组与对照无显著差异,说明高浓度阿维菌素暴露可能对鲤鱼机体造成不可逆损伤;阿维菌素暴露浓度与其对鲤鱼肝胰脏和肌肉CAT、GSTs活性抑制率之间具有显著的剂量-效应关系,可以考虑将其作为水体中阿维菌素类药物污染的生物标志物;同时,由于鲤鱼受污染胁迫时肌肉CAT、GSTs活性变化的显著性均远低于肝胰脏,因此当考虑...     

舞毒蛾谷胱甘肽S-转移酶对杨树次生物质协同溴氰虫酰胺的胁迫响应

为了明确舞毒蛾Lymantria dispar谷胱甘肽S-转移酶(GST)对杨树次生物质协同溴氰虫酰胺的胁迫响应机制,选择3种杨树次生物质(黄酮、槲皮素、芦丁)以及新型邻二苯甲酰胺类杀虫剂溴氰虫酰胺作为胁迫外源化合物,以舞毒蛾2龄幼虫为研究对象,通过人工饲料添加次生物质和溴氰虫酰胺的单剂和混剂,测定对舞毒蛾存活率、谷胱甘肽S-转移酶活性及其基因表达影响。结果表明,处理48 h后,3种联合处理组舞毒蛾幼虫的存活率显著低于对照组和各杨树次生物质单剂处理组,存活率依次为53.33%、60.00%和53.33%,各联合处理组幼虫存活率与溴氰虫酰胺处理组差异不显著。除处理6 h外,不同杨树次生物质单剂处理后GST活性均诱导增加。溴氰虫酰胺处理组在48 h内GST活性显著高于单剂处理组和对照组。除联合处理1在6 h、12 h的GST诱导活性低于溴氰虫酰胺处理组外,各联合处理组的GST诱导活性均高于溴氰虫酰胺处理组。舞毒蛾2龄幼虫取食含有不同处理的人工饲料后,其体内LdGSTe2、LdGSTs1、LdGSTs2和LdGSTz1均有所表达,且不同处理的诱导程度呈现差异。以上研究结果为杨树次生物质协同溴...     

南极衣藻谷胱甘肽-S-转移酶基因的表达及其对大肠杆菌低温耐受性的增强作用

谷胱甘肽-S-转移酶(Glutathione-S-transferase, GST, EC2.5.1.18)是生物体内一种重要的抗氧化酶, 为阐明GST在南极衣藻(Chlamydomonas sp. ICE-L)中的具体地位, 采用实时荧光定量PCR对不同温度下南极衣藻的GST基因的表达进行了分析; 并构建了原核表达载体pET28a(+)-GST, 转化至大肠杆菌BL21(DE3)中进行诱导表达, 通过平板培养法探讨了重组菌E. coli BL21(pET28a(+)-GST)对低温胁迫的耐受性。结果显示, GST在0℃时表达量最高, 最高可达对照组的两倍多; pET28a(+)-GST重组表达载体在E. coli BL21中实现了高效表达, 且主要以包涵体形式存在, 经HisTrap HP柱分离纯化获得高纯度的GST融合蛋白, 并通过SDS-PAGE及Western blot分析得以验证; 对低温胁迫实验发现南极衣藻GST蛋白的表达可以提高重组菌E. coli BL21对低温的耐受性, 说明GST基因对南极衣藻适应南极低温环境具有重要作用。     

高温胁迫对扶桑绵粉蚧成虫过氧化物酶和谷胱甘肽-S-转移酶活性的影响

为了研究高温胁迫对扶桑绵粉蚧的影响,将该虫分别置于35℃、38℃、41℃、44℃和47℃水浴处理2 h、3 h和4 h,然后置于26℃下恢复2 h,测定成虫的过氧化物酶(POD)和谷胱甘肽-S-转移酶(GSTs)活力的变化。结果表明在处理2 h、3 h和4 h下,对照(26℃)的POD活力值分别为(0.0527±0.0015)mmol/min、(0.0508±0.0015)mmol/min和(0.0483±0.0072)mmol/min,均高于高温各处理的POD活力值;5个温度处理下的POD活力值变化为低(35℃)-高(38℃)-低(41、44、47℃)的变化趋势;当处理2 h时,不同温度下的POD活力值分别是(0.0183±0.0009)(35℃)、(0.0480±0.0012)(38℃)、(0.0227±0.0012)(41℃)、(0.0197±0.0003)(44℃)、(0.0173±0.0007)(47℃)mmol/min。且在不同温度和时间处理下,成虫的GSTs活性变化趋势与POD活性变化趋势一致。即不同时间处理下,对照的GSTs活性均高于高温各处理的GSTs,其对照的GSTs活力值分别为(0.5537±0.0044)(2 h)、(0.5358±0.0078)(3 h)和(0.5291±0.0264)(4 h)mmol/min;5个温度处理下的GSTs活力值变化为低(35℃)-高(38℃)-低(41℃、44℃、47℃)的变化趋势;当处理3 h时,不同温度下的GSTs活力值分别是(0.5114±0.0116)(35℃)、(0.5426±0.0009)(38℃)、(0.4861±0.0073)(41℃)、(0.3657±0.0029)(44℃)、(0.3404±0.0156)mmol/min(47℃)。因此高温对扶桑绵粉蚧体内的POD和GSTs活力存在影响。     

