小菜蛾乙酰胆碱酯酶cDNA片段的克隆和序列分析

利用反转录-多聚酶链式反应(RT-PCR)的方法对小菜蛾Plutella xylostella的乙酰胆碱酯酶基因cDNA片段进行了克隆和序列分析。通过简并性上游引物和下游引物扩增出了小菜蛾乙酰胆碱酯酶基因281bp的cDNA片段。同源性分析表明, 该cDNA片段与其它昆虫乙酰胆碱酯酶基因序列具有较高的同源性。     

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。     

麦长管蚜羧酸酯酶基因cDNA片段克隆及吡虫啉胁迫对其表达的影响

羧酸酯酶在昆虫对杀虫剂的解毒代谢过程中发挥重要作用,本研究旨在分析吡虫啉胁迫对麦长管蚜羧酸酯酶基因表达的影响。采用同源克隆方法克隆麦长管蚜羧酸酯酶基因cDNA片段,实时荧光定量PCR技术检测羧酸酯酶基因在不同吡虫啉剂量下的表达量变化。扩增所得麦长管蚜羧酸酯酶基因cDNA片段大小为392 bp,命名为SaEST 3(GenBank登录号KY 441614),该片段编码130个氨基酸残基,分子量14 kD,等电点4.93。序列同源性比对及生物信息学分析表明SaEST3推导的氨基酸序列与豌豆长管蚜、麦双尾蚜、夹竹桃蚜、桃蚜的羧酸酯酶氨基酸序列相似性较高,分别为94%、85%、80%和80%。实时荧光定量PCR结果显示,不同吡虫啉处理剂量下,SaEST3 m RNA的相对表达量均上调。克隆得到的基因片段为麦长管蚜羧酸酯酶基因片段,吡虫啉对麦长管蚜羧酸酯酶基因SaEST3表达有一定的影响。     

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。     

甜菜夜蛾羧酸酯酶基因cDNA的克隆、表达及序列分析

羧酸酯酶是昆虫体内重要的解毒酶系之一,与昆虫抗药性产生相关。利用粉纹夜蛾Trichoplusia ni (Hübner)肠粘蛋白多克隆抗体免疫筛选甜菜夜蛾Spodoptera exigua (Hübner)中肠cDNA表达文库,得到编码羧酸酯酶的全长cDNA克隆。该cDNA克隆全长1 812 bp(GenBank登录号EF580101),开放阅读框长1 605 bp,编码535个氨基酸。该蛋白活性中心包括3个氨基酸残基（催化三联体）： Ser186, Glu319和His443, 3个N-联糖基化位点,具备羧酸酯酶的结构特征,属羧酸酯酶家族（EC: 3.1.1.-）。将该基因与pQE30载体重组,经IPTG诱导,spot-blot鉴定,蛋白获得了表达;以α-醋酸萘酯为底物,检测表达的羧酸酯酶活性为1.3 nmol/100 μL酶液。     

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。     

小麦Na+/H+反转运蛋白基因的克隆和特性

以水稻(Oryza sativa L.) Na+/H+反转运蛋白cDNA片段为探针,从小麦盐胁迫cDNA文库中筛选和克隆了2个小麦Na+/H+反转运蛋白基因,分别命名为TaNHX1 和 TaNHX2.序列分析表明TaNHX1为2 029 bp,包含一个完整的1 638 bp的ORF,编码546个氨基酸,其中含有DIFFIYLLPPI跨膜区.TaNHX2为1 693 bp,包含部分ORF及808 bp的3′-UTR.这2个基因与已知的水稻、拟南芥(Arabidopsis thialiana)和滨藜(Atriplex gmelini)中的同类基因NHX的相似性约为70%.RT-PCR分析表明小麦苗经400 mmol/L NaCl处理1 h后,TaNHX1的转录水平有所提高.     

