3种转基因成分检测蛋白芯片的研制

为研制一种可同时检测3种转基因成分BTCry1Ac蛋白、植酸酶蛋白、BTCry1Ah蛋白的蛋白芯片。本文将3种转基因成分蛋白质的单克隆抗体点于化学修饰的基片上,对芯片的表面修饰材料、点样缓冲液成分、封闭液种类、标记二抗使用浓度、反应时间进行优化,并对芯片检测的特异性、灵敏度进行测定。结果表明:研制芯片的表面为环氧修饰,使用含0.1%BSA和30%甘油的点样缓冲液和含1%BSA的封闭液,每次反应时间为30min时,有最佳反应结果;其检测灵敏度为:BTCry1Ac蛋白35ng/mL、植酸酶蛋白20ng/mL、BTCry1Ah蛋白30ng/mL,信号稳定;本研究建立的抗体蛋白芯片检测法可同时检测3种转基因蛋白,并具有较高的灵敏性、特异性和可靠性。     

Mature plant resistance of potato against some virus diseases. III. Mature plant resistance against potato virus YN,indicated by decrease in ribosome-content in ageing potato plants under field conditions

The concurrence of a decrease of the concentration of ribosomes and glycoproteins in leaves of potato plants under field conditions and mature plant resistance against potato virus Y^N was studied. On four of five dates with intervals of one or two weeks plants of randomly chosen plots in the field were inoculated. During plant growth sixth, tenth and fifteenth leaves of main stems were collected at regular intervals. With the aid of cellulose columns and polyethylene glycol-containing solvents the ribosome- en glycoprotein-contents (and in one experiment also the RNA-content) of these leaves were determined. Tubers were harvested from plants, inoculated fourteen days before. Lack of typical virus symptoms on plants grown from these tubers gave information on the presence of mature plant resistance.It was concluded that in plants which had developed about 20 leaves, there was a high degree of mature plant resistance when ribosome- and glycoprotein-contents in the fifteenth leaf was less than 2 and 4 OD₂₆₀/ml/g fresh weight respectively. When higher absorbance values are measured, mature plant resistance may or may not occur depending on environmental conditions. The ribosome- and glycoprotein-contents have a remote relation to the mature plant resistance. It is suggested that ribosome- and glycoprotein-contents of the youngest fully expanded leaves give the best indication of mature plant resistance.Samenvatting In veldproeven werde het verband tussen de concentraties van ribosomen en glycoproteinen in bladeren van aardappelplanten en de ouderdomsresistentie tegen aardappel Y^N-virus (PVY^N) bestudeerd. Op vier of vijf data met tussenpozen van een of twee weken werden planten in het veld met PVY^N geïnoculeerd. Gedurende de groeiperiode van de planten werden zesde, tiende en vijftiende bladeren op vastgestelde tijden verzameld. Chromatografische analyse met behulp van cellulosekolommen en solvents die polyethyleenglycol bevatten, leidde tot de bepalingen van de ribosoom- en glycoproteïnegehalten (en in één proef ook het RNA-gehalte) in de verzamelde bladeren. De oogst van de knollen vond plaats 14 dagen na inoculatie. Het niet tot ontwikkeling komen van de typische virussymptomen bij planten, die uit deze knollen waren gegroeid, was een aanwijzing voor het optreden van de ouderdomsresistentie.Geconcludeerd wordt dat waarschijnlijk enige mate van ouderdomsresistentie tegen PVY^N in een pootaardappelgewas aanwezig is, als het ribosoom- en glycoproteïnegehalte in het vijftiende blad van hoofdstengels met ongeveer twintig bladeren, leidt tot corresponderende extinctiewaarden bij 260 nm per ml en per gram vers gewicht van respectievelijk minder dan 2 en 4. Wanneer hogere extinctiewaarden worden gevonden kan ouderdomsresistentie wel of niet in belangrijke mate voorkomen, afhankelijk van de omstandigheden waaronder het gewas is gegroeid. Gesuggereerd wordt de jongste volledig ontwikkelde bladeren te gebruiken voor het aantonen van een gelijktijdig optreden van de afname in de ribosoom- en glycoproteïnegehalten en een praktisch waarneembare mate van ouderdomsresistentie.     

