Characterization of the chemosensory protein EforCSP3 and its potential involvement in host location by Encarsia formosa

Chemosensory proteins(CSPs) perform several functions in insects.This study performed the gene expression,ligand-binding,and molecular docking assays on the EforCSP3 identified in the parasitoid wasp Encarsia formosa,to determine whether EforCSP3 functions in olfaction,especially in host location and host preference.The results showed that EforCSP3 was highly expressed in the female head,and its relative expression was much higher in adults than in other developmental stages.The fluorescence binding assays suggested that the EforCSP3 exhibited high binding affinities to a wide range of host-related volatiles,among which dibutyl phthalate,1-octene,β-elemene,and tridecane had the strongest binding affinity with EforCSP3,besides α-humulene and β-myrcene,and should be assessed as potential attractants.Protein structure modeling and molecular docking predicted the amino acid residues of EforCSP3possibly involved in volatile binding.α-Humulene and β-myrcene attracted E.formosa in a previous study and exhibited strong binding affinities with EforCSP3 in the current study.In conclusion,EforCSP3 may be involved in semiochemical reception by E.formosa.

Structure,Binding Characteristics,and 3D Model Prediction of a Newly Identified Odorant-Binding Protein from the Cotton Bollworm,Helicoverpa armigera (Hübner)

The full-length sequence of the odorant binding protein 5 gene,HarmOBP5,was obtained from an antennae cDNA library of cotton bollworm,Helicoverpa armigera （Hübner）.The cDNA contains a 444 bp open reading frame,encoding a protein with 147 amino acids,namely HarmOBP5.HarmOBP5 was expressed in Escherichia coli and the recombinant protein was purified by affinity chromatography.SDS-PAGE and Western blot analysis demonstrated that the purified protein can be used for further investigation of its binding characteristics.Competitive binding assays with 113 odorant chemicals indicated that HarmOBP5 has strong affinity to some special plant volatiles,including （E）-β-farnesene,ethyl butyrate,ethyl heptanoate,and acetic acid 2-methylbutyl ester.Based on three-dimensional （3D） model of AaegOBP1 from Aedes aegypti,a 3D model of HarmOBP5 was predicted.The model revealed that some key binding residues in HarmOBP5 may play important roles in odorant perception of H.armigera.This study provides clues for better understanding physiological functions of OBPs in H.armigera and other insects.

Molecular and in vitro biochemical assessment of chemosensory protein 10 from brown planthopper Nilaparvata lugens at acidic pH

Chemosensory proteins(CSPs)are important molecular components of the insect olfactory system,which are involved in capturing,binding,and transporting hydrophobic odour molecules across the sensillum in sensillar lymph in regulating insect behavior.This protein family(CSPs)is also involved in many other systems that are not linked to olfactory receptors in olfactory sensilla.The brown planthopper(BPH)is a monophagous pest of rice that causes damage by sucking phloem sap and transmitting a number of diseases caused by viruses.In this study,fluorescence competitive binding assay and fluorescence quenching assay at acidic p H were performed as well as homology modelling to describe the binding affinity of Nlug CSP10.Fluorescence competitive binding assay(FCBA)demonstrated that Nlug CSP10 bound strongly to nonadecane,farnesene,and 2-tridecanone at acidic p H.The results of FCBA indicated that Nlug CSP10 bound different ligands at the physiological p H(5.0)of the bulk sensillum lymph.Fluorescence quenching assay demonstrated that Nlug CSP10 generated a stable complex with 2-tridecanone,while two ligands nonadecane and farnesene collided due to molecular collisions.The interaction of selected ligands with the modelled structure of Nlug CSP10 was also analyzed,which found the key amino acids(Gln23,Gln24,Gln25,Asn27,Met33,Ser34,Ile35,Tyr36,Asn42,Met43,Val45,Asn46,Asn93,Arg96,Ala97,Lys99,and Ala100)in Nlug CSP10 that were involved in binding of volatile compounds.The present study contributes to the binding profile of Nlug CSP10 that promotes the development of behaviorally active ligands based on BPH olfactory system.

