猪繁殖与呼吸综合征病毒Hn-1/06株GP3蛋白的原核表达与纯化

根据GenBank中PRRSV美洲株ATCC VR2332的ORF3基因序列,利用Primer 5.0软件设计合成了1对特异性引物,经RT-PCR从河南分离株Hn-1/06株扩增得到了大小为765 bp的片段,并将扩增的片段插入pTG19-T载体进行测序而获得含有ORF3的阳性克隆pTG19-T-GP3.将此基因亚克隆到原核表达载体pET-32a,经筛选获得了阳性重组质粒pET-GP3,进而在IPTG的诱导下成功表达,获得了大小约为45 kDa的融合蛋白GP3-His,纯化后经Western blot检测证实表达的融合蛋白具有良好的生物学活性.从而为进一步研究PRRSV次要结构蛋白GP3的免疫特性和功能奠定了基础.     

不同佐剂对猪传染性胃肠炎病毒S蛋白和猪流行性腹泻病毒S蛋白免疫原性的影响

本研究旨在探究不同佐剂对猪传染性胃肠炎病毒(TGEV)S蛋白和猪流行性腹泻病毒(PEDV)S蛋白免疫原性的影响。IPGT诱导重组表达菌株Rosetta(DE3)-pET28a-TGEV-S2/3和BL21(DE3)-pET28a-PEDV-S1/2/3,制备重组蛋白TGEV-S2、TGEV-S3、PEDV-S1、PEDV-S2和PEDV-S3,并进行SDS-PAGE和Western blot鉴定,将重组蛋白等量混匀分别与3种佐剂ISA 71VG、ISA 201VG和ISA 15AVG进行乳化制备免疫原免疫蛋鸡;Western blot检测蛋鸡血清抗体效价,免疫过氧化物酶单层细胞染色法(IPMA)检测抗体与病原的反应活性;监测鸡群平均日增重。结果显示,重组蛋白TGEV-S2、TGEV-S3、PEDV-S1、PEDV-S2和PEDV-S3的分子量依次为25 ku、28 ku、26 ku、24 ku和21 ku。Western blot分析显示,5种蛋白均具有良好的免疫原性;疫苗二次免疫后7天抗体效价达到高峰,ISA 201VG佐剂组、ISA 71VG佐剂和ISA 15AVG佐剂组组最高抗体效价依次为64000倍、32000倍和8000倍,对照组未检测到特异性抗体;ISA 71VG和ISA 201VG组对产蛋率下降相对较大,ISA15AVG 佐剂疫苗免疫组对产蛋率影响相对较小,免疫后7天后试验组产蛋率恢复正常。ISA 71VG佐剂组蛋鸡平均日增重显著降低,ISA 201VG和ISA 15AVG佐剂组蛋鸡平均日增重与对照组无显著差异;ISA 201VG佐剂组血清抗体与病毒反应活性良好,中和效价可达1:256。ISA 201VG佐剂配伍的疫苗免疫效果优于其他两种佐剂疫苗。     

玉米SSⅡb蛋白多克隆抗体的制备及其应用

抗体是蛋白质功能研究的重要试剂。玉米蛋白的功能研究因缺乏相应的抗体而难以开展。玉米淀粉合酶Ⅱb与淀粉合酶Ⅱa均属于淀粉合酶,其主要负责支链淀粉的延伸。但对淀粉合酶Ⅱb的报道较少,本研究以原核表达的淀粉合酶Ⅱb的C端(455～704 aa) GST融合蛋白为抗原,免疫新西兰大白兔,分离血清制备SSⅡb多克隆抗体。Western blot试验发现, SSⅡb多克隆抗体不但能识别SSⅡb-C (455～704 aa)抗原,而且能识别玉米不同发育阶段胚乳中SSⅡb蛋白。利用该多克隆抗体的Western blot试验表明,玉米SSⅡb蛋白的表达在授粉后10 d到30 d的胚乳中表达模式是先增后降,授粉后15 d表达量最高,而半定量RT-PCR的结果表明授粉后20 d SSⅡb的转录水平最高。这些结果表明成功制备了特异的玉米SSⅡb的多克隆抗体,并能在Western blot中特异识别玉米内源SSⅡb蛋白,为进一步深入研究SSⅡb蛋白功能奠定基础。     

