食虫沟瘤蛛钠离子通道基因cDNA片段的克隆和序列分析

昆虫对拟除虫菊酯类杀虫剂的敏感性下降主要与神经细胞膜上钠离子通道的敏感性降低有关。至今已在14种昆虫的para直向同源钠离子通道基因序列中发现了与击倒抗性有关的20个突变。作者利用反转录-多聚酶链式反应（RT-PCR）对食虫沟瘤蛛的钠离子通道基因ⅢS5-ⅣS6区域进行了克隆、序列分析及同源性比较分析。     

桃蚜电压门控钠离子通道基因cDNA克隆及其生物信息学分析

克隆获得桃蚜电压门控钠离子通道基因cDNA序列，明确钠离子通道的典型特征，为研究桃蚜抗性分子机理奠定基础。采用实验技术主要有RT-PCR和PCR，克隆桃蚜钠离子通道基因cDNA序列，利用相关软件对其序列进行生物信息学分析。克隆得到两段cDNA序列MpNa_v-1（NCBI登录号：MN124170）和MpNa_v-2（NCBI登录号：MN176136）。MpNa_v-1长度为2945 bp，包括2877 bp的完整开放阅读框，共编码958个氨基酸；MpNa_v-2长度为3546 bp，包括3486 bp的完整开放阅读框，共编码1161个氨基酸。MpNa_v-1和MpNa_v-2共同组成桃蚜的钠离子通道α亚基，MpNa_v-1包含同源结构域Ⅰ和同源结构域Ⅱ，MpNa_v-2包含同源结构域Ⅲ和同源结构域Ⅳ。同源比对发现，桃蚜与豌豆蚜和高粱蚜钠离子通道基因相似度分别高达97.67%和97.65%，所克隆序列包含昆虫钠离子通道α亚基典型特征，具有MFM模块，并含有蚜虫类钠通道特有模块DENS。成功地克隆桃蚜钠离子通道基因，为阐明其对拟除虫菊酯类药剂产生靶标抗性的分子机制奠定基础。     

麦长管蚜电压门控钠离子通道基因克隆及序列分析

克隆获得麦长管蚜电压门控钠离子通道基因cDNA序列,明确其典型特征,为研究麦长管蚜抗性分子机理奠定基础。采用RT-PCR技术,克隆麦长管蚜钠离子通道基因cDNA序列,利用DNASTAR Lasergene 7.10软件对其序列进行分析。克隆得到两条cDNA序列SaNa_v1和SaNa_v2（GenBank登录号分别为MN176137和MN161584）,SaNa_v1序列长3423 bp,共编码1141个氨基酸;SaNa_v2序列长2874 bp,共编码958个氨基酸。同源比对发现,SaNav1与禾谷缢管蚜和桃蚜的第一部分钠离子通道基因相似度分别高达97.81%和97.91%;SaNav2与禾谷缢管蚜和桃蚜的第二部分钠离子通道基因相似性高达97.90%和96.43%。所克隆序列包含4个同源结构域,每个结构域6个跨膜片段（S1～S6）,存在钠通道选择性关键残基“DENS”。本文成功克隆了麦长管蚜中钠离子通道基因,为麦长管蚜钠离子通道抗性机制的研究提供依据。     

南京小菜蛾对拟除虫菊酯的抗性机制

通过解毒酶活性测定与钠离子通道基因片段的克隆、测序,研究了南京小菜蛾对拟除虫菊酯产生高水平抗性的生化和分子机制。结果表明,南京抗性种群的酯酶和多功能氧化酶O-脱甲基活性分别是敏感种群的1.06和1.16倍,无显著差异;以2,4二硝基氯苯(CDNB)和1,2-二氯-4-硝基苯(DCNB)为底物,南京抗性种群的谷胱甘肽S-转移酶活性分别是敏感种群的0.68倍和1.03倍;进一步利用聚合酶链式反应(PCR)分别从敏感和抗性小菜蛾基因组DNA中扩增出了位于钠离子通道结构域IIS6的232bp和248bp的DNA片段;与敏感种群相比,南京抗性种群钠离子通道基因存在一个保守性点突变,即C突变为T,并导致亮氨酸突变为苯丙氨酸,表明神经不敏感性是南京小菜蛾对拟除虫菊酯产生高水平抗性的主要机制。     

