棉蚜2个乙酰胆碱酯酶cDNA片段的基因克隆与序列分析

采用RT－PCR技术,利用简并引物从棉蚜（Aphis gossypii Glover)中克隆出2个乙酰胆碱酯酶基因的cDNA片段,Ag,ace 1l和Ag.ace2.Ag.ace1基因的cDNA片段为282bp,编码94个氨基酸;Ag.ace2基因的cDNA片段为264bp,编码88个氨基酸。扩增获得的2个乙酰胆碱酯酶基因cDNA片段所编码的氨基酸序列均与其他昆虫的乙酰胆碱酯酶基因有很高的同源性。首次从一种昆虫中克隆出2个乙酰胆碱酯酶基因片段,为同一种昆虫中存在多个乙酰胆碱酯酶基因的假设提供了直接的分子生物学证据。     

小菜蛾酯酶基因的克隆

根据已知的酯酶基因的保守性氨基酸序列设计简并引物 ,通过逆转录 -聚合酶链反应 (RT PCR)扩增出小菜蛾PlutellaxylostellaL .酯酶基因片段 ,然后按照测序结果再设计 1对特异引物 ,利用PCR方法 ,筛选小菜蛾的cDNA文库。将RT PCR获得的 1条长度为 3 3 0bp的目的条带 ,亚克隆入T -载体 ,测序结果表明共得到了 1 0个不同的酯酶基因片段。利用特异引物对小菜蛾的cDNA文库进行初筛 ,显示文库中存在有小菜蛾的酯酶基因。     

程序化设计简并引物与克隆小菜蛾酯酶基因

利用遗传密码简并性 ,针对特定的氨基酸序列设计简并引物 ,是克隆蛋白质家族cDNA的常规方法。文章介绍了利用blastp ,blockmaker,CodeHop ,SwissProt,SpTrEMBL等网络工具及数据库设计昆虫抗性酯酶的简并引物。用这对引物从抗有机磷杀虫剂的小菜蛾Plutellaxylostella中克隆了 1段cDNA。经blastx检索genebank,发现此cDNA产物与其它昆虫抗性酯酶基因有高度的相似性。研究表明 ,程序化设计的简并引物可信性强 ,阳性率高 ,能迅速得到满意结果     

褐飞虱乙酰胆碱酯酶基因全长cDNA的克隆及序列分析

利用反转录聚合酶链式反应(RT_PCR)结合快速扩增cDNA末端(RACE)技术克隆了褐飞虱Nilaparvata lugens 乙酰胆碱酯酶基因cDNA。该cDNA全长2 467 bp,包含一个1 938 bp的开放阅读框(GenBank登录号AJ852420); 在推导出的646个氨基酸残基的前体蛋白中, N端的前30个氨基酸残基为信号肽,随后的616个氨基酸残基是成熟的乙酰胆碱酯酶序列,其预测的分子量为69 418 D。在一级结构中,形成催化活性中心的3个氨基酸残基(Ser242,Glu371和His485),以及在亚基内形成二硫键的6个半胱氨酸完全保守; 位于催化功能域的14个芳香族氨基酸中有10 个完全保守。该酶的氨基酸序列与黑尾叶蝉的同源性最高,达83%。对来自23种昆虫（包括褐飞虱）的30个乙酰胆碱酯酶的聚类分析显示,褐飞虱的乙酰胆碱酯酶与其中6个Ⅱ型乙酰胆碱酯酶（AChE2）同属一个支系; 此外,只存在于昆虫AChE2中的超变区及特异的氨基酸残基,也存在于褐飞虱的乙酰胆碱酯酶中。以上结果表明,所克隆的褐飞虱的乙酰胆碱酯酶基因是一个与黑腹果蝇的orthologous型基因同源的AChE2基因。     

小菜蛾羧酸酯酶基因的克隆及其序列分析

利用CODEHOP设计简并引物,通过反转录多聚酶链式反应(RTPCR)克隆小菜蛾Plutellaxylostella抗性种群中抗性相关的羧酸酯酶基因,随机挑取测序5个阳性克隆进行测序,测序结果经过blastx比较,发现所获得的大约420bp的基因片断均为羧酸酯酶基因,与双翅目昆虫蚊子的氨基酸序列同源性达85%以上 。     

