Induction of disease resistance in Norway spruce (Picea abies) by necrotizing fungi

Norway spruce trees ( Picea abies ) preinoculated with the root rot fungus Heterobasidion annosum , Nectria fuckeliana or a pathogenic strain of the blue-stain fungus Ceratocystis polonica were more efficiently protected against a subsequent massive inoculation with pathogenic C. polonica than trees pretreated with nonpathogenic C. polonica or sterile malt agar. Control trees that received no pretreatment were extensively colonized by the mass inoculation. There was a strong negative correlation between the length of the phloem necroses induced by the pretreatment inoculations and the extent of host symptoms caused by mass inoculation with pathogenic C. polonica . The degree of induced resistance in Norway spruce thus depended on the amount of host tissue destroyed by the pretreatment.     

Developmental and tissue-specific expression of CHS1 from Plutella xylostella and its response to chlorfluazuron

The cDNA of chitin synthase-1 gene of Plutella xylostella (PxCHS1) was characterized and expression patterns of the two splice variants PxCHS1A and PxCHS1B were investigated in various developmental stages and in major body tissues by RT-PCR and real-time quantitative PCR (qPCR). The PxCHS1 cDNA was 5461 bp in length with an open reading frame of 4701 bp that encoded a putative protein of 1567 amino acids with predicted molecular mass of 179 kDa. The two splice variants were expressed from the mutually exclusive exons which were same in size (177 bp) but showed only 66% identity at the nucleotide level. Both splice variants were expressed in all developmental stages. The qPCR data suggested an uneven expression of the two variants in the body where expression of PxCHS1A was 3.7-fold higher than that of PxCHS1B. The expression of PxCHS1A was 1.5-fold higher in the head than the body whereas in case of PxCHS1B the difference between head and body was 6.3-fold. Chlorfluazuron did not change the expression of PxCHS1 in larvae.     

Adult Drosophila melanogaster glutathione S-transferases: Effects of acute treatment with methyl parathion

The effect of acute treatment of methyl parathion (MP) on the expression of BSP/GSH-agarose purified glutathione S-transferases (GSTs) in Drosophila melanogaster was investigated. Using 2-D gel analysis of the identified Epsilon-class, only DmGSTE6 (100%) and DmGSTE7 (72%) demonstrated significant increases in expression, suggesting the possibility that both may be involved in MP metabolism. A smaller percentage increase was also observed in DmGSTD1 (18%), a known DDT dehydrochlorinase, DmGSTE3 and DmGSTE9 and a putative Epsilon-class GST, CG16936, were shown not to be responsive to the challenge. Our finding demonstrates that not all member of the Epsilon-class GST, which are known for their role in insecticide resistance, are immediately responsive to this toxic challenge.     

Transformation a mutant Monochoria vaginalis acetolactate synthase (ALS) gene renders Arabidopsis thaliana resistant to ALS inhibitors

Acetolactate synthase (ALS) genes from Monochoria vaginalis resistant (R) and susceptible (S) biotypes against ALS inhibitors found in Korea revealed a single amino acid substitution of Proline (CCT), at 169th position based on the M. vaginalis ALS sequence numbering, to serine (TCT) in conserved domain A of the gene (equal to the proline 197 in Arabidopsis thaliana ALS gene sequence). A. thaliana plants transformed with the single mutated (Pro₁₆₉ to Ser) M. vaginalis ALS gene (including transit signal peptide) showed cross-resistance patterns to ALS-inhibiting herbicides, like as sulfonylurea-herbicide bensulfuron methyl (R/S factor of 9.5), imidazolinone-herbicide imazapyr (R/S factor of 5.1), and triazolopyrimidine-herbicide flumetsulam (R/S factor of 17.6) when measuring hypocotyls’ length of A. thaliana. The ALS activity from the transgenic A. thaliana plants confirmed the cross-resistance pattern to these herbicides like as R/S factor of 8.3 to bensulfuron methyl, 2.3 to imazapyr, and 13.2 to flumetsulam.     