Functional Characteristics of a Novel Chemosensory Protein in the Cotton Bollworm Helicoverpa armigera (Hübner)

A chemosensory protein named HarmCSP5 in cotton bollworm Helicoverpa armigera （Hvbner） was obtained from antennal eDNA libraries and expressed in Escherichia coll. The real time quantitative PCR （RT-qPCR） results indicated that HarmCSP5 gene was mainly expressed in male and female antennae but also expressed in female legs and wings. Competitive binding assays were performed to test the binding affinity of recombinant HarmCSP5 to 60 odor molecules including some cotton volatiles. The resules showed that HarmCSP5 showed strong binding abilities to 4-ehtylbenzaldehyde and 3,4-dimethlbenz aldehyde, whereas methyl phenylacetate, 2-decanone, 1-pentanol, carvenol, isobomeol, nerolidol, 2- nonanone and ethyl heptanoate have relatively weak binding affinity. Moreover, the predicted 3D model of HarmCSP5 consists of six α-helices located among residues 33-38 （αl）, 40-48 （α2）, 62-72 （α3）, 80-96 （α4）, 98-108 （α5）, and 116-119 （α6）, two pairs of disulfide bridges Cys49-Cys55, Cys75-Cys78. The two amino acid residues, Ile94 and Trpl01, may play crucial roles in HarmCSP5 binding with ligands and need further study for confirmation.

In vitro examining the existing prognoses how TBP binds to TATA with SNP associated with human diseases

We in vitro examined the existing prognoses of the dissociation constant, KD, between ТАТА- Binding Protein (TBP) and ТАТА box with single nucleotide polymorphism (SNP) associated with human diseases. Five SNPs of the genes for cytochrome P450 2A6 (associated with lung cancer), β-globin (associated with β-thalassemia), mannose binding lectin (associated with variable immunodeficiency), superoxide dismutase 1 (associated with amyotrophic lateral sclerosis) and triosephosphate isomerase (associated with anemia) fell within the range of –ln(KD;M/KD;WT) between –1.5 and –1 (here KD;WT and KD;M denote the normal ТАТА box and with SNP). The mea-surements using EMSA demonstrated that: 1) all the predictions stating that the affinity between ТВР and ТАТА boxes with SNPs would be reduced were correct;2) the departures of three predictions from the measurements fell within the confidence interval;3) all the predictions consistently underestimated actual mutational damage caused to ТАТА boxes with SNPs (a < 0.05;binomial law) and two of these predictions did so significantly (a < 0.05, Student’s t-test). This consistent underestimation seems to be associated with the damage to the context that modulates ТВP/ТАТА affinity, for example, the contact between the nucleosomal histone H3-Н4 dimer and the core promoter immediately near ТАТА boxes.

Development of a universal phosphorylated peptide-binding protein for simultaneous assay of kinases

This study describes the development of a universal phosphorylated peptide-binding protein designed to simultaneously detect serine, threonine and tyrosine kinases. The Escherichia

Effects of insulin-like growth factor binding protein 7 on cell cycle and proliferation of gastric cancer in vitro and in vivo

Objective To investigate the effects of insulin-like growth factor binding protein 7(IGFBP7)on the proliferation,cell cycle of gastric cancer cell and the expression of cynlin D1,cyclin-dependent kinase(CDK)4,and to observe the effects of IGFBP7 on the growth of gastric tumor xenografts in nude mice.Methods The MKN-28cell line was interfered by small interfere ribonucleic acid(siRNA)(interfered group),and blank control group,

不同浓度过氧化氢对Peroxiredoxin Ⅱ与血红蛋白结合的影响

为探究不同浓度的过氧化氢对抗氧化蛋白(Peroxiredoxin Ⅱ)与血红蛋白结合的影响,利用蛋白质体外结合实验,将带有GST标签的鼠重组Prx Ⅱ蛋白和鼠血红蛋白进行结合,以及利用蛋白质免疫印迹法检测目的蛋白,检验在不同浓度的过氧化氢存在时,Peroxiredoxin Ⅱ(Prx Ⅱ)与血红蛋白的结合情况。结果表明,随着过氧化氢浓度的增加,Prx Ⅱ与血红蛋白的结合程度受到抑制,同时发现血红蛋白只与Prx Ⅱ的二聚体形式结合。为过氧化氢存在时Prx Ⅱ保护血红蛋白提供了基础理论。