粘质沙雷氏菌武汉株PLA1基因的克隆和序列

通过鸟枪法构建了粘质沙雷氏菌SerratiamarcescensCW W 90 3菌株的基因组文库。使用LB 卵黄平板 ,从中筛选出 1条含磷酯酶基因的 3 0 10bp的EcoRⅠ片段。通过测序及亚克隆分析 ,发现 1个编码磷酯酶的基因phlA ,长度为 96 3bp ,编码 1个由 32 0个氨基酸组成 ,分子量为 33ku的磷酯酶PHL。PHL的氨基酸序列与多种细菌产生的磷酯酶A1的氨基酸序列有很高的同源性。在 phlA下游发现 1个 75 6bp的ORF phlB ,编码 1条 2 5 1个氨基酸组成的蛋白质 ,分子量为 2 7ku ,将其命名为PHLS ,此基因的功能有待于进一步研究。     

拟南芥春化相关基因VRN2的克隆及其植物表达载体的构建

本文利用特异性引物,从拟南芥RNA中提取春化相关基因VRN2 cDNA序列,GenBank登录号AY063047。该基因序列大小为1 354 bp,编码区为1 323 bp,编码氨基酸441个。将克隆片段插入中间载体pBPFΩ7,经PstI酶切回收带有P35S启动子和nos终止子片段,连接pBI121载体,构建VRN2基因的植物表达载体。     

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.     

Two different genes encoding acetylcholinesterase existing in cotton aphid (Aphis gossypii).

Two acetylcholinesterase (AChE) genes, Ace1 and Ace2, have been cloned from cotton aphid, Aphis gossypii Glover, using the rapid amplification of cDNA ends (RACE) technique. To the best of our knowledge, this should be the first direct molecular evidence that multiple AChE genes exist in insects. The Ace1 gene was successfully amplified along its full length of 2371 bp. The open reading frame is 2031 bp long and encodes 676 amino acids (GenBank accession No. AF502082). The Ace2 gene was amplified as a mega-fragment of 2130 bp lacking part of 5'-end untranslated region (UTR). The open reading frame is 1992 bp long and ecodes a protein of 664 amino acids (GenBank accession No. AF502081). Both genes have the conserved amino acids and features shared by the AChE family, but share only 35% identity in amino acid sequence. The Ace1 gene is highly homologous to the AChE gene of Schizaphis graminum (AF321574) with 95% identity, and Ace2 to that of Myzus persicae (AF287291) with 92% identity. Phylogenetic analysis showed that the two cloned AChEs of A. gossypii are different in evolution. The phylogenetic tree generated by the PHYLIP program package inferred that AChE2 of A. gossypii is a more ancestral form of AChE. Homology modeling of structures using Torpedo californica (2ACE_) and Drosophila melanogaster (1Q09:A) native acetylcholinesterase structure as main template indicated that the two AChEs of Aphis gossypii might have different three-dimensional structures. Alternative splicing of Ace1 near the 5'-end resulting in two proteins differing by the presence or absence of a fragment of four amino acids is also reported.     

Mutations of acetylcholinesterase1 contribute to prothiofos-resistance in Plutella xylostella (L.)

Insensitive acetylcholinesterase (AChE) is involved in the resistance of organophosphorous and carbamate insecticides. We cloned a novel full-length AChE cDNA encoding ace1 gene from adult heads of the diamondback moth (DBM, Plutella xylostella). The ace1 gene encoding 679 amino acids has conserved motifs including catalytic triad, choline-binding site and acyl pocket. Northern blot analysis revealed that the ace1 gene was expressed much higher than the ace2 in all examined body parts. The biochemical properties of expressed AChEs showed substrate specificity for acetylthiocholine iodide and inhibitor specificity for BW284C51 and eserine. Three mutations of AChE1 (D229G, A298S, and G324A) were identified in the prothiofos-resistant strain, two of which (A298S and G324A) were expected to be involved in the prothiofos-resistance through three-dimensional modeling. In vitro functional expression of AChEs in Sf9 cells revealed that only resistant AChE1 is less inhibited with paraoxon, suggesting that resistant AChE1 is responsible for prothiofos-resistance.     