双叉犀金龟表皮蛋白Td14144表达纯化及性质研究

表皮蛋白是昆虫表皮的重要组成成份，在昆虫的生长发育过程中起着重要作用，有可能成为农业害虫的防治靶标。双叉犀金龟Trypoxylus dichotomus是铁皮石斛的一种重要害虫，且对其表皮蛋白的研究较少。本文通过研究双叉犀金龟表皮蛋白Td14144的表达纯化及与几丁质结合的相关性质明确Td14144功能的重要性。根据双叉犀金龟转录组测序数据，利用RT-PCR获得表皮蛋白Td14144基因（GenBank登录号：MZ463195），并对其进行生物信息学分析。序列分析表明表皮蛋白Td14144属于CPR家族中RR-2亚族，含有R&R保守结构域；系统进化分析结果表明，Td14144与光肩星天牛Anoplophora glabripennis的AgCP的亲缘关系最近。随后将基因片段与pET-28a载体同源重组构建表达载体pET28a-14144，在大肠杆菌Escherichia coli BL21中原核表达，并使用金属螯合层析进行分离纯化，SDS-PAGE及Western blot验证重组蛋白的表达纯化，成功获得95%以上纯度的Td14144蛋白。利用几丁质结合试验评估Td14144与不同类型几丁质结合的能力，发现Td14144可以与壳聚糖、α-几丁质、β-几丁质和胶体几丁质结合，其中对壳聚糖和α-几丁质的结合能力最强。     

Insecticidal activity of scorpion toxin (ButaIT) and snowdrop lectin (GNA) containing fusion proteins towards pest species of different orders

BACKGROUND: The toxicity of a fusion protein, ButalT/GNA, comprising a venom toxin (ButaIT) derived from the red scorpion, Mesobuthus tamulus (F.), and Galanthus nivalis agglutinin (GNA), was evaluated under laboratory conditions against several pest insects. Insecticidal activity was compared with SFI1/GNA, a fusion comprising a venom toxin (SFI1) derived from the European spider Segestria florentina (Rossi) and GNA, which has been previously demonstrated to be effective against lepidopteran and hemipteran pests, and to GNA itself. RESULTS: Injection assays demonstrated that both fusion proteins were toxic to lepidopteran larvae, dipteran adults, coleopteran adults and larvae and dictyopteran nymphs. ButalT/GNA was more toxic than SFI1/GNA in all cases. GNA itself made a minor contribution to toxicity. Oral toxicity of ButalT/GNA towards lepidopteran pests was confirmed against neonate Spodoptera littoralis (Boisd.), where incorporation at 2% dietary protein resulted in 50% mortality and > 85% reduction in growth compared with controls. ButaIT/GNA was orally toxic to Musca domestica L. adults, causing 75% mortality at 1 mg mL^?1 in aqueous diets and, at 2 mg g^?1 it was orally toxic to Tribolium castaneum (Herbst.), causing 60% mortality and a 90% reduction in growth. CONCLUSIONS: Toxicity of the ButaIT/GNA recombinant fusion protein towards a range of insect pests from different orders was demonstrated by injection bioassays. Feeding bioassays demonstrated the potential use of the ButaIT/GNA fusion protein as an orally active insecticide against lepidopteran, dipteran and coleopteran pests. These experiments provide further evidence that the development of fusion protein technology for the generation of new, biorational, anti‐insect molecules holds significant promise. © Crown Copyright 2009. Reproduced with permission of Her Majesty's Stationery Office. Published by John Wiley & Sons, Ltd.     

一种促进植物根系生长的极细链格孢菌蛋白质分离、纯化和生物功能

通过硫酸铵沉淀、有机溶剂沉淀、离子交换和分子筛连用的方法，从极细链格孢菌JH505菌株中分离纯化到分子量约为35kD的植物激发子。利用固相梯度凝胶双向电泳方法测定了该蛋白的等电点为4．22。将该纯化蛋白稀释液浸泡小麦种子8h，7d后小麦根系琥珀酸脱氢酶活性提高65．27％，经蛋白处理的小麦根长比对照提高13．1％。     

稻瘟病菌几丁质结合蛋白MoChtbp1的功能初步研究

稻瘟病是一种由子囊真菌稻瘟病菌引起的危害粮食安全的世界性病害.多数效应蛋白对于稻瘟病菌的毒力起到重要作用,在与寄主植物的相互作用中至关重要,因此,筛选并鉴定稻瘟病菌效应蛋白,阐释其基因功能对于稻瘟病的防治具有重要意义.本研究根据已有的稻瘟病菌效应蛋白相关研究方法以及生物信息学软件筛选出了一个几丁质结合蛋白Mo-Chtb...     