The clone of laccase gene and its potential function in cuticular penetration resistance of Culex pipiens pallens to fenvalerate

Decreased insecticides cuticular penetration, as one of resistant mechanisms in insect, has been extensively documented. Laccases, are enzymes with p-diphenol oxidase activity, was related to the cuticular tanning in insect. In this study, one laccase 2 gene (CpLac2) was cloned from Culex pipiens pallens. The CpLac2 contains an open reading frame (ORF) of 2289 bp and encodes a putative 762 amino acid protein. The deduced protein of CpLac2 was more similar to laccase 2 than other insect laccases, and shared the highest identity with laccases from the same family mosquito, Aedes aegypti and Anopheles gambiae. The developmental expression model of CpLac2 in C. pipiens pallens was measured by RT-PCR. The result showed the CpLaC2 was abundantly expressed in egg, the 4th instar larva and pupa, which suggested the role of CpLac2 for egg chorion tanning and cuticular sclerotization. Meanwhile, the expression of CpLac2 in fenvalerate-susceptible and -resistant strains of C. pipiens pallens was measured by real-time PCR. The result revealed the CpLac2 was significant higher expressed in resistant strain than in susceptible strain. The overexpression of CpLac2 in resistant strain suggested that resistance could derive from reinforcement of the cuticle, which decreased the penetration of insecticide in cuticle.     

Cytochrome P450 monooxygenase-mediated neonicotinoid resistance in the house fly Musca domestica L

Neonicotinoids play an essential role in the control of house flies Musca domestica. The development of neonicotinoid resistance was found in two field populations. 766b was 130- and 140-fold resistant to imidacloprid and 17- and 28-fold resistant to thiamethoxam in males and females, respectively. 791a was 22- and 20-fold resistant to imidacloprid and 9- and 23-fold resistant to thiamethoxam in males and females, respectively. Imidacloprid selection of 791a increased imidacloprid resistance to 75- and 150-fold in males and females, respectively, whereas selection with thiamethoxam had minimum impact. Neonicotinoid resistance was in all cases suppressed by PBO. The cytochrome P450 genes CYP6A1, CYP6D1 and CYP6D3 were constitutively over-expressed in resistant strains and CYP6D1 and CYP6D3 differentially expressed between sexes. The highest level of CYP6A1 expression was observed in both gender of the imidacloprid-selected strain after neonicotinoid exposure. CYP6D1 expression was increased after neonicotinoid exposure in resistant males. CYP6D3 expression was induced in both sexes upon neonicotinoid exposure but significantly higher in females.     

三叶草斑潜蝇hsp70的克隆及其表达量在高低温胁迫下的变化

为了明确高低温胁迫对三叶草斑潜蝇hsp70表达量的影响,采用RT-PCR和RACE技术获得了1条三叶草斑潜蝇诱导型热激蛋白基因hsp70,命名为Lthsp70-1,并利用实时荧光定量PCR技术检测其在温度胁迫后的表达量.该基因的开放阅读框为1923 bp,编码640个氨基酸.氨基酸序列中含有HSP70家族的签名序列IFDLGGGTFDVSIL和IVLVGGSTRIPK、DnaK特征基序IDLGTT(Y)S(C)V、非细胞器基序RARFEEL,以及C末端的保守序列EEVD.实时荧光定量PCR结果显示:成虫在31～33℃范围内,其hsp70表达量随温度升高而上升,33℃时达到最高峰;35～39℃时,hsp70表达量迅速下降;蛹经0℃胁迫0.5～2.0 h,其hsp70表达量随时间延长呈上升趋势.由此可见,高低温胁迫均能诱导三叶草斑潜蝇hsp70的表达.     

