Cloning of a rice tau class GST isozyme and characterization of its substrate specificity

The GST cDNA was successfully cloned from an Oryza sativa cDNA library by PCR using oligonucleotide primers based on the OsGSTU5 (GenBank Accession No. AF309377) sequence. The cDNA was composed of a 687-bp open reading frame encoding for 228 amino acids. The deduced amino acid sequence of this gene shared over 60% sequence identity with the sequences of the tau class ZmGSTU6 and ZmGSTU19. This gene was expressed in Escherichia coli with the pET vector system and the gene product was purified to homogeneity by GSH-Sepharose affinity column chromatography. The expressed OsGSTU5 formed a homo-dimer composed of 25 kDa subunit and its pI value was approximately 7.5. The OsGSTU5 displays high activities towards 1-chloro-2,4-dinitrobenzene and 1,2-epoxy-3-(p-nitrophenoxy)propane. The activity of the OsGSTU5 was significantly inhibited by hematin and ethacrynic acid. The OsGSTU5 shows the highest activity towards chloro-s-triazine and acetanilide herbicides.     

Molecular cloning, expression, and characterization of a phi-type glutathione S-transferase from Oryza sativa

The structural gene for glutathione S-transferase in Oryza sativa was successfully cloned from a cDNA library by the polymerase chain reaction method. The deduced amino acid sequence of this gene showed 44-66% similarity to the sequences of the class phi GSTs from Arabidopsis thaliana and Zea mays. This gene was expressed in Escherichia coli with the pET vector system and the gene product was purified to homogeneity by GSH-Sepharose affinity column chromatography. The expressed OsGSTF3-3 was a homo-dimer composed of 24 kDa subunit and its pI value was approximately 7.3. The OsGSTF3-3 was retained on GSH affinity column and its K_m value for GSH was 0.28 mM. The OsGSTF3-3 displayed high activity toward 1-chloro-2,4-dinitrobenzene, a general GST substrate and also had high activities towards acetanilide herbicides, alachlor, and metolachlor. The OsGSTF3-3 was highly sensitive to inhibition by benastatin A and S-hexyl-GSH. From these results, the expressed OsGSTF3-3 is a phi class GST and seems to play an important role in the conjugation of the chloroacetanilide herbicides.     

Purification and characterisation of a family of glutathione transferases with roles in herbicide detoxification in soybean (Glycine max L.); selective enhancement by herbicides and herbicide safeners

Glutathione transferases in soybean (GmGSTs) involved in herbicide detoxification in cell suspension cultures were purified by S-hexylglutathione affinity chromatography and resolved by a combination of HPLC and SDS-PAGE into 11 polypeptides. Analysis by Western blotting using antisera raised to three previously characterised tau (GmGSTU) class subunits demonstrated that five polypeptides were related to GmGSTU1, three to GmGSTU2, and one to Gm GSTU3. Plants contained a simpler profile of polypeptides, with a single GmGSTU2-like polypeptide predominating. With respect to herbicide detoxification, two GmGSTU2-related polypeptides dominated the activity toward the chloroacetanilide acetochlor, while an unclassified subunit was uniquely associated with the detoxification of diphenyl ethers (acifluorfen, fomesafen). The inducibility of the different GST subunits was determined in soybean plants exposed to photobleaching diphenyl ethers and the safeners naphthalic anhydride and dichlormid. GmGSTU3, a GmGSTU1-like polypeptide, and thiol (homoglutathione) content were induced by all chemical treatments, while two uncharacterised subunits were only induced in plants showing photobleaching.     

Molecular characterization of two acetylcholinesterase genes from the brown planthopper, Nilaparvata lugens (Hemiptera: Delphacidae)

Acetylcholinesterase (AChE), which is encoded by the ace gene, catalyzes the hydrolysis of the neurotransmitter acetylcholine to terminate nerve impulses at the postsynaptic membrane. AChE is a primary target of many insecticides including organophosphates (OP) and carbamates (CB). In this study, full-length cDNA sequences of two ace genes (Nlace1 and Nlace2) were sequenced from the brown planthopper (BPH) Nilaparvata lugens, the most destructive insect pest of rice crops. Nlace1 cDNA is 2842 nucleotides long and contains an ORF potentially encoding a 790 amino acid peptide. Nlace2 cDNA is 2852 bp in length and contains an ORF that potentially encodes a 672 amino acid peptide. NlAChE1 has an identity of 40% with NlAChE2 at the amino acid sequence level. Phylogenetic analysis of 59 AChEs from 32 animal species showed that NlAChE1 is most closely related to AChE1s from Blattella germanica and Nephotettix cincticeps, while NlAChE2 is most closely related to AChE2 from N. cincticeps. Quantitative RT-PCR analysis showed that Nlace1 is expressed at a much higher level than Nlace2 in all developmental stages and tissues, demonstrating that NlAChE1 may be the dominant AChE form of the two enzymes. This result will help reveal the resistance mechanism of N. lugens to organophosphorous and carbamate insecticides and promote development of more selective insecticides targeting the main NlAChE1.     