斜纹夜蛾对茚虫威的抗性机制

以斜纹夜蛾对茚虫威相对敏感种群和抗性倍数为15.63倍的抗性选育种群为材料,通过解毒酶活性测定与钠离子通道基因片段的克隆、测序,研究了斜纹夜蛾对茚虫威的抗性机制.结果表明,与相对敏感种群相比,抗性种群酯酶活性提高了2.27倍,但抗性和敏感种群谷胱甘肽S-转移酶和多功能氧化酶O-脱甲基活性无显著差异;3龄幼虫酯酶活性随着抗性指数的上升而逐渐提高;从相对敏感和抗性斜纹夜蛾种群3龄幼虫基因组DNA中扩增出位于钠离子通道IIS5-IIS6的341 bp DNA片段;与相对敏感种群相比,茚虫威抗性种群钠离子通道基因没有发生突变.     

小菜蛾GluCl受体α亚基cDNA基因克隆和序列分析

利用RT-PCR和RACE技术对小菜蛾[Plutella xylostella(L.)]4龄幼虫谷氨酸门控的氯离子通道(GluCl)受体cDNA全长进行了克隆和序列分析。通过设计简并性上、下游引物扩增出小菜蛾GluCl受体α亚基基因192bp的cDNA片段,根据得到的片段序列设计3′和5′特异性引物采用RACE技术进行3′和5′末端的克隆,获得了小菜蛾GluCl受体的cDNA的完整序列,GenBank登录号为GQ221939。该基因全长2144bp,其开放阅读框(ORF)1344bp,编码447个氨基酸。相似性分析表明:它与果蝇和赤拟谷盗的相似性均为73%,与埃及伊蚊的相似性为75%,与东亚飞蝗的相似性为79%;氨基酸分析后显示它具有GluClα亚基的典型特征,表明克隆的cDNA序列是小菜蛾GluClα亚基基因的序列。     

海南普通大蓟马对拟除虫菊酯药剂抗性监测及钠通道突变检测

普通大蓟马Megalurothrips usitatus在海南省对豇豆造成严重危害且抗药性逐渐增强。本研究测定了2019年至2021年海南省普通大蓟马对氯菊酯和甲氰菊酯的抗性。结果表明,海口、乐东和三亚3个地理种群对甲氰菊酯处于极高水平抗性,对氯菊酯处于高水平抗性,且抗性逐年增强。对普通大蓟马钠离子通道序列分析发现存在M283R突变,该突变位于钠离子通道同源结构域Ⅰ。突变频率检测显示,2019年至2021年连续3年海口种群该突变位点的突变频率分别为1/30、1/30、3/30,有升高趋势。本研究发现海南省普通大蓟马从2019年到2021年对拟除虫菊酯类药剂的抗药性呈逐年上升趋势。     