芽胞杆菌CQBS03抑菌蛋白TasA基因的克隆及原核表达

为了探讨柑桔溃疡病生防菌芽胞杆菌Bacillus CQBS03菌株TasA基因的功能,采用PCR方法从CQBS03基因组DNA中扩增出编码TasA基因的全长DNA序列,并构建pEASY-E1/TasA原核表达载体,经大肠杆菌Escherichia coli表达获得TasA基因的融合表达蛋白,纸碟法检验融合蛋白对柑桔溃疡病菌Xanthomonas citri citri的抑制作用。结果显示,CQBS03菌株的TasA基因包含1个786 bp的完整开放阅读框（GenBank登录号为JQ309841）,编码261个氨基酸残基;该序列与来源于解淀粉芽胞杆菌B.amyloliquefaciens的1个已知同源TasA基因序列FJ713580的相似性达99.75%。原核表达产物经SDS-PAGE分析,检测到约31 kD的融合蛋白;纯化后的融合蛋白对柑桔溃疡病菌有明显的抑制作用,72 h后抑菌圈直径达11.5 mm。研究表明TasA基因是生防菌芽胞杆菌CQBS03抑制柑桔溃疡病菌的功能基因之一,并且该基因对原核表达宿主没有抑制作用,具有较好的开发利用前景。     

水稻条纹病毒胁迫对灰飞虱mucin基因表达量的影响

为分析灰飞虱mucin基因在水稻条纹病毒(Rice stripe virus, RSV)侵染过程中的作用,采用荧光定量PCR的方法分析了RSV 胁迫条件下灰飞虱mucin基因mRNA表达量的变化。饲喂RSV病叶1天和2天时灰飞虱mucin基因的表达量没有明显变化,而在3天和7天时灰飞虱mucin基因的表达量增加了3.60倍和1.97倍。饲喂健康水稻叶片1、2、3和7天后,灰飞虱mucin基因的表达量分别为饲喂前的1.07、1.12、0.78和0.34倍,而饲喂病叶1、2、3和7天后,mucin基因的表达量分别为饲喂前的1.15、1.19、3.19和1.01倍。结果表明,病毒胁迫使灰飞虱体内mucin基因的表达量增加,暗示mucin基因在RSV与灰飞虱互作过程中起着重要作用。     

大豆对灰斑病菌15号小种的抗病基因定位及标记检测

为明确大豆对灰斑病菌15号小种的抗性位点,以大豆抗病品种垦丰16、感病品种绥农10及其杂交F₂、F₃代群体为试验材料,在接种鉴定的基础上,运用SSR标记技术及分离群体组群分析法(BSA法)对垦丰16抗病基因进行了定位,并应用108份大豆新品系对标记进行了符合性检测。结果表明,垦丰16对15号小种的抗性受1对显性基因控制,抗病基因位于大豆染色体组的J连锁群上,将该基因定名为Rcs15。用Mapmaker/Exp 3.0 b进行连锁分析,获得了5个与抗病基因紧密连锁的SSR标记:Satt 529、Satt 431、Sat_151、Satt 547和Sat_224,标记与抗病基因间的排列顺序和遗传距离为Sat_151-10.7 cM-Satt 529-18.5 cM- Rcs15-6.7 cM-Satt 547-7.8 cM-Sat_224-10.7 cM-Satt 431。标记符合性检测结果显示,Satt 547和Sat_224的检测准确率达到85%以上,可用于分子标记辅助选择育种和抗源筛选。     