我国部分地区沙地葡萄茎痘相关病毒分离物外壳蛋白序列变异分析

为明确我国葡萄中沙地葡萄茎痘相关病毒（GRSPaV）的感染情况及病毒外壳蛋白（coat protein,CP）基因的变异特点,从而为其致病性、病害的防治以及抗病毒基因工程等研究提供依据,本研究对采自我国16个省市自治区的65个葡萄品种305株葡萄样品中的GRSPaV进行RT-PCR检测,根据地区与品种差异选取了24个阳性样品进行cp基因克隆与测序分析,并对不同RNA提取方法进行了比较。结果显示,114株样品被GRSPaV侵染,平均带毒株率为37.4%;分离物间及同一分离物不同克隆间的序列差异较大,从24个分离物克隆获得的37条cp基因序列与来源于不同国家的12个GRSPaV分离物的核苷酸序列同源性为80.5%~99.7%,氨基酸序列同源性为88.8%~100%;各个分离物的遗传距离无明显地域差异;SiO₂吸附法比SDS法和CTAB法更适宜葡萄样品RNA的提取。     

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。     

Purification and partial characterization of a glutathione S-transferase from the two-spotted spider mite, Tetranychus urticae

Glutathione S-transferases (GSTs) are known to catalyze conjugations by facilitating the nucleophilic attack of the sulfhydryl group of endogenous reduced glutathione on electrophilic centers of a vast range of xenobiotic compounds, including insecticides and acaricides. Elevated levels of GSTs in the two-spotted spider mite, Tetranychus urticae Koch, have recently been associated with resistance to acaricides such as abamectin [Pestic. Biochem. Physiol. 72 (2002) 111]. GSTs from acaricide susceptible and resistant strains of T. urticae were purified by glutathione-agarose affinity chromatography and characterized by their Michaelis-Menten kinetics towards artificial substrates, i.e., 1-chloro-2,4-dinitrobenzene and monochlorobimane. The inhibitory potential of azocyclotin, dicumarol, and plumbagin was low (IC₅₀ values > 100 μM), whereas ethacrynic acid was much more effective, exhibiting an IC₅₀ value of 4.5 μM. GST activity is highest in 2-4-day-old female adults and dropped considerably with progressing age. Furthermore, molecular characteristics were determined for the first time of a GST from T. urticae, such as molecular weight (SDS-PAGE) and N-terminal amino acid sequencing (Edman degradation). Glutathione-agarose affinity purified GST from T. urticae strain WI has a molecular weight of 22.1 kDa. N-terminal amino acid sequencing revealed a homogeneity of ≈50% to insect GSTs closely related to insect class I GSTs (similar to mammalian Delta class GSTs).     

Cloning and expression of an atrazine inducible cytochrome P450, CYP4G33, from Chironomus tentans (Diptera: Chironomidae)

Previous studies performed in our laboratory have measured the effect of atrazine exposure on cytochrome P450-dependent monooxygenase activity and have found increased activity in midge larvae (Chironomus tentans) as a result of atrazine exposure (1-10 ppm). Here we report the cloning and expression of a specific C. tentans CYP4 gene that is responsive to atrazine induction with an open reading frame of 1678 bp which encodes a putative protein of 559 amino acid residues. Alignments of deduced amino acid sequences with other insect P450 genes and phylogenetic analysis indicated a high degree of similarity to other insect CYP4 genes. Northern blotting analysis employing a fragment of 1200 bp from the CYP4 gene as a probe indicated that the CYP4 gene was expressed in all developmental stages, but was expressed at highest levels in late instar larvae. Additionally, over-expression of CYP4 in C. tentans exposed to atrazine (10 mg/l) confirms the ability of atrazine to induce specific P450 genes and provides insight into potential consequences of atrazine exposure in aquatic organisms.     