猪圆环病毒2型和猪繁殖与呼吸综合征病毒混合感染的流行病学调查

研究2009-2010年我国部分地区猪圆环病毒2型(PCV2)和猪繁殖与呼吸综合征病毒(PRRSV)混合感染的情况。采用PCR和RT-PCR方法对采集自我国安徽、江苏、山东、浙江等7省的227份组织和血清样品进行PCV2和PRRSV检测。PCV2a感染率为15.9%,PCV2b感染率为44.5%,PRRSV感染率为45.8%,其中17份样品表现为PCV2a和PRRSV的混合感染,50份样品表现为PCV2b和PRRSV的混合感染,10份样品表现为PCV2a,PCV2b和PRRSV的混合感染,分别占样品总数的7.5%,22.0%和4.4%。猪群中PCV2和PRRSV的感染比较普遍,混合感染率也较高,加重了疫病防控的难度。     

BYDV-GAV外壳蛋白和复制酶基因酵母双杂诱饵载体的构建及表达

在植物病毒侵染过程中,病毒蛋白能够与寄主蛋白发生相互作用以利于病毒的复制、积累和扩散。酵母双杂交系统是体外研究蛋白相互作用的有利工具,其在致病机制研究中得到广泛的利用。本研究利用大麦黄矮病毒(BYDV)外壳蛋白(CP)和依赖RNA的复制酶(RdRp)基因的全长序列,构建了这2个基因的酵母双杂诱饵载体pG-BKT7-CP和pGBKT7-RdRp,并转化酵母进行表达分析。含有pGBKT7-CP和pGBKT7-RdRp诱饵载体的酵母,在SD/-Trp固体培养基和SD/-Trp/Kan液体培养基中都能正常生长,但在SD/-Ade/-Trp/X-Gal和SD/-His/-Trp/X-Gal培养基上不能生长。结果表明,这2个载体表达的融合蛋白对酵母没有毒害作用,且自身没有激活性,可作为酵母双杂系统的诱饵载体,用来分析与外壳蛋白和复制酶相互作用的蛋白。     

异构系统中检测蛋白质相互作用方法的原理及应用

基因组测序的完成并没有真正解释出细胞生命活动的本质。科学家们逐渐发现所有细胞功能都涉及蛋白质相互作用,许多疾病的发病也是由于蛋白质相互作用的改变,因此研究蛋白质相互作用具有重要的意义,也成为蛋白质组学研究的重点。目前已有许多实用且成熟的技术应用于蛋白质相互作用的检测。异构系统中检测蛋白质相互作用的方法包括细菌、酵母、哺乳动物细胞的双杂交系统和免疫共沉淀技术。文章将对这些技术的原理及应用作以综述。     

甘蓝自交不亲和信号传导元件ARC1的体外表达及其与SRK相互作用验证

在甘蓝自交不亲和信号传导中ARC1和上游因子SRK之间可能存在相互作用。为进一步证实该相互作用,以甘蓝E1为材料,采用RT-PCR技术扩增ARC1的编码序列, 构建ARC1原核表达质粒pET43.1a-ARC1,转化宿主菌大肠杆菌BL21,通过SDS-PAGE检测该蛋白的表达。利用免疫共沉淀原理及pET43.1a-ARC1融合蛋白序列中的6×His标签与Ni⁺结合的特点建立了体外检测蛋白质相互作用的新方法, 并用该方法对ARC1与SRK的相互作用进行了检测。结果表明,在体外ARC1能与SRK相互作用并形成复合体,这为深入分析ARC1与SRK相互作用机理以及探讨ARC1与下游传导元件的相互作用提供了理论和技术基础。     