甘薯茎线虫乙酰胆碱酯酶基因Dd-ace-2全长cDNA的克隆和序列分析

利用RT-PCR、RACE技术克隆了甘薯茎线虫(Ditylenchus destructor)乙酰胆碱酯酶基因(Dd-ace-2) cDNA (GenBank 登录号EF583058), 用DNAMAN5.0、MEGA3.0进行了序列分析。克隆的Dd-ace-2 基因cDNA全长2425 bp, 包含一个2205 bp的开放阅读框, 编码734个氨基酸。Dd-ace-2基因推导的氨基酸序列与南方根结线虫(Meloidogyne incognita)、秀丽小杆线虫(Caenorhabditis elegans)和动物寄生线虫胎生网尾线虫(Dictyocaulus viviparous)ace-2的氨基酸序列同源性分别达48.0%、42.7%和42.1%。在推导的734个氨基酸残基的前体蛋白中, 前面的701个氨基酸残基是成熟的乙酰胆碱酯酶序列, 其预测的分子量为79240.38 D。在一级结构中, 形成催化活性中心的3个氨基酸残基(Ser291, Glu442和His574)、胆碱结合位点Trp(177), 以及在亚基内形成二硫键的6个半胱氨酸完全保守; 在电鳐乙酰胆碱酯酶分子的催化功能域中存在14个保守的芳香族氨基酸残基, 其中10个在甘薯茎线虫乙酰胆碱酯酶中完全保守。与其它线虫和物种乙酰胆碱酯酶的聚类分析显示, 甘薯茎线虫的乙酰胆碱酯酶与其它线虫乙酰胆碱酯酶ACE-2同属一个支系。     

小菜蛾及菜蛾绒茧蜂乙酰胆碱酯酶敏感性的相关变化

用生物测定和生化检测的方法,对福州地区小菜蛾Plutella xylostella和菜蛾绒茧蜂Apanteles plutellae的抗药性及两种昆虫乙酰胆碱酯酶对杀虫剂的敏感性进行了田间监测。结果显示,从1998年9月至1999年4月,小菜蛾乙酰胆碱酯酶对6种有机磷和氨基甲酸酯杀虫剂敏感性逐渐恢复,寄生于同一虫源的菜蛾绒茧蜂乙酰胆碱酯酶敏感性的变化也呈明显的相关性,但菜蛾绒茧蜂乙酰胆碱酯酶的敏感性高于其寄主小菜蛾。脱离选择压力后,两种昆虫对杀虫剂的敏感性迅速恢复,乙酰胆碱酯酶的K_i值显著增高。对乙酰胆碱酯酶的K_m、V_max和K_i值测定结果表明,两种昆虫对有机磷和氨基甲酸酯杀虫剂的抗性与乙酰胆碱酯酶对杀虫剂的不敏感性有关。此外还研究了不同发育期小菜蛾乙酰胆碱酯酶活性及其K_i值的变化。探讨了在杀虫剂选择压力下,两种昆虫乙酰胆碱酯酶敏感性的环境适应性变化机制。     

桃蚜MpAChE基因RNAi表达载体构建及转化

通过害虫取食植物表达害虫发育关键基因dsRNA的转基因植株,分析能否通过抑制害虫特定基因的表达来防控害虫。本研究利用RT-PCR技术从桃蚜中克隆乙酰胆碱酯酶基因383 bp cDNA片段,命名为MpAChE。进一步利用该MpAChE基因片段构建植物RNAi表达载体RNAi-MpAChE,并通过浸花法转化野生型拟南芥,通过卡那霉素抗性筛选转化植株,PCR及Southern杂交进一步鉴定转基因植株。结果表明:克隆的cDNA片段与桃蚜中已克隆的乙酰胆碱酯酶(GenBank登录号AY147797)cDNA序列核苷酸一致性为99%。卡那霉素抗性初步筛选和PCR进一步鉴定,获得25株阳性转基因植株。从25株中随机选择的5株阳性植株,Southern杂交均为阳性。经接种桃蚜初步鉴定,转基因植株对蚜虫的抗性效果不显著。     

粉纹夜蛾类Cry1Ac受体氨肽酶N cDNA片段的克隆与分析

设计简并引物,采用RT-PCR方法对粉纹夜蛾Trichoplusia ni (Hübner)细胞系BTI-TN5B-4的氨肽酶N (aminopeptidase N, APN)基因cDNA片段进行了克隆和序列分析, 通过两对引物扩增出了两种氨肽酶N基因的cDNA片段, 大小分别为188 bp 和564 bp,分别命名为AS188（GenBank登录号: CD809324）和AS564（GenBank登录号: CD809326）。对这两个片段推导的氨基酸序列进行同源性分析, 结果表明两者与已报道的鳞翅目昆虫中肠的Cry1Ac 毒素受体氨肽酶N有较高的同源性。     