浙江省杨梅园果蝇及其寄生性天敌种类多样性调查

首次系统地调查了浙江省杨梅主要产区果蝇及其寄生性天敌种群资源,明确了为害杨梅的果蝇主要类群及其寄生性天敌优势种类。采用形态学鉴定与COI分子生物学方法相结合,分析发现我省杨梅果蝇种类主要有6种：黑腹果蝇Drosophila melanogaster、斑翅果蝇D.suzukii、拟果蝇D.simulans、高桥氏果蝇D.takahashii、伊米果蝇D.immigrans和黑果蝇D.virile;浙江省杨梅果蝇寄生蜂有9种,分属于4个科,它们分别是茧蜂科Braconidae的开臂反颚茧蜂属Asobara、环腹瘿蜂科Figitidae的丽匙胸瘿蜂属Ganaspis和小环腹瘿蜂属Leptopilina、锤角细蜂科Diapriidae的毛锤角细蜂属Trichopria和金小蜂科Pteromalidae的蝇蛹金小蜂属Pachycrepoideus。同时,不同年份杨梅果园果蝇种群数量变动较大,其中2018年各地区斑翅果蝇比例明显高于2019年。大部分地区（杭州除外）开臂反颚茧蜂属Asobara寄生蜂数量明显多于其他寄生蜂,为浙江省果蝇优势寄生蜂。本文最后就各类寄生蜂生物学特性及其生防潜力进行了讨论,旨在为杨梅果园及其他果园果蝇害虫的生物防治提供基础资料。     

黔东南州丹寨县不同品种蓝莓主要病虫害发生情况调查研究

[目的]调查研究黔东南州丹寨县甲脚村、烧茶村蓝莓基地灰霉病、锈病、果蝇、巢蛾发生危害情况,拟在为品种合理布局,病虫害防治提供科学依据.[方法]通过2020年4—8月份调查灰霉病、锈病及使用引诱剂诱杀果蝇和巢蛾成虫.[结果]甲脚村4—8月份,中、早熟品种夏普蓝灰霉病、锈病平均病株率及诱集果蝇数量高于晚熟品种灿烂和布里吉塔;夏普蓝诱集巢蛾成虫与灿烂品种相同,高于布里吉塔.烧茶村中、早熟品种夏普蓝灰霉病、锈病平均病株率及诱集果蝇成虫数量高于灿烂.[结论]夏普蓝品种灰霉病、锈病平均病株率、诱杀果蝇成虫数量高于灿烂和布里吉塔.生产上建议,3种品种合理布局,并加强对中、早熟品种夏普蓝灰霉病、锈病、果蝇和巢蛾的防治.     

山东临朐不同品种樱桃上果蝇发生种类及种群动态调查

为了解不同品种樱桃果蝇的发生情况,于2019年在山东临朐应用糖醋液诱捕器监测果蝇成虫发生动态,并调查了不同品种樱桃受果蝇为害情况及相关品质指标.结果表明,不同樱桃品种间果蝇成虫发生量及发生高峰期不同,以黄蜜和美早上果蝇的发生数量最多,监测成虫发生高峰期较先锋、红灯、拉宾斯晚.樱桃果实成熟期,美早和黄蜜上果蝇幼虫数量和蛀...     

Detection of cyclodiene insecticide resistance-associated mutations by single-stranded conformational polymorphism analysis

Cyclodiene insecticide resistance is associated with replacements of a single amino acid within the putative lining of a δ-aminobutyric acid (GABA)-gated chloride ion channel gene Resistance to dieldrin (Rdl). Only two resistance-associated amino acid replacements have been identified; alanine to serine in Drosophila melanogaster, D. simulans, Aedes aegypti, and Tribolium castaneum and alanine to glycine as a second allele in D. simulans. Here we report that single stranded conformational polymorphism (SSCP) analysis of genomic DNA, amplified by the polymerase chain reaction (PCR) for exon 7 of the Rdl gene, can be used to genotype strains or individuals of all of these insects. This technique also appears simultaneously to distinguish between D. melanogaster and D. simulans, sibling species only reliably identifiable by examination of male genitalia. The relative advantages of this genotyping technique against other PCR-mediated techniques in monitoring for insecticide resistance are discussed.     