结球甘蓝雌蕊调控因子SPT与HEC1的克隆及相互作用分析

为研究SPT和HECs及其相互作用对甘蓝雌蕊发育的影响, 以结球甘蓝自交不亲和系E1为材料, 提取雌蕊总RNA, 根据拟南芥中SPT和HEC1基因设计引物, 采用同源克隆的方法克隆SPT基因, 其序列1085 bp, 开放阅读框(ORF) 1062 bp; HEC1基因ORF 696 bp。通过cDNA推导得到的氨基酸序列表明, SPT编码353个氨基酸残基, 预测分子量为37.67 kD, pI为6.83; HEC1编码231个氨基酸残基, 预测分子量为25.26 kD, pI为10.2。分析表明, 两基因在各器官中均表达, 但SPT在果实和雌蕊中表达量最高, 而HEC1在根和花蕾中的表达量最高。为检测两者的相互作用, 构建原核表达质粒pCold I-SPT和pGEX-HEC1, Pull-down试验表明两蛋白能够在体外相互作用。同时构建pGBKT7-SPT、pGADT7-HEC1及互换载体pGBKT7-HEC1和pGADT7-SPT酵母表达载体, 分别转化酵母Y2HGold和Y187感受态细胞后均未出现自激活和毒性现象, 融合后的二倍体酵母均能在SD/–Trp–Leu–Ade–His/X-α-gal/AbA板上长出蓝色菌斑, 表明SPT和HEC1能够相互作用激活下游的HIS3、AUR1-C、MEL1和ADE2报告基因, 酵母双杂交试验结果与Pull-down检测一致, 说明SPT和HEC1能够相互作用以调控雌蕊的发育。     

结球甘蓝雌蕊发育调控因子SPT与HEC1的基因克隆及相互作用

为研究SPT和HEC及其相互作用对甘蓝雌蕊发育的影响,以结球甘蓝自交不亲和系E1为材料,提取雌蕊总RNA,根据拟南芥中SPT和HEC1基因设计引物,采用同源克隆方法克隆SPT基因,其序列1085 bp,开放阅读框(ORF)1062 bp;HEC1基因ORF 696 bp。通过cDNA推导得到的氨基酸序列表明,SPT编码353个氨基酸残基,预测分子量为37.67 kD,pI为6.83;HEC1编码231个氨基酸残基,预测分子量为25.26 kD,pI为10.2。两基因在各器官中均表达,但SPT在果实和雌蕊中表达量最高,而HEC1在根和花蕾中的表达量最高。为检测两者的相互作用,构建原核表达质粒pCold I-SPT和pGEX-HEC1,pull-down试验表明两蛋白能够在体外相互作用。同时构建pGBKT7-SPT、pGADT7-HEC1及互换载体pGBKT7-HEC1和pGADT7-SPT酵母表达载体,分别转化酵母Y2HGold和Y187感受态细胞后均未出现自激活和毒性现象,融合后的二倍体酵母均能在SD/–Trp–Leu–Ade–His/X-α-gal/AbA板上长出蓝色菌斑,表明SPT和HEC1能够相互作用激活下游的HIS3、AUR1-C、MEL1和ADE2报告基因,酵母双杂交试验结果与Pull-down检测一致,说明SPT和HEC1能够相互作用以调控雌蕊的发育。     

齿兰环斑病毒CP基因的原核表达及其产物抗血清制备

本研究在于探讨侵染兰花的重要病毒之一齿兰环斑病毒（Odontoglossum ringspot virus,ORSV）的检测技术。从福建省漳州市采集感染齿兰环斑病毒的建兰病样,设计一对特异性引物扩增并克隆得到该病毒的外壳蛋白基因,该基因开放阅读框长477 bp,编码158 aa约合18.0 kDa的蛋白质,随后将目的基因插入pET-29a（+）中构建相应的原核表达载体进行诱导表达,目的蛋白经纯化后免疫家兔获得了特异性抗血清。Western blot检测结果表明,抗血清与诱导表达的ORSV外壳蛋白发生特异性反应。间接酶联免疫吸附检测结果表明,抗血清可检测病叶的最低稀释度达1:25600（v/v）,最佳工作浓度为1:12800（v/v）,病汁液灵敏度为0.195 mg/mL（w/v）,而与TMV等11种同源或异源病毒均无明显的血清学交叉反应。     

Location of a high-lysine gene and the DDT-resistance gene on barley chromosome 7

Summary The high-lysine gene in Risø mutant 1508 conditions an increased lysine content in the endosperm via a changed protein composition, a decreased seed size, and several other characters of the seed. The designation lys3a, lys3b, and lys3c, is proposed for the allelic high-lysine genes in three Risø mutants, nos 1508, 18, and 19. Linkage studies with translocations locate the lys3 locus in the centromere region of chromosome 7. A linkage study involving the loci lys3 and ddt (resistance to DDT) together with the marker loci fs (fragile stem), s (short rachilla hairs), and r (smooth awn) show that the order of the five loci on chromosome 7 from the long to the short chromosome arm is r, s, fs, lys3, ddt. The distance from locus r to locus ddt is about 100 centimorgans.     