Identification and expression profiles analysis of odorant-binding proteins in soybean aphid,Aphis glycines(Hemiptera:Aphididae)

The soybean aphid,Aphis glycines,is an extreme specialist and an important invasive pest that relies on olfaction for behaviors such as feeding,mating,and foraging.Odorant-binding proteins(OBPs)play a vital role in olfaction by binding to volatile compounds and by regulating insect sensing of the environment.In this work we used rapid amplification of complementary DNA ends technology to identify and characterize 10 genes encoding A.glycines OBPs(AglyOBPs)belonging to 3 subfamilies,including 4 classic OBPs,5 Plus-C OBPs,and one Minus-C OBP.Quantitative real-time polymerase chain reaction demonstrated variable specific expression patterns for the 10 genes based on developmental stage and aphid tssue sampled.Expression levels of 7 AglyOBPs(2,3,4,5,7,9,and 10)were highest in the 4th instar,indicating that the 4th nymphal instar is an important developmental period during which soybean aphids regulate feeding and search for host plants.Tissue-specific expression results demonstrated that AglyOBP2,7,and 9 exhibited significantly higher expression levels in antennae.Meanwhile,ligand-binding analysis of5 OBPs demonstrated binding of AglyOBP2 and AglyOBP3 to a broad spectrum of volatiles released by green leaf plants,with bias toward 6-to 8-carbon chain volatiles and strong binding of AglyOBP7 to trans-B-farnesene.Taken together,our findings build a foundation of knowledge for use in the study of molecular olfaction mechanisms and prov ide insights to guide future soybean aphid research.

Effects of point mutation C→T at - 64 of human δ globin gene promoter on DNA binding proteins

By electrophoretic mobility shift assay (EMSA), the effect of point mutation C→T at - 64 of human δ-globin gene on its binding proteins has been studied. Two segments of 36 bp from - 83- - 48 bp of the 6 globin gene promoter, named WOG and MOG, were synthesized. WOG includes wild type CAAT-like box (CCAAC), while MOG includes the mutant CAAT-like box (CCAAT, -64 C→T). Results indicate that: ( i ) in erythroid cell lines MEL, K562 and Hemin induced K562, the affinity of MOG with CCAAT binding protein (CBF) and GATA-1

Molecular and functional characterization of three odorant-binding proteins from the wheat blossom midge,Sitodiplosis mosellana

Sitodiplosis mosellana,a periodic but devastating wheat pest,relies on wheat spike volatiles as a cue in sclecing hosts for oviposition.Insect odorant-binding proteins(OBPs)are thought to play essential roles in filtering,binding and transporting hydropho-bic odorant molecules to specific receptors.To date,the molecular mechanisms underlying S.mosellana olfaction are poorly understood.Here,three S.mosellana antenna-specific OBP genes,SmosOBPII,16 and 21,were cloned and bacterially expressed.Binding properties of the recombinant proteins to 28 volatiles emitted from wheat spikes were in-vestigated using fluorescence competitive binding assays.Sequence analysis suggested that these SmosOBPs belong to the Classic OBP subfamily.Ligand-binding analysis showed that all three SmosOBPs preferentially bound alcohol,ester and ketone com-pounds,and SmosOBP11 and 16 also selectively bound terpenoid compounds.In par-ticular,the three SmosOBPs had high binding affinities(Ki<20μmol/L)to 3-hexanol and cis-3-hexenylacetate that elicited strong electroantennogram(EAG)response fromfemale antennae.In addition,SmosOBP11 displayed significantly higher binding(Ki<8μmo/L)than SmosOBP16 and 21 to l-octen-3-ol,D-panthenol,a-pinene and heptyl acetate which elicited significant EAG response,suggesting that SmosOBP11 plays a ma-jor role in recognition and transportation of these volatiles.These findings have provided important insight into the molecular mechanism by which S.mosellana specifically rec-ognizes plant volatiles for host selection,and have facilitated identification of effective volatile attractants that are potentially useful for pest monitoring and trapping.