Molecular characterization of two acetylcholinesterase genes from the oriental tobacco budworm, Helicoverpa assulta (Guenée)

Acetylcholinesterase (AChE) has been known to be the target of organophosphorous and carbamate insecticides. Only a single AChE, however, existed in insects and was involved in insecticide resistance, recently another AChE is reported in mosquitoes and aphids. We have cloned cDNAs encoding two ace genes, designated as Ha-ace1 and Ha-ace2 by a combined degenerate PCR and RACE strategy from adult heads of the oriental tobacco budworm, Helicoverpa assulta. The Ha-ace1 and Ha-ace2 genes encode 664 and 647 amino acids, respectively and have conserved motifs including a catalytic triad, a choline-binding site and an acyl pocket. Both Ha-AChEs were determined to be secretory proteins based on the existence of a signal peptide. The Ha-ace1 gene, the first reported ace1 in lepidopterans, belongs to the ace1 subfamily whereas the Ha-ace2 gene showed high similarity to those in the ace2 subfamily. Phylogenetic analysis showed that the Ha-ace1 gene was completely diverged from the Ha-ace2, suggesting that the Ha-ace genes are duplicated. Quantitative real time-PCR revealed that expression level of the Ha-ace1 gene was much higher than that of the Ha-ace2 in all body parts examined. The biochemical properties of purified proteins by affinity chromatography showed substrate specificity for acetylthiocholine iodide, and inhibitor specificity for BW284C51 and eserine and their peptide sequences partially identified by a MALDI-TOF mass spectrometer demonstrated that two Ha-AChEs were expressed in vivo.     

叶绿体超氧化物歧化酶（ChlSOD）基因的构建及序列分析

构建叶绿体超氧化物歧化酶基因（ChlSOD),采用RT-PCR方法分离豌豆RUBP羧化酶小亚基导肽基因（TP）,定向克隆至pUC19测序,定向克隆烟草MnSOD成熟蛋白基因（SODm)至pUC19;采用平粘端连接法将二者在pUC19中构成嵌合基因ChlSOD,并对此基因进行序列分析,序列分析表明：TPcDNATP,12bp的Linker及615bp SODm。TP与ChlSOD基因的序列分析与国外报道序列完全吻合。     

斑地锦RT-qPCR内参基因的筛选

实时荧光定量PCR(RT-qPCR)的前提条件之一是具有合适的内参基因。为筛选斑地锦(Euphorbia maculata)合适的RT-qPCR内参基因,该文利用同源克隆法克隆斑地锦GAPDH、EF-1α、act、UBQ、TUB-α、eIF-4A、CYP等基因片段,RT-qPCR检测7个候选内参基因在斑地锦不同生长期根、茎、叶和果实中的表达情况,并用geNorm、NormFinder和BestKeeper等生物学软件对各候选基因表达稳定性进行评价。结果表明:(1)克隆的GAPDH、EF-1α、act、UBQ、TUB-α、eIF-4A、CYP基因片段为729、808、753、422、233、656、313 bp,分别编码242、269、250、140、77、218、103个氨基酸,与其他植物相应氨基酸序列的最高同源性均在85%以上。(2)综合3个分析软件分析内参基因表达稳定性得出,表达稳定性排名为UBQ>EF-1α>TUB-α>eIF-4A>GAPDH>CYP>act。因此,可以选取UBQ作为斑地锦RT-qPCR分析的内参基因,用于不同生长期基因组织特异性表达研究。     

Characterization and regulation of Schizosaccharomyces pombe gene encoding thioredoxin