Comparative study on the susceptibility of cutworms (Lepidoptera: Noctuidae) to Agrotis segetum nucleopolyhedrovirus and Agrotis ipsilon nucleopolyhedrovirus

The common cutworm (Agrotis segetum) and the black cutworm (Agrotis ipsilon) are serious soil pests of many vegetable and field crops all over the world. We have demonstrated the cross-infectivity of two baculoviruses, A. segetum nucleopolyhedrovirus (AgseNPV) and A. ipsilon nucleopolyhedrovirus (AgipNPV) for these two insect pests. The susceptibility of A. segetum to AgipNPV was confirmed by DNA restriction endonuclease analyses of DNA isolated from virus harvested from infected A. segetum larvae. For an initial comparison of both viruses, partial polyhedrin sequences were amplified by PCR, cloned, and sequenced. Both viruses shared a very similar polyhedrin gene sequence resulting in only three amino acid substitutions. Phylogenetic analyses clearly demonstrated that both viruses belong to NPV group II and are most closely related to a clade consisting of Spodoptera exigua NPV, Spodoptera frugiperda NPV, and Spodoptera littoralis NPV. Since AgipNPV shows high virulence for both cutworm species, it appears to be a suitable candidate as a single biological control agent of A. segetum and A. ipsilon.     

小地老虎几丁质酶基因的克隆与序列分析

利用昆虫几丁质酶对几丁质的调控作用破坏几丁质新陈代谢的平衡来防治害虫, 在生物防治策略中具有很大的发展潜力。从处于预蛹期的小地老虎Agrotls ipsilon (Hufnagel)体中肠内提取总的RNA, 经反转录, 利用cDNA末端快速扩增技术（RACE）获得了几丁质酶基因的cDNA序列。该基因序列已经登录GenBank并获得登录号为EU035316。该序列长度为2 823个碱基, 含有一个1 674个碱基的开放读码框。开放读码框编码558个氨基酸残基, 预测的分子量为62.5 kDa, 等电点5.12。推导得到的氨基酸序列含有2个N-位糖基化位点,20个O-位糖基化位点, 含有2个几丁质酶所具有的保守序列：N-端的催化区和C-端的几丁质结合区。氨基酸序列与其他昆虫, 特别是鳞翅目昆虫的几丁质酶高度同源。     

Leaf biomechanical properties as mechanisms of resistance to black cutworm (Agrotis ipsilon) among Poa species

Biomechanical properties can be important parameters in resistance of plants to herbivorous insects. As plants age, however, there can be dramatic changes in physical defenses that can then influence their susceptibility to insect herbivores. We measured changes in leaf biomechanical properties during ontogeny of Poa species and the relationship of these changes to the development of a generalist herbivore, the black cutworm, Agrotis ipsilon Hufnagel (Lepidoptera: Noctuidae), was investigated. Larvae were reared on two representative age classes, i.e., young (<60 days after planting) and old (>1 year after planting), of foliage in laboratory assays. Foliage generally reaches a peak fracture force between 80 and 109 days after planting depending on grass type. Foliage from old plants was significantly tougher than that of young plants, and black cutworm larvae reared on foliage from young plants gained significantly (ca. four times) more weight than those fed on foliage from old Poa plants. In addition, fracture force has a negative relationship with black cutworm development. Plant fiber, particularly neutral detergent fiber accounted for 65 and 46% of the variation in fracture force and larval development, respectively. These results provide additional insight into how plant ontogeny influences physical defenses to an insect herbivore in a grass system. Likewise, this is seemingly the first study to suggest a mechanism for host plant resistance to black cutworm. Plant fiber may be a useful trait to explore in plant improvement programs in which black cutworm is a primary pest (e.g., managed turfgrass).     

Microbial control of black cutworm (Lepidoptera: Noctuidae) in turfgrass using Agrotis ipsilon multiple nucleopolyhedrovirus

Agrotis ipsilon multiple nucleopolyhedrovirus (family Baculoviridae, genus Nucleopolyhedrovirus, AgipMNPV), a naturally occurring baculovirus, was found infecting black cutworm, Agrotis ipsilon (Hufnagel) (Lepidoptera: Noctuidae), on central Kentucky golf courses. Laboratory, greenhouse, and field studies investigated the potential of AgipMNPV for managing black cutworms in turfgrass. The virus was highly active against first instars (LC50 = 73 occlusion bodies [OBs] per microl with 2-microl dose; 95% confidence intervals, 55-98). First instars that ingested a high lethal dose stopped feeding and died in 3-6 d as early second instars, whereas lethally infected fourth instars continued to feed and grow for 4-9 d until death. Sublethal doses consumed by third or fifth instars had little or no effect on subsequent developmental rate or pupal weight. Horizontal transmission of AgipMNPV in turfgrass plots was shown. Sprayed suspensions of AgipMNPV (5 x 10(8) - 6 x 10(9) OBs/m2) resulted in 75 to > 93% lethal infection of third or fourth instars in field plots of fairway-height creeping bentgrass, Agrostis stolonifera (Huds.), and on a golf course putting green collar. Virus spray residues (7 x 10(9) OBs/m2) allowed to weather on mowed and irrigated creeping bentgrass field plots significantly increased lethal infection of implanted larvae for at least 4 wk. This study, the first to evaluate a virus against a pest in turfgrass, suggests that AgipMNPV has potential as a preventive bioinsecticide targeting early instar black cutworms. Establishing a virus reservoir in the thatch and soil could suppress successive generations of that key pest on golf courses and sport fields.     