Comparison of Different MARs(Matrix Attachment Regions) Effect on Transgene Expression

Three MARs(matrix attachment regions)fragments were cloned from tobacco(Nicotiana tabacum)(MAR1), yeast(Saccharomyces cerevisiae)(MAR3)and kidney bean(Phaseolus vulgaris)(MAR5)which ranged 984, 822 and 782bp, respectively. Sequence analysis showed that all thefragments had fairly high A/T content (73, 62 and 75%, respectively),harbored differentnumber and different type of some characteristic motifs of MARs, such as A-box and T-box,etc. The results of in vitro binding assay showed that the three MARs fragments derivedfrom different organisms could bind specifically to the matrix extracted from the tobacconuclei with different strength, which also demonstrated that these MARs fragments arefunctionally conserved during evolution. By using these MARs fragments to flank the β-glucuronidase (GUS) reporter gene and bialaphos resistance(bar) selectable marker gene,and then introducing the resulting plant expression vectors containing MARs-uidA-bar-MARs into tobacco through Agrobacterium mediated procedures, the effects of MARs sequenceson the expression of transgenes in tobacco were investigated and compared. The GUSactivity in individual transformants showed that, comparing to the controls withoutadditional MARs, the overall transgene expression level in transformants with MARs hadbeen greatly increased while the variations in transgene expression among transformantswere decreased in different degrees. In accordance with the results of sequence analysisand in vitro binding assay in which MAR1 fragment showed the strongest binding strength,this MARs fragment also showed the greatest effect in increasing transgene overallexpression level.

Mechanisms mediating cholinergic antral circular smooth muscle contraction in rats

AIM: To investigate the pathway (s) mediating rat antral circular smooth muscle contractile responses to the cholinomimetic agent, bebhanechol and the subtypes of muscarinic receptors mediating the cholinergic contraction. METHODS: Circular smooth muscle strips from the antrum of Sprague-Dawley rats were mounted in muscle baths in Krebs buffer. Isometric tension was recorded. Cumulative concentration-response curves were obtained for (+)-cisdioxolane (cD), a nonspecific muscarinic agonist, at 10^-8-10^-4mol/L, in the presence of tetrodotoxin (TTX, 10^-7mol/L). Results were normalized to cross sectional area. A repeat concentration-response curve was obtained after incubation of the muscle for 90min wibh antagonists for M1(pirenzepine), M2(methoctramine) and M3(darifenacin) muscarinic receptor subtypes. The sensitivity to PTX was tested by the ip injection of 100mg/kg of PTX 5d before the experiment. The antral circular smooth muscles were removed from PTX-treated and non-treated rats as strips and dispersed smooth muscle cells to identify whether PTX-linked pathway mediated the contractility to bethanechol. RESULTS: A dose-dependent contractile response observed with bethanechol, was not affected by TTX. The pretreatment of rats with pertussis toxin decreased the contraction induced by bethanechol. Lack of calcium as well as the presence of the L-type calcium channel blocker, nifedipine, also inhibited the cholinergic contraction, with a reduction in responsefrom 2.5±0.4g/mm^2 to 1.2±0.4g/mm^2(P<0.05), The doseresponse curves were shifted to the right by muscarinic antagonists in the following order of affinity: darifenacin (M3)>methocramine (M2)>pirenzepine (M1). CONCLUSION: The muscarinic receptors-dependent contraction of rat antral circular smooth muscles was linked to the signal transduction pabhway(s) involving pertussis-toxin sensitive GTP-binding proteins and to extracellular calcium via L-type voltage gated calcium channels. The presence of the residual contractile response after the treatment with nifedipine, suggests that an additional pathway could mediate the cholinergic contraction. The involvement of more than one muscarinic receptor (functionally predominant type 3 over type 2) also suggests more than one pathway mediating the cholinergic contraction in rat antrum.