A cDNA coding thioredoxin (TRX) was isolated from a cDNA library of Schizosaccharomyces pombe by colony hybridization. The 438 bp EcoRI fragment, which was detected by Southern hybridization, reveals an open reading frame which encodes a protein of 103 amino acids. The genomic DNA encoding TRX was also isolated from S. pombe chromosomal DNA using PCR. The cloned sequence contains 1795 bp and encodes a protein of 103 amino acids. However, the C-terminal region obtained from the cDNA clone is -Val-Arg-Leu-Asn-Arg-Ser-Leu, whereas the C-terminal region deduced from the genomic DNA appears to contain -Ala-Ser-Ile-Lys-Ala-Asn-Leu. This indicates that S. pombe cells contain two kinds of TRX genes which have dissimilar amino acid sequences only at the C-terminal regions. The heterologous TRX 1C produced from the cDNA clone could be used as a subunit of T7 DNA polymerase, while the TRX 1G from the genomic DNA could not. The upstream sequence and the region encoding the N-terminal 18 amino acids of the genomic DNA were fused into the promoterless beta-galactosidase gene of the shuttle vector YEp357 to generate the fusion plasmid pYKT24. Synthesis of beta-galactosidase from the fusion plasmid was found to be enhanced by hydrogen peroxide, menadione and aluminum chloride. It indicates that the expression of the cloned TRX gene is induced by oxidative stress.     

A novel acetylcholinesterase gene in mosquitoes codes for the insecticide target and is non-homologous to the ace gene in Drosophila

Acetylcholinesterase (AChE) is the target of two major insecticide families, organophosphates (OPs) and carbamates. AChE insensitivity is a frequent resistance mechanism in insects and responsible mutations in the ace gene were identified in two Diptera, Drosophila melanogaster and Musca domestica. However, for other insects, the ace gene cloned by homology with Drosophila does not code for the insensitive AChE in resistant individuals, indicating the existence of a second ace locus. We identified two AChE loci in the genome of Anopheles gambiae, one (ace-1) being a new locus and the other (ace-2) being homologous to the gene previously described in Drosophila. The gene ace-1 has no obvious homologue in the Drosophila genome and was found in 15 mosquito species investigated. In An. gambiae, ace-1 and ace-2 display 53% similarity at the amino acid level and an overall phylogeny indicates that they probably diverged before the differentiation of insects. Thus, both genes are likely to be present in the majority of insects and the absence of ace-1 in Drosophila is probably due to a secondary loss. In one mosquito (Culex pipiens), ace-1 was found to be tightly linked with insecticide resistance and probably encodes the AChE OP target. These results have important implications for the design of new insecticides, as the target AChE is thus encoded by distinct genes in different insect groups, even within the Diptera: ace-2 in at least the Drosophilidae and Muscidae and ace-1 in at least the Culicidae. Evolutionary scenarios leading to such a peculiar situation are discussed.     

蜻蜓凤梨花发育相关B类MADS-box基因克隆及表达分析

为探索MADS-box基因在凤梨花发育过程中的调控机制,通过设计简并引物,利用RACE技术,从蜻蜓凤梨花蕾中分离得到2个花发育相关B类MADS-box基因,分别命名为AfAP3和AfPI;AfAP3cDNA全长957bp,编码区编码226个氨基酸;AfPI cDNA全长808bp,编码区编码198个氨基酸,二者均具有典型的植物MADS-box蛋白结构.RT-PCR分析结果表明,AfAP3和AfPI基因主要在花器官中表达,在根系中也有微量表达;乙烯诱导后7d,AfPI基因在茎尖处开始有表达,表明此时蜻蜓凤梨花芽分化可能已经完成,AfAP3基因表达晚于AfPI.     

内蒙古白绒山羊IGF-IR基因cDNA克隆及组织表达特异性分析

旨在克隆内蒙古白绒山羊IGF-IR基因并分析其基本表达模式.采用RT-PCR克隆基因,将得到的IGF-IR基因cDNA片段的核苷酸序列及其编码的氨基酸序列进行生物信息学分析.半定量RT-PCR进行组织特异性表达检测.获得了内蒙古白绒山羊IGF-IR基因3’端编码区2118 bp的cDNA序列(JN200823),编码705个氨基酸残基.核苷酸序列与牛的IGF-IR( XM606794.3)基因同源性为98％,相应的氨基酸序列同源性为99％.SMART分析表明,推导出的编码蛋白具有跨膜域,酪氨酸激酶催化域.半定量RT-PCR检测表明,IGF-IR基因在绒山羊脑、胰腺、肝、肾组织中均有表达.