A note on the origin of Kalanchoë x vadensis Boom & Zeilinga (Crassulaceae)

Summary K x vadensis is a hybrid of K. blossfeldiana and K. marmorata obtained after doubling the number of chromosomes.     

Simultaneous Determination of Four Phenolic Acids in Radix Salviae Miltiorrhizae Formula Granules by QAMS

[Objectives]This study aimed to establish a QAMS(quantitative analysis of multi-components by single-marker)method for simultaneous determination of four phenol...     

Avoidance of leaf rust fungi in wild relatives of cultivated cereals

Summary Avoidance of rust fungi that was based on poor appressorium induction was previously found in Hordeum chilense. In the present study 95 accessions of Triticeae were screened for avoidance of Puccinia hordei. The percentage of appressorium formation per germinated spore ranged from 6 to 90%. On none of the 41 accessions of Aegilops, Agropyron, Elymus, Secale, Thinopyrum or Triticum studied was the rate of appressorium formation lower than 25%. Lower rates of appressorium formation were, however, found on accessions of wild barley species Hordeum brachyantherum, H. marinum, H. parodii and H. secalinum. Its implications in cereal breeding are discussed.     

Present status and future strategy in breeding faba beans (Vicia Faba L.) for resistance to biotic and abiotic stresses

Progress is being made, mainly by ICARDA but also elsewhere, in breeding for resistance to Botrytis, AScochyta, Uromyces, and Orobanche; and some lines have resistance to more than one pathogen. The strategy is to extend multiple resistance but also to seek new and durable forms of resistance. Internationally coordinated programs are needed to maintain the momentum of this work.Tolerance of abiotic stresses leads to types suited to dry or cold environments rather than broad adaptability, but in this cross-pollinated species, the more hybrid vigor expressed by a cultivar, the more it is likely to tolerate various stresses.     

Water Extraction Process of Chinese Herbal Compound Man Gan Ning

[Objectives]To optimize the water extraction process of Chinese Herbal Compound Man Gan Ning and establish a method for its extraction and content determination...     

Pollen and pollination experiments. III. The viability of apple and pear pollen as affected by irradiation and storage

Summary Apple and pear pollen was irradiated with doses of 0, 50, 100, 250 and 500 krad (gamma rays) and stored at 4°C and 0–10% r.h. From the in-vitro germination percentages an average LD 50 dose of about 220 krad was estimated. For both irradiated and untreated pollen a close and corresponding lineair relationship existed between germination percentage and pollen tube growth.Irradiated pollen was much more sensitive to dry storage conditions than untreated pollen, resulting in less germination and more bursting. Apparently, irradiation caused the pollen cell membrane to lose its flexibility faster than normal. Rehydration of dry-stored, irradiated pollen in water-saturated air restored germination percentages up to their initial levels. The importance of this procedure in germination trials is stressed.     

Cytoplasmic male sterility,resistance to gall mite and mildew,and season of leafing out in black currants