体外靶向TP53BP2基因shRNA慢病毒载体的构建及功能鉴定

目的 本研究旨在构建靶向肿瘤蛋白p53结合蛋白2(TP53BP2)基因的短发夹RNA(shRNA)慢病毒载体,以抑制肝癌细胞TP53BP2的表达。方法 设计了2对针对TP53BP2基因的RNA干扰序列,并合成相应的shRNA序列。shRNA退火形成双链oligo序列后,应用基因重组技术构建重组质粒,经菌落PCR和测序鉴定,将重组正确的质粒进行慢病毒包装和滴度测定,并采用Western Blot、qRT-PCR和激光共聚焦技术观察慢病毒Lenti-shTP53BP2对HepG2细胞TP53BP2基因的干扰效果。结果 测序比对结果显示,各重组慢病毒载体与设计参考序列一致,提示各重组慢病毒体构建成功;重组慢病毒载体经慢病毒包装后,显示pHS-ASR-LW429、pHS-ASR-LW512和pHS-ASR-LW513的滴度分别为9.7×108TU/mL、6.1×108TU/mL和6.4×108TU/mL;用慢病毒Lenti-shTP53BP2(pHS-ASR-LW512和pHS-ASR-LW513)感染HepG2细胞后,与对照慢病毒(pHS-ASR-LW429)比,经Western Blot、qRT-PCR和激光共聚焦结果显示两个Lenti-shTP53BP2均能显著下调HepG2细胞TP53BP2基因水平和蛋白表达量。结论 本研究成功构建了靶向TP53BP2基因shRNA慢病毒载体,其能有效下调HepG2细胞TP53BP2的表达,为进一步研究TP53BP2在肝癌发生发展过程中的机制研究奠定了基础。

Isolation of a strong matrix attachment region (MAR) and identification of its function in vitro and in vivo

Inclusion of MARs in transgene cassettes enhances their expression and reduces position-effect variations in the transgenic host. Four new MARs (TM2, TM3, AM1 and AM2) were isolated from tobacco and Arabidopsis by PCR method. The nuclei isolated from suspension-cultured cells of rice were used to prepare nuclear matrix. With a characterized MAR (TM1) as a positive control, the Matrix-MAR interactions were tested by an in vitro binding assay to identify the DNA sequences as MARs and their binding strength to nuclear matrix in vitro was compared. The results showed that TM2 and TM3 had stronger binding strength than TM1. To determine the functions of the four new MARs in vivo, binary vectors pBI121 carrying a uidA GUS reporter gene were modified with direct repeat MARs inserted on both sides of the reporter gene cassette and were transferred into tobaccos via Agrobacterium-mediated transformation procedure. Quantitative GUS assays of the transgenic tobaccos showed that when flanking a GUS reporter gene

棉铃虫气味结合蛋白HarmOBP16的组织表达谱及配基结合特性分析

【目的】揭示棉铃虫Helicoverpa armigera气味结合蛋白(odorant binding protein,OBP)基因Harm OBP16的组织表达谱及其重组蛋白与气味化合物的结合特性。【方法】基于棉铃虫触角转录组数据,利用PCR技术从棉铃虫成虫触角中PCR克隆气味结合蛋白基因,并进行生物信息学和系统进化分析;采用qPCR对其进行组织[头部(去除触角和喙)、胸部、腹部、足、翅、触角和喙]表达谱分析;进一步采用原核表达系统表达和纯化重组蛋白;最后采用荧光竞争结合实验测定该重组蛋白与85种候选气味物质的结合能力。【结果】从棉铃虫成虫触角中克隆得到一个Atypical OBP家族基因Harm OBP16(Gen Bank登录号:JQ753074),其开放阅读框长441 bp,编码146个氨基酸,推断的编码蛋白等电点为6.87,具有10个保守的半胱氨酸。组织表达谱结果表明,Harm OBP16在雌成虫翅中高表达。纯化后的重组蛋白Harm OBP16对植物花的挥发物香叶基丙酮(Ki=14.2μmol/L)、β-紫罗兰酮(Ki=15.2μmol/L)、辛醛(Ki=15.3μmol/L)、芳樟醇(Ki=16.8μmol/L)、(R)-(+)-柠檬烯(Ki=14.9μmol/L)和β-蒎烯(Ki=17.3μmol/L)有较强的结合能力。【结论】Harm OBP16可能在棉铃虫识别寄主植物的过程中发挥一定的作用,可为基于气味物质的棉铃虫引诱剂或驱避剂的研发提供潜在的分子靶标。

