鸭瘟病毒的PCR检测及其产物分析

根据基因库中已有的鸭瘟 Ul6的序列 ,合成一对引物 ,对 3株鸭瘟病毒进行 PCR扩增 ,均扩增出大小约 42 0 bp的特异性片段。进行测序 ,结果表明 3个毒株扩增后的片段均为 42 1bp。经重复实验后确定最佳反应条件 ,按该条件对两例临床病料进行检测 ,结果均出现大小约为 42 0 bp的特异片段。PCR产物与 L ake Andes株的相关序列比较 ,广东毒株与 L ake Andes株同源性较高     

T7 RNA多聚酶基因的克隆、序列测定及其在E.coli中的表达

从BL21(DE3)E．coli菌株中以PCR的方法扩增得到了与T7RNA多聚酶(T7RNApolymerase，T7pol)基因大小一致的DNA片断。将PCR产物纯化后直接克隆到pGEM—T载体中，经酶切鉴定和DNA序列分析表明克隆得到了正确的T7pol基因。将T7pol基因亚克隆入pET-28b( )中，构建得到原核表达质粒pET28T7。该质粒的BL21(DE3)pLysS转化菌在IPTG的诱导下可表达约98800的蛋白，这与T7pol的相对分子质量一致。将该质粒转化DH5α、JMl09、HBl01、BL21(DE3)和BL21(DE3)pLysS等5种不同的宿主菌，仅有转化T7pol酶活性受到抑制的宿主菌BL21(DE3)pLysS才能得到转化子，而其余4种T7T7pol酶活性不受抑制的E．coli宿主菌不能得到转化子。pET28T7原核表达质粒这种仅能在T7pol酶活性受到抑制的宿主菌中才能存活的现象说明本试验所克隆的T7pol基因能正确表达出具有RNA转录酶活性的蛋白。     

Changes in the glutathione thiol-disulfide status in wheat grain by foliar sulphur fertilization: consequences for the rheological properties of dough

Storage proteins and glutathione in wheat play an important role in gluten network formation and can be modified by supplementation of nitrogen (N) and sulphur (S) in wheat plants. The glutathione thiol-disulfide status and its relationship to the molecular weight distribution wheat polymeric protein and dough rheological properties have been examined after different foliar S fertilizations (S derived from micronized elemental S and NS, a mixture of N urea and elemental S) applied at the post-anthesis stage. Changes in levels of reduced glutathione (GSH), glutathione disulfide (GSSG), polymeric protein-glutathione mixed disulfide (PPSSG) were analysed by reversed phase high performance liquid chromatography, during grain development using the wheat cultivars, Soissons and Trémie. During the grain desiccation phase, S supplementation (i) increased the GSSG/GSH ratio by 23–25% (ii) induced PPSSG accumulation, and (iii) decreased the formation of SDS-unextractable polymeric protein (UPP) and its molecular mass distribution. However, simultaneous N and S supplementation results in: (i) a decrease in PPSSG formation by 20–30% and (ii) an increase of UPP by 7–18% by enhancing both the branching of the aggregated proteins and their molecular weight. The mixograph parameters show that all forms of endogenous glutathione are linked to dough weakening and are negatively correlated with dough mixing tolerance, dough strength and consistency, while UPP is positively correlated with dough strength and consistency. These findings indicate that S nutrition influences dynamics of the glutathione forms in the grain and results in modification the degree of polymerization of storage protein. Thus both the changes in the form of glutathione and protein polymerization influence the rheological properties of dough.     

RT-PCR及RFLP分析技术对鸡传染性支气管炎病毒的诊断和S1基因分型的研究

本研究使用分别扩增整个S1糖蛋白基因 (引物A)和S1糖蛋白基因N_端高变区Ⅰ (引物B)的 2对引物对 3个IBV标准株M41、Connecticut、Arkansas及 5个地方分离株 (C60 ,D41A ,D41B ,A112 1,A1171)进行RT_PCR扩增。用引物A时 ,有 5个IBV毒株扩增到目的片段 (172 0bp) ;用引物B时 ,所有 8个IBV毒株均得到与预计大小相符的目的产物 (2 2 8bp)。对 172 0bp的PCR产物用限制性内切酶HaeⅢ进行酶切 ,结果得出 3个不同的RFLP图谱 ,其中M41、Connecticut、D41B具有相同的HaeⅢ酶切图谱。Arkansas和D41A则分别具有互不相同的图谱 ;对 2 2 8bp的PCR产物进行DdeⅠ、RsaⅠ限制性内切酶消化 ,根据它们的RFLP图谱 ,8个IBV毒株可分为 5个基因型。综合 2对引物的PCR产物的RFLP分析结果 ,8个IBV毒株可分为 7个基因型 ,分型的结果与传统的血清学方法吻合。本研究建立的方法和技术具有快速、简单、特异、灵敏等优点 ,为现场流行毒株的定型(基因型 /血清型 )及其S1基因变异的跟踪研究以及更有效防制传染性支气管炎奠定了基础。     