Bt菌株FB的生物学特性及其cry、vip基因型鉴定

菌株FB是1株对小菜蛾Plutella xylostella幼虫具有高毒力的苏云金芽胞杆菌Bacillus thuringiensis （Bt）。本研究通过扫描电子显微镜、大质粒电泳、总蛋白SDS-PAGE及菌株生长特征观察的方式研究了菌株FB特征,克隆得到了基因cry1Ia、cry1Ea、cry1Ab、cry2Ab和vip3Aa全长,依据全基因组测序结果得到了1个cry8基因部分片段,首次在Bt菌株中同时发现基因cry1类和cry8类,这五种基因推导的氨基酸序列与已知基因序列相比,最高相似性分别为99%、98%、99%、100%、99%,而cry8半长基因与已知基因仅为63%。生测结果表明,蛋白Cry1Ia、Cry1Ea和Cry1Ab对小菜蛾幼虫具有较高杀虫活性。     

Linkage of genes for sodium channel and cytochrome P450 (CYP6B10) in Heliothis virescens

Genetic linkage of hscp (heliothis sodium channel protein) and CYP6B10 was discovered in Heliothis virescens. The hscp gene encodes the sodium channel target of pyrethroid insecticides and cytochrome P450 genes encode important enzymes involved in detoxication of various pesticides. Previously, two mechanisms, nerve insensitivity due to sodium channel and synergism by propynyl aryl ethers, were observed in pyrethroid-resistant H virescens and were not separated by repeated back-crossing. We hypothesized genetic linkage of target site insensitivity and monooxygenase-mediated detoxication. Single nucleotide polymorphisms were discovered in IIS6 of hscp; Hpy of hscp and CYP6B10. Segregation of these and other markers was tested in backcrosses. We observed cosegregation of hscp to CYP6B10, but both genes assorted independently of y, ye and sex. Genes y and ye assorted independently of each other. This was the first observation of linkage between genes controlling detoxication and sodium ion channel insensitivity in a species known to express high levels of pyrethroid resistance. Linkage was not likely because this species has 31 chromosomes; therefore, we will investigate the possibility of a resistance cassette. We expect similar linkage in other noctuid pests.     

The sodium channel gene in Tetranychus cinnabarinus (Boisduval): identification and expression analysis of a mutation associated with pyrethroid resistance

BACKGROUND: The carmine spider mite (CSM), Tetranychus cinnabarinus, is the most harmful mite pest of various crops and vegetable plants. Pyrethroid insecticide fenpropathrin has been used to control insects and mites worldwide, but CSM has developed resistance to this compound. RESULTS: Three synergists together eliminated about 50% resistance against fenpropathrin in the CSM. A point mutation was identified from the sodium channel gene of fenpropathrin‐resistant CSM (FeR) by comparing cDNA sequences between FeR and susceptible (S) sodium channel genes, which caused a phenylalanine (F) to isoleucine (I) change at amino acid 1538 position in IIIS6 of the sodium channel and has been proven to confer strong resistance to pyrethroid in other species. The mRNA expression of the sodium channel gene in the FeR and abamectin‐resistant strain (AbR), which was included as a control, were both relatively lower than in the S. CONCLUSION: These results demonstrate that a mutation (F1538I) is present in the sodium channel gene in FeR of CSM, likely playing an important role in fenpropathrin resistance in T. cinnabarinus, but that decrease in the abundance of sodium channel did not confer this resistance. The F1538I mutation could be used as a molecular marker for detecting kdr resistance in Arachnida populations. Copyright © 2011 Society of Chemical Industry     

Turnip mosaic virus induces expression of the LRR II subfamily genes and regulates the salicylic acid signaling pathway in non-heading Chinese cabbage

In order to study the defense response to turnip mosaic virus (TuMV) infection in non-heading Chinese cabbage (Brassica campestris ssp. chinensis Makino), we cloned the LRR II subfamily genes which comprises six members. They were high homologous to the function-known LRR II genes of Arabidopsis. We investigated their expression through quantitative real-time PCR analysis. TuMV infection induced the expression of these genes locally and systematically, and regulated the endogenous accumulation of salicylic acid (SA). Exogenous SA spraying was able to induce resistance to the susceptibility of the TuMV-infected plants, which might function via inhibiting the viral duplication. Though TuMV-induced SA accumulation was not the determinant in regulating gene expression, it mediated the reaction oxygen species (ROS) burst as a channel of defense.