紫云英非特异性转脂蛋白AsIB259的体外酶活性及在共生固氮中的功能

将紫云英AsIB259基因编码的一个预测的非特异性转脂蛋白AsIB259（nsLTP,non-specific lipid transfer protein）在大肠杆菌表达菌株Rosetta 2（DE3）中进行了诱导表达,体外测定AsIB259蛋白的脂质结合动力学特征,检测它对不同碳链长度脂肪酸及其衍生物的结合能力,进一步考查AsIB259超表达对紫云英毛根结瘤情况的影响。结果表明：AsIB259具有转脂蛋白的典型特征,与16～22碳链长度脂肪酸的结合活性较强,对茉莉酸也表现出一定的结合活性;AsIB259超表达导致根瘤数量增加1.5倍且固氮酶活性降低,说明该基因在根瘤形成和固氮过程中发挥重要作用。

CRH通过PKA途径调节大鼠下丘脑CRH表达的研究

目的 探讨促肾上腺皮质激素释放激素 (corticotropin releasinghormone ,CRH)刺激下丘脑神经元表达CRH的信号调控机制。方法 通过大鼠下丘脑脑片实验模型 ,运用免疫组织化学、Westernblot技术 ,观察CRH激活下丘脑促肾上腺皮质激素释放激素Ⅰ型受体 (corticotropin releasinghormonetype 1receptor ,CRH1R)后蛋白激酶A(proteinkinaseA ,PKA)信号通路的活性变化 ,及其与CRH表达的关系。结果 CRH可引起下丘脑脑片磷酸化PKA、磷酸化CREB及CRH含量明显增加 ,而CP 15 45 2 6、H89可显著抑制磷酸化PKA、磷酸化CREB及CRH含量的增加。结论 在严重创伤应激反应中 ,CRH通过PKA途径实现对下丘脑神经元CRH的表达的超短正反馈调节。

乙型肝炎病毒e抗原结合蛋白2原核表达载体的构建及诱导表达

目的体外构建乙型肝炎病毒e抗原结合蛋白2原核表达载体并观察其表达情况。方法以含有HBEBP2全长序列的质粒为模板,通过PCR扩增获得HBEBP2基因片段,将HBEBP2连接到pGEM-T载体,在测序证实正确后将其插入至原核表达载体pET-32a(+)中,转化BL21大肠埃希菌,经IPTG诱导,并通过SDS-PAGE和Western blot分析鉴定融合蛋白的表达。结果扩增获得的HBEBP2基因片断被成功构建到大肠埃希菌原核表达载体中。经IPTG诱导,得到了融合蛋白的表达,经Western blot分析证实其分子量为34kD,具有抗原性。结论体外成功表达HBEBP2蛋白,为进一步研究HBEBP2蛋白的免疫原性和生物学特性奠定了基础。

气相色谱法测定甘草酸α体、β体及其苷元的人血浆蛋白结合率

目的研究甘草酸立体异构体及其苷元的血浆蛋白结合率。方法用气相色谱法测定人血浆中甘草酸立体异构体(α体和β体)及其苷元的浓度,并计算蛋白结合率。结果当血浆浓度大于50%时,α体的血浆蛋白结合率大于β体;血浆浓度小于50%时,α体的血浆蛋白结合率小于β体。而苷元的血浆蛋白结合率始终是α体小于β体。结论甘草酸立体异构体及其苷元在不同浓度血浆中的蛋白结合率存在差异。