Molecular characterisation of two plasmids from paulownia witches’-broom phytoplasma and detection of a plasmid-encoded protein in infected plants

Two plasmids were cloned from paulownia witches’-broom phytoplasma-Nanyang strain (PaWBNy). Southern blotting using pPaWBNy-1ORF1 probe confirmed the existence of the two plasmids. The 4485 bp plasmid, designated pPaWBNy-1, had a nucleotide content of 24 mol% G+C and contained six putative open reading frames (ORFs). The 3837 bp plasmid, designated pPaWBNy-2, had a nucleotide content of 25.9 mol% G+C and contained five putative ORFs which showed similarity with ORFs in pPaWBNy-1. The two plasmids contained a series of tandem repeats and encoded a replication-associated protein (RepA) and a single-stranded DNA-binding protein (SSB), which were necessary for the replication of plasmids. Seven putative proteins encoded by two plasmids were predicted to contain one or more hydrophobic transmembrane domains, respectively, and presumably to be localised to the membrane. ORF4 from pPaWBNy-1 was partially cloned and the recombinant protein with His-tag expressed in Escherichia coli. The fusion protein was used for immunisation and the polyclonal antiserum to ORF4 protein detected the native expression of ORF4 protein in Western blot analysis from infected but not healthy plants.     

桃小食心虫成虫GOBPs与PBPs的基因克隆及表达谱分析

基于桃小食心虫Carposina sasakii触角转录组测序数据,利用RACE技术克隆了桃小食心虫2个普通气味结合蛋白GOBPs和3个性信息素结合蛋白PBPs全长基因,分别命名为CsasGOBP1、CsasGOBP2、CsasPBP1、CsasPBP2和CsasPBP3。5个基因的核苷酸序列全长分别为617、811、755、795和561bp,开放阅读框分别为504、492、507、492和498bp;蛋白分子量分别为16.6、16.34、16.5、15.76和16.66kDa,等电点分别为4.85、4.92、5.41、4.91和4.93,并具有的6个典型保守的半胱氨酸位点。进化树分析结果表明,5个基因能够与其他鳞翅目昆虫中的GOBPs和PBPs聚在不同的分支。qPCR对其雌雄不同部位表达谱分析发现,5个基因均在雄虫触角中显著表达,在头、胸、腹、足和翅中低量表达。该研究为更好地了解其在桃小食心虫嗅觉识别过程中的作用奠定了基础。     

SGT1 contributes to maintaining protein levels of MEK2DD to facilitate hypersensitive response-like cell death in Nicotiana benthamiana

Gene silencing revealed that the mitogen-activated protein kinase (MAPK) cascade in Solanaceae consisted with MEK2-WIPK/SIPK, is required for R protein-induced hypersensitive response (HR) cell death and/or resistance. Overexpression of MEK2^DD results in HR-like cell death. MEK2^DD is a phospho-mimic and constitutive active form harboring mutations at putative phosphorylation sites of upstream MAPKKK. The molecular mechanism that induces HR-like cell death is unknown. Here we report SGT1 is required for the accumulation of MEK2^DD protein, not MEK2^WT. Virus-induced gene silencing of SGT1 resulted in low protein accumulation of MEK2^DD. This result suggests that SGT1 has a positive role in the accumulation of the MEK2 active form protein to facilitate signal transduction.     

Cry1Ac的绿色荧光蛋白GFP标记及其在棉铃虫幼虫中肠的定位分布

为了明确Cry1Ac蛋白在棉铃虫体内与中肠组织的相互作用,采用重叠PCR方法将Bt-cry1Ac基因和绿色荧光蛋白GFP基因融合,构建含Cry1Ac毒蛋白和绿色荧光蛋白GFP原核表达载体,并在大肠杆菌大量表达。利用荧光显微镜观察发现,表达Cry1Ac-GFP融合蛋白的大肠杆菌在蓝光激发下发出绿色荧光。将含有融合蛋白的菌液拌入人工饲料饲喂3龄棉铃虫幼虫96h,取棉铃虫幼虫中肠做冰冻切片并在荧光显微镜下观察。结果显示,取食含有Cry1Ac-GFP融合蛋白饲料的棉铃虫幼虫中肠能够发出强烈荧光。比较Cry1Ac杀虫蛋白敏感和抗性棉铃虫幼虫中肠的发光部位,敏感棉铃虫幼虫的中肠围食膜已经消失,肠壁细胞发出强烈的荧光,而抗性棉铃虫的围食膜较健全并发出荧光。     

不同氮肥运筹对冬小麦产量、蛋白质及其组分的影响

2003～2004年在大田节水栽培条件下,研究了3个施氮水平(157.5 kg/hm2、226.5 kg/hm2、295.5kg /hm2)下氮肥一次性底施与分次施用(底肥 拔节期追肥)对两个小麦品种(强筋麦济南17与中筋麦石家庄8号)品质和产量的影响.结果表明:节水栽培条件下不同氮素水平及等量氮肥一次性基施与分次施用两小麦品种产量差异不显著,过量施用氮肥产量反而有所下降;施氮越多,氮肥生产力越低;不同处理对两品种小麦籽粒蛋白质及其各组分的影响也同样没有达到显著水平.     