Summary Cytoplasmic male sterility (cms) is described in the F₁ hybrids Ribes × carrierei (R. glutinosum albidum × R. nigrum) and R. sanguineum × R. nigrum. In backcrosses to R. nigrum, progenies with R. glutinosum cytoplasm were either all male sterile, or segregated for full male fertility (F) and complete (S) and partial (I) male sterility. Ratios of F:I+S suggested that two linked genes controlled cms, F plants being dominant for one (Rf ₁) and recessive for the other (Rf ₂).Segregation for cms in relation to three linded genes, Ce (resistance to the gall mite, Cecidophyopsis ribes), Sph ₃(resistance to American gooseberry mildew, Sphaerotheca mors-uvae) and Lf ₁(one of two dominant additive genes controlling early season leafing out) indicated that Rf ₁and Rf ₂were in this linkage group. The gene order and approximate crossover values appeared to be: % MathType!MTEF!2!1!+-% feaafiart1ev1aaatCvAUfeBSjuyZL2yd9gzLbvyNv2CaerbuLwBLn% hiov2DGi1BTfMBaeXafv3ySLgzGmvETj2BSbqef0uAJj3BZ9Mz0bYu% H52CGmvzYLMzaerbd9wDYLwzYbItLDharqqr1ngBPrgifHhDYfgasa% acOqpw0xe9v8qqaqFD0xXdHaVhbbf9v8qqaqFr0xc9pk0xbba9q8Wq% Ffea0-yr0RYxir-Jbba9q8aq0-yq-He9q8qqQ8frFve9Fve9Ff0dme% aabaqaciGacaGaamqadaabaeaafaaakeaacaWGdbGaamyzamaamaaa% baGaaiiiaiaacccacaGGWaGaaiOlaiaacgdacaGG0aGaaiiiaiaacc% caaaGaaiiiaiaacccacaGGGaGaamOuaiaadAgaliaaigdakmaamaaa% baGaaiiiaiaacccacaGGGaGaaiiiaiaaccdacaGGUaGaaiOmaiaacs% dacaGGGaGaaiiiaiaacccacaGGGaGaaiiiaaaacaWGsbGaamOzaSGa% aGOmaOWaaWaaaeaacaGGGaGaaiiiaiaacccacaGGGaGaaiiiaiaacc% cacaGGGaGaaiiiaiaacccaaaGaamitaiaadAgaliaaigdakmaamaaa% baGaaiiiaiaacccacaGGGaGaaiiiaiaacccacaGGGaGaaiiiaiaacc% cacaGGGaGaaiiiaiaacccacaGGGaaaaiaadofacaWGWbGaamiAaSGa% aG4maaaa!6E4D!\[Ce\underline { 0.14 } Rf1\underline { 0.24 } Rf2\underline { } Lf1\underline { } Sph3\]. Crossover values of 0.36 for Ce-Lf ₁, and 0.15 for Lf ₁-Sph ₃were estimated from the relative mean differences in season of leafing out between seedlings dominant and recessive for Ce and Sph ₃.It is suggested that competitive disadvantage of lf ₁-carrying gametes and/or zygotes at low temperatures may be implicated in the almost invariable deficit of plants dominant for the closely linked mildew resistance allele Sph ₃. Poor performance of lf ₁- (and possibly lf ₂-) carrying gametes and young zygotes during periods of low temperature at flowering might also account for the liability of some late season cultivars and selections to premature fruit drop (running off).     

Optimization of Extraction Technology and Determination of Content of Total Phenols of Leaves in Acanthopanax giraldii Harms

[Objectives] To determine the optimum extraction technology for total phenols of leaves in Acanthopanax giraldii Harms.[Methods]The single factor test and ortho...     

Screening techniques and sources of resistance to parasitic angiosperms

Parasitic angiosperms cause great losses in many important crops under different climatic conditions and soil types. The most widespread and important parasitic angiosperms belong to the genera Orobanche, Striga, and Cuscuta. The most important economical hosts belong to the Poaceae, Asteraceae, Solanaceae, Cucurbitaceae, and Fabaceae. Although some resistant cultivars have been identified in several crops, great gaps exist in our knowledge of the parasites and the genetic basis of the resistance, as well as the availability of in vitro screening techniques. Screening techniques are based on reactions of the host root or foliage. In vitro or greenhouse screening methods based on the reaction of root and/or foliar tissues are usually superior to field screenings and can be used with many species. To utilize them in plant breeding, it is necessary to demonstrate a strong correlation between in vitro and field data. The correlation should be calculated for every environment in which selection is practiced. Using biochemical analysis as a screening technique has had limited success. The reason seems to be the complex host-parasite interactions which lead to germination, rhizotropism, infection, and growth of the parasite. Germination results from chemicals produced by the host. Resistance is only available in a small group of crops. Resistance has been found in cultivated, primitive and wild forms, depending on the specific host-parasite system. An additional problem is the existence of pathotypes in the parasites. Inheritance of host resistance is usually polygenic and its transfer is slow and tedious. Molecular techniques have yet to be used to locate resistance to parasitic angiosperms. While intensifying the search for genes that control resistance to specific parasitic angiosperms, the best strategy to screen for resistance is to improve the already existing in vitro or greenhouse screening techniques.