鸡沙门氏菌弱毒冻干苗的研制

由减毒鸡沙门氏苗97A疫苗株作为制苗用菌种，经普通琼脂培养，冷冻真空干燥等工艺生产鸡沙门氏菌弱毒疫苗4批，每批疫苗分别以免疫剂量接种6日龄AA肉鸡，免疫第14天时，用强毒株Sg9和Sp4攻击，免疫组死亡保护率均达90％以上，试验结果表明，该苗具有良好的安全性和免疫效果。     

猪传染性胃肠炎病毒南京株纤突S蛋白基因的克隆与序列分析

参考 Genbank收录的 TGEV- Miller株的基因序列 ,自行设计合成 1对引物 (TGEVP5 /P6 ) ,对不同代次的 TGEV疫苗弱毒 STC3及种毒 、种毒 进行了 RT- PCR扩增 ,产物经琼脂糖凝胶电泳分析 ,均出现 1条大约 12 6 2 bp的目的条带 ,经 Eco R 酶切 ,都产生了 871bp和391bp左右的两个片段 ,与预期大小相符。将种毒 RT- PCR扩增目的条带回收纯化后克隆入PMD18- T载体中 ,转化宿主菌 DH5 α,挑选阳性克隆 (命名为 PTs) ,提取重组质粒 ,用 Hpa 、Eco R 对重组质粒进行酶切鉴定以及 PCR扩增 ,然后进行序列测定 ,并进行了序列分析 ,证实与国外标准毒株 Miller、Fs772 / 70、Purdue、TO14等有较高的同源性     

A method to test wheat leaves for their reactions to inoculation with Septoria species

The construction and application is described of a polystyrol humidity box in which wheat leaves, while continuing to function as parts of living plants, can be tested for their reactions toSeptoria spp. in an atmosphere nearly saturated with water, as is required for successful infection. The method is simple, accurate ans inexpensive.Samenvatting Voor dit doel is een z.g. vochtdoos geconstrueerd (Fig. 1A). Het te toetsen blad wordt daar doorheen geleid en in de doos geïnoculeerd met een druppel conidiën-suspensie van de schimmel. Onder het blad staat wat water in de doos (Fig. 1B). Na afsluiting ontstaat in de doos de hoge luchtvochtigheid van bijna 100% die nodig is voor de infectie. Dit wordt op deze wijze eenvoudig en goedkoop gerealiseerd. De bladeren die zo worden getoetst blijven onbeschadigd functioneren aan de plant. Na enkele dagen kan de doos worden geopend en kan de symptoom-ontwikkeling worden afgewacht en gevolgd (Fig. 3A, 3B en 4). De methode leent zich voor nauwkeurig werk en vereist zeer weinig infectiemateriaal. De Technische en Fysische Dienst voor de Landbouw (TFDL), Wageningen, ontwierp en construeerde een statief voor het gebruik van de vochtdozen in serie (Fig. 2).     

The effect of some Frescon analogs on the aquatic snail,Lymnaea stagnalis: II. Correlation of neurotoxicity to Molluscicidal effectiveness

Although the molluscicide Frescon is a strong neurotoxin to the Lymnaea stagnalis central nervous system in vitro, it is probable that the exposure of the whole animal to this molluscicide fails to result in central nervous system abnormalities: Frescon does not appear to reach the brain in sufficient quantity to disrupt its normal activity. However, only those Frescon analogs found to be neurotoxic were molluscicidal, suggesting some related mode, if not site, of action. Frescon and its analogs may act by affecting excitable tissues other than the nervous system (e.g., the snail musculature) by altering certain functional and/or structural membrane properties.     

梨自交不亲和强度不同品种花柱S糖蛋白含量的差异

 提取梨( Pyrus) 自交不亲和强度不同品种花柱可溶性蛋白质, 应用等电聚焦电泳法在凝胶板上分离出S 糖蛋白, 用图像解析法进行定量。结果表明, 花柱总S 糖蛋白含量因品种而异, 自交不亲和强、弱及自交亲和的品种每1 􀀁g 花柱可溶性蛋白中含总S 糖蛋白分别为􀀂1. 3 ng 、0. 7 ~ 0. 9 ng、 0. 7 ng 。不同的S 基因所表达的S 糖蛋白量差异显著, 即使是同一S 基因所对应的S 糖蛋白量也因品种而异。