稻曲病菌Six1类效应蛋白UvSix1-1的功能研究

 稻曲病菌在侵染过程中会分泌大量的效应蛋白来帮助其侵染。本研究鉴定到一个稻曲病菌效应蛋白UvSix1-1,同源序列比对及系统发育分析发现其具有Six1蛋白保守的氨基酸位点,并与多个病原菌中的Six1蛋白具有同源性。进一步研究发现UvSix1-1可以抑制Bax、XEG1和INF1引起的烟草叶片细胞坏死,并且其预测的信号肽区域具有分泌功能,在烟草上瞬时表达UvSix1-1可以促进辣椒疫霉的定殖。对不同稻曲菌株中的序列进行分析,没有发现序列多态性,说明该基因非常保守。以上研究结果表明,UvSix1-1在病原菌侵染过程中发挥作用,研究结果为稻曲病菌效应蛋白的研究提供参考。     

致病疫霉诱导的马铃薯酵母双杂交文库构建及无毒蛋白PiAVR3b寄主靶标筛选

致病疫霉是引起番茄、马铃薯晚疫病的植物病原卵菌,其分泌的效应蛋白在抑制寄主免疫方面发挥重要功能。鉴定效应蛋白的寄主靶标对研究植物与病原菌互作具有重要意义。本研究构建了高效马铃薯酵母双杂交cDNA文库,并通过筛选致病疫霉无毒蛋白PiAVR3b的互作蛋白来评价构建文库质量。分别提取接种致病疫霉后24 h和48 h的马铃薯栽培种‘合作88’叶片RNA,等量混合后构建cDNA文库。次级文库容量为1.6×10~7 cfu/mL,重组率为100%,插入片段平均在1 000 bp以上。以PiAVR3b为诱饵,通过酵母双杂交筛选初步获得10个候选靶标蛋白。经点对点验证,进一步确定了4个与PiAVR3b互作的靶标蛋白,分别是马铃薯MYB-like蛋白、线粒体外膜孔蛋白、碳分解代谢抑制蛋白、肽基脯氨酰异构酶。本研究构建的酵母双杂交文库为致病疫霉效应蛋白靶标筛选及植物与病原菌互作研究奠定了基础。     

李属坏死环斑病毒怀柔分离物CP基因的克隆、原核表达及其抗血清制备

本研究通过RT-PCR技术获得了包含李属坏死环斑病毒怀柔分离物CP蛋白基因的DNA片段,构建了针对pET29a载体的两种表达质粒;〖JP2〗转化大肠杆菌BL21(DE3),并经IPTG诱导表达了带不同融合肽段的PNRSV1（25 ku）〖JP〗和PNRSV2（29 ku）融合蛋白。经过Ni NTA亲和柱与SDS PAGE分离纯化,获得大量表达融合蛋白,并免疫家兔制备了融合表达蛋白的特异性抗体。间接ELISA测定其效价分别为8×103和3.2×104。     

大麦条纹花叶病毒中国株CP基因克隆分析、原核表达及抗血清制备

 首次用PCR方法,自大麦条纹花叶病毒中国株(BSMV-CH)的RNA₂(GenBank登录号为:AY789694,未报道)中扩增出外壳蛋白(coat protein,CP)基因,并亚克隆到pMD18-T载体上进行测序分析。结果表明BSMV-CH的CP全长597bp,它与BSMV其它株系CP的核苷酸同源性和氨基酸同源性分别为93.1%~93.8%和91.4%~92.4%;与同属的早熟禾半潜病毒(PSLV)和剪秋罗环斑病毒(LRSV)的核苷酸同源性分别为53.0%和41.8%,氨基酸同源性分别为48.1%和40.0%。根据大麦病毒属病毒已经报道的CP氨基酸序列绘制系统发育树,分析表明分离的各毒株在进化上存在明显地理位置的相关性。把CP基因亚克隆到GST融合表达载体pEGX-6p-1上,优化IPTG诱导终浓度后,在37℃ IPTG终浓度为1.0mmol/L时,融合蛋白GST-CP在大肠杆菌BL21中得到了特异表达。12% SDS-PAGE表明,表达产物大小为48.5kDa,主要以包含体形式存在。对蛋白进行定量后,用冰冷的KCl显色,分离表达的蛋白质特异条带制备抗原,免疫家兔得到抗血清,酶联法(enzyme-linked immunosorbant assay,ELISA)测定的抗血清效价为1/6 400。用抗血清对感病大麦和健康大麦进行检测,结果表明抗血清有很高特异性。     