Orientation Behavior and Regulatory Gene Expression Profiles in Migratory Agrotis ipsilon (Lepidoptera: Noctuidae)

Journal of Insect Behavior - Many insects such as the crop pest Agrotis ipsilon (black cutworm) migrate seasonally to exploit suitable breeding and winter habitats. The migration of a crop pest...     

Molecular characterization of Agrotis segetum nucleopolyhedrovirus from Poland

The turnip moth, Agrotis segetum (Lepidoptera, Noctuidae), is an important pest insect in Europe, Asia, and Africa. We have genetically characterized and classified a nucleopolyhedrovirus isolated from A. segetum larvae in Poland (AgseNPV-P). The restriction pattern of AgseNPV-P was distinct from an isolate from England/France (AgseNPV-UK and AgseNPV-F). Sequence analysis of three conserved baculovirus genes, polyhedrin, lef-8 and pif-2, revealed that AgseNPV-P differs substantially from the already described NPVs isolated from A. segetum and possibly represents a new NPV species. Phylogenetic analysis placed AgseNPV-P among group II NPVs and showed the closest relationship to Agrotis ipsilon (Agip) NPV and Spodoptera exigua (Se) MNPV.     

Biology of Microplitis kewleyi (Hymenoptera: Braconidae): A parasite of Agrotis ipsilon (Lepidoptera: Noctuidae) larvae

Microplitis kewleyi Muesebeck is a gregarious internal parasite of larvae of the black cutworm Agrotis ipsilon (Hufnagel). Studies of the biology of the parasite revealed that there was an inverse relationship between host instar and parasite preference. Duration of development from egg to pupa ranged from 18 days at 27°C to 68.7 days at 16°C. Development from egg to pupa took 13.5–21.6 days when fourth and first instar host larvae, respectively, were parasitized. A larger number of parasites emerged from hosts parasitized in the fourth instar (22.4) than the first instar (11.5). Parasite pupation occurred when the host was in the fifth/sixth instar, depending on the instar parasitized. Thirty‐nine per cent of host larvae exposed as first instars to parasites died before parasite emergence. This decreased to 0% for host larvae exposed as fourth instars. The sex ratio was 1:1.2 (M:F). Thirty‐seven per cent of hosts exposed diurnally were stung, compared to 24% exposed nocturnally. Mean daily progeny was highest (12) on the first day, decreasing to zero after 20 days. Percent host parasitism was also highest on the first day (35%) decreasing to nearly 0% after 18 days. There appear to be three parasite larval instars. Host larvae often remained alive after parasite emergence.     

昆虫病原线虫防治烟草小地老虎技术优化研究

小地老虎Agrotis ipsilon是烟草Nicotiana tabacum移栽期主要的切根害虫,严重危害烟草生产。为研判烟草生产中符合双减（减肥、减药）要求的生物和生态防控技术,集成适合推广的虫害综合治理模式,本研究在云南玉溪华宁县以昆虫病原线虫（EPN）Steinernema carpocapsae All粉剂和化学药剂敌杀死（溴氰菊酯）处理,配合2种幼虫食诱方法（传统引诱剂、人工引诱剂）以及2种增效助剂（激健1、2）组合,调查移栽后烟草幼苗受小地老虎危害死亡率,评价生物药剂、食诱剂和助剂对烟草幼苗的保护效果。结果表明,无论是否存在食诱剂,移栽4 d各施药处理均能显著降低烟草幼苗死亡率,随时间的推移,所有处理的烟草幼苗死亡率均显著上升;无食诱剂情况下,EPN+激健1和敌杀死于移栽7 d仍具显著保苗效果;传统的糖醋酒液食诱法本身具备一定保苗效果,配合施药,初期效果最好,4 d时,烟草幼苗危害率最低达0.83%±0.008%（传统食诱剂+昆虫病原线虫）;人工食诱剂持效性最好,移栽10 d时配合EPN、EPN+激健2和敌杀死仍具明显保苗效果。施用化学药剂时,食诱剂本身对保苗效果无显著贡献;使用EPN时,传统食诱法较空白具有显著保苗效果,但与人工食诱法间差异不显著。移栽4 d时,EPN+激健1较单独施放EPN具显著保苗效果,之后调查中添加助剂小区与EPN小区无显著差异。综上所述,EPN粉剂配合人工食诱剂和激健助剂,可长期较好地抑制小地老虎对烟草幼苗危害,为集成烟草全程绿色生态治理模式提供了有效参考。