亚洲玉米螟6-磷酸葡萄糖异构酶基因(Pgi)全序测定及分析

为进一步从核酸水平上研究亚洲玉米螟Pgi基因相关特性,采用反转录PCR及RACE等技术对该基因编码序列及DNA全序列进行了测定,与Gen Bank中其它昆虫Pgi基因相关信息进行比较分析,并构建了系统发育树。结果表明:亚洲玉米螟Pgi基因编码区序列长为1 671 bp,共编码556个氨基酸;其DNA序列全长为10 078 bp(短序列为9 311 bp),由12个外显子与11个内含子镶嵌而成;各外显子长度与鳞翅目大部分昆虫相同,介于95~188 bp之间,内含子序列总长度为8 407 bp(短序列为7 640 bp),各内含子长度介于418~1 547 bp之间,且在内含子3、4、5、11上均发现杂合现象。系统发育结果显示,大部分昆虫Pgi基因c DNA严格按物种聚类,除了双翅目的家蝇Musca domestica与黑森瘿蚊Mayetiola destructor出现一定的交叉现象外,其余没有出现交叉现象。     

南方根结线虫Col基因分子克隆及其VIGS效应分析

为了明确线虫角质层胶原蛋白基因(Col)与根结线虫病害的关系,利用从线虫基因组中预测的Col设计特异引物,克隆了南方根结线虫Meloidogyne incognitacol基因MiCol.该基因完整编码区长903bp,编码300个氨基酸.克隆的MiCol与预测的MiCol基因序列一致性高达99.67％,氨基酸序列一致性高达100％.利用病毒介导的基因沉默技术,将其导入番茄植株并接种南方根结线虫,60 d后,沉默载体pTV-MiColi处理番茄植株的根结数比空载体对照及清水对照分别减少49.4％和49.2％,表明MiCol基因沉默显著降低了南方根结线虫的侵染数量.     

韭菜迟眼蕈蚊紫外敏感视蛋白基因的克隆及光强度对其表达量影响

为明确韭菜迟眼蕈蚊Bradysia odoriphaga紫外敏感视蛋白基因Bo-uv的作用及其与趋光性的关系,利用常规PCR方法克隆获得Bo-uv基因的全长cDNA序列,分析了其敏感视蛋白的氨基酸序列与其它12种昆虫同源蛋白氨基酸序列之间的系统进化关系,运用qPCR技术检测了不同发育阶段、不同组织及不同光强度下Bo-uv基因的相对表达量。结果表明,Bo-uv基因cDNA全长2 757 bp,开放阅读框1 542 bp,编码514个氨基酸。韭菜迟眼蕈蚊紫外敏感视蛋白的氨基酸序列与其它12种昆虫同源蛋白的氨基酸序列一致性为21.93%～43.00%,与橘小实蝇Bactrocera dorsalis的氨基酸序列同源性最高。Bo-uv基因在韭菜迟眼蕈蚊蛹末期、成虫期表达,在成虫头部的相对表达量较高。在0～10 000 lx光强范围内雌、雄成虫体内该基因的相对表达量均呈先增高后降低趋势。与对照相比,1 000 lx光强度下其相对表达量显著升高,10 000 lx时相对表达量显著降低。表明光强度能够有效地调控Bo-uv基因的表达,该基因在韭菜迟眼蕈蚊感知外界光刺激过程中具有重要作用。     

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.     

Analysis of two acetylcholinesterase genes in Bombyx mori

Two acetylcholinesterases (AChE, EC 3.1.1.7) cDNAs were identified and cloned from silkworm, Bombyx mori. One of those, BmAChE-o cDNA, is comprised of 3197 nucleotides which encode 638 amino acids, having an amino acid sequence homology of 72% with Drosophila melanogaster Ace-orthologous AChE (AO-AChE). In some species, another AChE group based on the sequence, Drosophila Ace-paralogous AChE (AP-AChE) has been recognized in relation to organophosphate- or carbamate-resistance, but there have been few reports of AP-AChE among lepidopteran species. However, we isolated the AP-AChE from lepidopteran silkworm, and cloned full ORF as BmAChE-p, which cDNA consisted of 2465 nucleotides that encode 683 amino acids. The homologies with other AP-AChEs were over 60% when compared. Although silkworm is not a target of pesticides, the genomic information obtained in this study will contribute to insecticide-resistance study on lepidopteran pest species.     

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.     

禾谷缢管蚜RpGST1基因的克隆、序列分析及原核表达

为揭示禾谷缢管蚜耐药性的分子机理,采用RT-PCR方法克隆了禾谷缢管蚜谷胱甘肽-S-转移酶(glutathione S-transferases,GSTs)的cDNA序列,命名为RpGST1(GenBank登录号KP192850),并构建原核表达载体pET32-RpGST1,对RpGST1基因进行原核表达、SDS-PAGE和Western blotting检测。结果显示,禾谷缢管蚜RpGST1基因的编码区长651 bp,编码216个氨基酸,分子量约为24.06 kD,理论等电点为6.20;RpGST1基因在大肠杆菌中成功表达出一个分子量约为45 kD的融合蛋白,与预测的融合蛋白分子量大小一致。通过克隆RpGST1基因的cDNA序列并进行序列比对分析,表明构建了GST基因的原核表达载体并成功表达。