小麦小GTP结合蛋白基因TaRop3克隆及其表达分析

利用同源克隆法从小麦抗条锈病基因Yr5近等基因系（Taichung29*6/Yr5）克隆到编码Rop蛋白基因的全长cDNA序列,并将基因命名为TaRop3（Triticum aestivum Rop3）,聚类分析其所在Rop蛋白家族中的亚组,并利用半定量RT-PCR方法分析其组织表达特异性和不同诱导条件下表达动态。序列分析表明,TaRop3开放阅读框为639 bp,预测编码含213个氨基酸残基Ⅱ型Rop蛋白,C末端具有膜定位基序,与大麦HvRop4和水稻OsRac4聚类在同一亚组。TaRop3基因在小麦幼苗和成株根、茎、叶片和茎节、柱头及花药中均有表达,其中在幼苗及成株茎部表达水平较高。非亲和条锈菌小种CYR17侵染和水杨酸处理诱导TaRop3表达增强,而干旱、高温胁迫以及脱落酸、乙烯利和茉莉酸甲酯处理均降低该基因表达水平。     

Characterization of imidacloprid resistance in the housefly Musca domestica (Diptera: Muscidae)

In recent years, imidacloprid was introduced to control the housefly in China and it was documented that the housefly indeed showed signs of resistance to imidacloprid somewhere but not in China. Therefore, a housefly population collected from filed (IFS) was selected continuously with imidacloprid to establish the resistant strain (IRS) and the basic characteristics were investigated in this study. After continuous selection over 21 generations, the resistance ratio increased from 9.01 to 140, and different levels of cross-resistance were developed to beta-cypermethrin, chlorpyrifos, chlorfenapyr, acetamiprid and azamethiphos in the IRS strain. The realized heritability of resistance was 0.10. The synergistic ratios for IRS pretreated with DEF, DEM and PBO were 1.68, 1.52 and 2.53, and the corresponding ones for IFS were 3.17, 1.87 and 2.67, respectively. Synergistic and biochemical assays suggested that the cytochrome P450 may play an important role in the imidacloprid resistance comparing with GSTs- and carboxylesterases-mediated detoxification in the IRS strain, and there might be additional mechanisms (e.g. reduced target-site sensitivity) contributed to imidacloprid resistance in the IRS strain.     

Detection of insecticide resistance-associated mutations in cat flea Rdl by TaqMan-allele specific amplification

We are interested in correlating the performance of fipronil in populations of cat fleas (Ctenocephalides felis) with potential cross-resistance conferred by point mutations in the Resistance to dieldrin gene, Rdl. Here we report the sequencing of exon 7 of the cat flea Rdl gene and the documentation of a putative resistance-associated mutation predicting the replacement of alanine302 with a serine. We describe two polymerase chain reaction (PCR) based diagnostics for the detection of this mutation. First, PCR mediated detection of a restriction endonuclease polymorphism (PCR REN) using a BsmAI site created by the resistance-associated mutation. Second, a TaqMan assay using allele specific fluorogenic probes and the TaqMan 5^′ specific nuclease. We describe how such diagnostics can be used in the diagnosis of resistance in the field and laboratory.     

Regulation of cytochrome P450 expression in Drosophila: Genomic insights

Genomic tools such as the availability of the Drosophila genome sequence, the relative ease of stable transformation, and DNA microarrays have made the fruit fly a powerful model in insecticide toxicology research. We have used transgenic promoter-GFP constructs to document the detailed pattern of induced Cyp6a2 gene expression in larval and adult Drosophila tissues. We also compared various insecticides and xenobiotics for their ability to induce this cytochrome P450 gene, and show that the pattern of Cyp6a2 inducibility is comparable to that of vertebrate CYP2B genes, and different from that of vertebrate CYP1A genes, suggesting a degree of evolutionary conservation for the “phenobarbital-type” induction mechanism. Our results are compared to the increasingly diverse reports on P450 induction that can be gleaned from whole genome or from “detox” microarray experiments in Drosophila. These suggest that only a third of the genomic repertoire of CYP genes is inducible by xenobiotics, and that there are distinct subsets of inducers/induced genes, suggesting multiple xenobiotic transduction mechanisms. A relationship between induction and resistance is not supported by expression data from the literature. The relative abundance of expression data now available is in contrast to the paucity of studies on functional expression of P450 enzymes, and this remains a challenge for our understanding of the toxicokinetic aspects of insecticide action.