新型真菌源激活蛋白对豌豆幼苗生理特性影响的初步研究

为探讨真菌源激活蛋白在促进植物生长和增产方面的作用机理,研究了不同浓度激活蛋白处理对豌豆幼苗生理特性的影响。结果表明,经1、2、5μg/mL激活蛋白处理后第7d,豌豆幼苗根系活力比对照分别增加了46.0%、58.5%和21.0%,硝酸还原酶活性比对照分别增加64.38%、140.62%和35.31%;处理后第15d,植物叶片叶绿素含量比对照分别增加了13.69%、18.49%和6.16%,可溶性糖含量比对照分别增加20.68%、32.03%和17.28%,可溶性蛋白含量比对照分别增加18.59%、32.05%和22.10%。不同浓度激活蛋白均能促进与植物生长相关的生理指标的提高,其中以2μg/mL的作用效果最好。     

Cry1Ba3蛋白对亚洲玉米螟杀虫活性测定及其与BBMVs的结合分析

提取并纯化Cry1Ba3蛋白截短片段,用饲喂人工饲料的方法对亚洲玉米螟（Ostrinia furnacalis）初孵幼虫进行生物活性测定。结果显示,Cry1Ba3截短片段对亚洲玉米螟有良好的杀虫活性（LC50=5.29 μg/g,95%置信限为3.96～7.10 μg/g）。而纯化的Cry1Ac蛋白60 ku的活性片段对亚洲玉米螟初孵幼虫的杀虫活性LC50为0.83 μg/g。提取亚洲玉米螟5龄幼虫的BBMVs,用纯化的Cry1Ba3和Cry1Ac蛋白进行竞争结合试验,结果显示Cry1Ba3与Cry1Ac在亚洲玉米螟中肠的结合没有竞争作用,说明两种毒素在亚洲玉米螟BBMVs上可能存在不同的受体。     

马铃薯Y病毒衣壳蛋白的表达、纯化与结晶条件筛选

为研究马铃薯Y病毒（potato virus Y,PVY）衣壳蛋白（coat protein,CP）在体外表达及CP重组蛋白的晶体生长条件,通过Eco R Ⅰ/Hind Ⅲ酶切及连接技术构建pET-32a-PVY CP原核表达载体,对PVY CP的诱导剂浓度进行优化,利用蛋白质纯化及脱盐技术对PVY CP进行纯化和脱盐,并分析PVY CP重组蛋白的晶体生长条件。结果表明,成功构建了pET-32a-PVY CP原核表达载体;PVY CP在大肠杆菌Escherichia coli感受态细胞BL21(DE3)原核表达系统中,于16℃下利用0.8 mmol/L异丙基-β-D-硫代吡喃半乳糖苷（isopropyl-beta-D-thiogalactopyranoside,IPTG）诱导表达可获得高浓度的PVY CP可溶性蛋白;PVY CP重组蛋白在二水甲酸镁处理下可生长出棒状结构晶体。     

番茄抗病毒基因Tm-2~2及其等位基因tm-2氨基酸差异分析

番茄抗病蛋白Tm-2~2与其等位感病蛋白tm-2在序列上相对保守但也有一定的差异性,研究这些差异对于了解Tm-2~2抗病蛋白的结构和功能起着重要作用。本研究构建了不同的Tm-2~2/tm-2置换突变体,在本生烟草上利用农杆菌注射法共表达突变体及其效应因子MP或TMV-GFP载体。通过对植物坏死表型以及病毒量变化的观察,发现当Tm-2~2ARC结构域中RNBS-D保守基序中的第413位甘氨酸突变成tm-2中的丝氨酸后,Tm-2~2介导的对烟草花叶病毒属病毒的抗性减弱。这说明RNBS-D保守基序对于Tm-2~2行使其抗病功能是至关重要的。