粉纹夜蛾类Cry1Ac受体氨肽酶NcDNA片段的克隆与分析

设计简并引物,采用RT-PCR方法对粉纹夜蛾Trichoplusia ni(Hubner)细胞系BTI-TN-5B1-4的氨肽酶N(aminopeptidase N,APN)基因cDNA片段进行了克隆和序列分析,通过两对引物扩增出了两种氨肽酶N基因的cDNA片段,大小分别为188 bp和564 bp,分别命名为AS188(GenBank登录号:CD809324)和AS564(GenBank登录号:CD809326).对这两个片段推导的氨基酸序列进行同源性分析,结果表明两者与已报道的鳞翅目昆虫中肠的Cry1Ac毒素受体氨肽酶N有较高的同源性.     

cDNA sequence analysis of ribosomal protein S13 gene in Plutella xylostella （Lepidoptera： Plutellidae）

Ribosomal protein S 13 gene has been cloned and analyzed in many organisms,but there are few documents relating to insects. In this communication, the full-length cDNA sequence of ribosomal protein S 13 gene in the diamondback moth, Plutella xylostella(Lepidoptera: Plutellidae), was determined by using PCR amplification technique. The features of the ribosomal protein S 13 gene sequence were analyzed and the deduced amino acids sequence was compared with those from other insects. The results of multi-alignment of the amino acid sequences between the diamondback moth and other insect species revealed that this gene sequence is highly conserved in insects. Based on maximum likelihood method, a phylogenetic tree was constructed from 10 different species using PHYLIP software. It showed that nematode is one separate lineage and the five insect speciesbe long to another lineage, whereas those species higher than insects form the third one. The pattern of this phylogenetic tree evidently represented the evolution of different species.     

小菜蛾化学感受蛋白基因PxylCSP1的克隆和表达

利用RT-PCR和RACE技术克隆到小菜蛾Plutella xylostella 化学感受蛋白（CSP）基因PxylCSP1（GenBank登录号: FJ361903）,其核苷酸序列全长405 bp,编码134个氨基酸残基,预测N-末端包含19个氨基酸组成的信号肽序列,估测其成熟蛋白分子量为13.56 kD,等电点为6.12。该基因编码氨基酸序列和其他鳞翅目昆虫CSP的氨基酸序列比对同源性较高（70%~80%）。RT-PCR结果表明PxylCSP1不仅存在于小菜蛾的触角中,还存在于头、足、腹和翅中。Real-time PCR结果表明PxylCSP1的表达水平因被测小菜蛾的性别、日龄、组织不同和交配与否而异。     

Bacillus thuringiensis insecticidal Cry1Aa toxin binds to a highly conserved region of aminopeptidase N in the host insect leading to its evolutionary success.

Bacillus thuringiensis insecticidal protein, Cry1Aa toxin, binds to a specific receptor in insect midguts and has insecticidal activity. Therefore, the structure of the receptor molecule is probably a key factor in determining the binding affinity of the toxin and insect susceptibility. The cDNA fragment (PX frg1) encoding the Cry1Aa toxin-binding region of an aminopeptidase N (APN) or an APN family protein from diamondback moth, Plutella xylostella midgut was cloned and sequenced. A comparison between the deduced amino acid sequence of PX frg1 and other insect APN sequences shows that Cry1Aa toxin binds to a highly conserved region of APN family protein. In this paper, we propose a model to explain the mechanism that causes B. thuringiensis evolutionary success and differing insect susceptibility to Cry1Aa toxin.     

Genomic organization of the para-sodium channel alpha-subunit genes from the pyrethroid-resistant and -susceptible strains of the diamondback moth

We examined the genomic organization of the para-sodium channel alpha-subunit gene of the diamondback moth, Plutella xylostella (L.). The nucleotide sequence contained 34 putative exons, which covered almost the entire coding region of the gene producing 1,889 amino acid residues. Deduced amino acid identity to the hscp locus of Heliothis virescens was 84%. Comparison of deduced amino acid sequences of the permethrin-resistant and -susceptible strains showed two substitutions other than kdr and super-kdr-like substitutions. They were Ala to Thr (A1060T) and Pro to Ser (P1836S) at the linker region of the domains II-III and the carboxyl terminus, respectively. Furthermore, we developed PCR amplification protocols for the rapid detection of both substitutions.     

Primary structure of acetylcholinesterase: implications for regulation and function

A cDNA encoding acetylcholinesterase (AChE) (EC 3.1.1.7) from Torpedo californica was isolated and from its nucleotide sequence the entire amino acid sequence of the processed protein and a portion of the leader peptide has been deduced. Approximately 70% of the tryptic peptides from the catalytic subunit of the 11 S form have been sequenced, and a comparison of the peptide sequences with the sequence inferred from the cDNA suggests that the cDNA sequence derives from mRNA for the 11 S form of the enzyme. The amino acid sequence is preceded by a hydrophobic leader peptide and contains an open reading frame encoding for 575 amino acids characteristic of a secreted globular protein. Eight cysteines, most of which are disulfide linked, are found along with four potential sites of N-linked glycosylation. The active-site serine is located at residue 200. Local homology is found with other serine hydrolases in the vicinity of the active site, but the enzyme shows striking global homology with the COOH-terminal portion of thyroglobulin. Further comparison of the amino acid sequences of the individual enzyme forms with other cDNA clones that have been isolated should resolve the molecular basis for polymorphism of the AChE species.     

Cloning and expression of male-specific protein (MSP) from the hemolymph of greater wax moth, Galleria mellonella L

Male-specific protein (MSP) is a soluble protein that accumulates in high amounts in the hemolymph and other organs of adult male wax moth. The MSP was purified from adult male wax moth by gel filtration and reversed phase column chromatography, and its amino acid sequence was determined. Because of blocked N-terminus, several internal amino acid sequences of MSP were obtained by the in-gel digestion method using trypsin. RT-PCR was conducted using degenerate primers designed from the internal amino acid sequences. 5'-RACE PCR was used to obtain the complete coding region and 5'-UTR sequence. The full length MSP cDNA sequence encodes a 239 amino acid polypeptide with an 18 amino acid signal peptide. The putative mature MSP has a molecular mass of 24,317 Da and an isoelectric point (pI) of 6.00, but shows a molecular mass of 27 kDa on SDS-PAGE. Sequence alignment showed a significant similarity between MSP and juvenile hormone binding proteins (JHBPs) of several lepidopteran species, including G. mellonella.     

Absence of protein polymorphism attributable to insecticide-insensitivity of acetylcholinesterase in the green rice leafhopper, Nephotettix cincticeps

The cDNA sequence of acetylcholinesterase (AChE) from the green rice leafhopper, Nephotettix cincticeps, was amplified, based on conserved peptide sequences of AChEs. A 2.3 kb contiguous sequence, containing an ORF encoding an AChE precursor with 677 amino acid residues was obtained. The deduced protein sequence showed the most similarity to that of AChE in the Colorado potato beetle, having common features in the primary AChE structure. cDNA sequences of individual leafhoppers from an insecticide susceptible strain and the resistant strain Nakagawara, whose methylcarbamate-insensitive AChEs show 10(2) or more I(50) ratio for propoxur, were compared. No fixed inter-strain difference was identified in the protein sequence, though amino acid substitution polymorphism was found at one position in the susceptible strain. Insecticide-insensitivity of leafhopper AChE does not result from changes in the protein primary structure that is encoded by the AChE gene sequence isolated in this study.     

Cloning and complete sequence characterization of two gypsy moth aminopeptidase-N cDNAs, including the receptor for Bacillus thuringiensis Cry1Ac toxin.

The complete cDNAs corresponding to two distinct gypsy moth (Lymantria dispar) larval gut aminopeptidases, APN1 and lambda APN2, were cloned and sequenced. The 3.4 kilobasepair cDNA of APN1 which encodes a 1017 amino acid prepro-protein corresponds to the previously-identified gypsy moth APN (APN-1) that specifically binds the Cry1Ac delta-endotoxin of Bacillus thuringiensis. Analysis of the primary structure of APN1 revealed a cluster of five potential N-linked glycosylation sites near the N-terminus and a C-terminal sequence characteristic of a putative glycosylphosphatidyl-inositol (GPI) anchor signal sequence. The cDNA of APN1 encodes the N-terminal peptide sequence and nine internal sequences obtained from the purified brush border membrane vesicle Cry1Ac receptor by protein sequencing. The lambda APN2 cDNA encodes a shorter protein with 51% similarity to APN1 that also appears to have a GPI anchor signal sequence. Expression of the APN1 cDNA in a baculovirus vector was confirmed by immunoblotting.