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杀虫晶体蛋白
    Studies on the Enzyme-linked Immunosorbent Assay for Detection of Bacillus thuringiensis Insecticidal Protein Expressed in Transgenic Plants Ⅱ.Preparation of Polyclonal Antibody to Bt Insecticidal Protein
    转Bt基因植物中杀虫蛋白的酶联免疫检测技术研究Ⅱ.Bt杀虫晶体蛋白抗体的制备
    UV-Protection of Bt Melanin to Insecticidal Protein
    Bt黑色素对杀虫晶体蛋白的紫外保护
    In this paper, vegetative( vip83 ) and crystal(cry1Ac10 and cry1Ca) insecticidal protein genes from Bacillus thuri ngiensis were simultaneously electrospored into the plasmid-free strain BMB17 1. By the means of the specific P CR detection, the recombinant strains BMB2830-171 contained cry 1Ac10 and vip83, and BMB2 882-171 had cry1Ca and vip83 , were obtained respectively.
    将构建的营养期杀虫蛋白基因vip83表达质粒pBMB2 32 8和含杀虫晶体蛋白基因(cry1Ac1 0或cry1Ca)质粒同时电转化无质粒突变株BMB1 71并双抗筛选。 经PCR特异引物扩增验证 ,分别得到含cry1Ac1 0和vip83、cry1Ca和vip83的双基因重组菌BMB2 830 1 71和BMB2 882 1 71。
    A cryllel gene cloned from a Bacillus thuringiensis isolate in China and designated as by International Bt insecticidal Protein Gene Nomenclatural Committee was silent in host cell and encoded a protein consisting of 719 amino acids which was highly toxic to Plutella xylostella, Ostrinia furnacalis and Leguminivora glycinivorella larvae.
    cry1Ie1基因是我国鉴定分离并经国际命名的一种新型的苏云金芽孢杆菌(Bacillus thuringiensis,Bt)杀虫晶体蛋白基因。 该基因在Bt菌株中沉默,编码719个氨基酸组成的蛋白,对小菜蛾(Plutella xylostella)、玉米螟(Ostrinia furnacalis)和大豆食心虫(Leguminivora glycinivorella)等害虫具有较高的杀虫活性。
    The crySCa gene from HBF-1 strain was expressed in Bt HD-73". The expressed product showed toxic to Ahomala corpulenta larvae with 68.3% corrected mortality in 160pig/g soil concentration. A novel cryS gene was cloned from Bt strain 185. The homology between the sequence of it and known cry8 genes was less than 78%, so it was designed as crySEal gene (AY329081) by International Bt insecticidal Protein Gene Nomenclatural Committee.
    该基因与已知cry8基因的同源性均低于78%,已在GenBank登录(AY329081),并被国际Bt杀虫晶体蛋白基因命名委员会命名为cry8Ea1基因。 这是我国发现的第一个第二等级的Bt杀虫基因,杀虫活性的研究正在进行。
    1. Bt insecticidal protein which was of high purity with a molecular weight of 130KD was prepared by using several biochemical techniques. 2. specific antibody was acquired by immunizing white rabbits with purified Bt insecticidal protein as antigen.
    首先用碱液(50mM Na_2CO_3,50mM EDTA,3%巯基乙醇)裂解孢晶混合物,调节PH至等电点沉淀出晶体杀虫蛋白,再用聚丙烯酰胺凝胶制备电泳进一步纯化粗提的杀虫晶体蛋白,经SDS-PAGE电泳分析显示只有一条带,说明获得的分子量约为130KD的杀虫晶体蛋白达到了电泳纯,为制备特异性抗体提供了条件保障。
    Acording to the amino acids sequence of Bacillus thuringiensis insecticidal protein Cry1A(b) and Cry1A(c), A fusion insecticidal gene GFM Cry1A with 1824bp has been synthesized. Plant expression vector pGB I 1214AB containing multi regulation elements has been constructed. In addition, another plant expression vector pGB I 1214ABC carrying bivalant insecticidal genes has been constructed, which harboring modified CpT I and GFM CrylA gene.
    根据苏云金芽胞杆菌杀虫晶体蛋白CrylA(b)和CrylA(c)的氨基酸序列,采用植物偏爱密码子,人工合成了全长1824bp的融合GFM CrylA Bt杀虫基因,并构建了带有多个表达调控元件的该基因高效植物表达载体pGBI1214AB,以及该基因和修饰豇豆胰蛋白酶抑制剂(CpTI)基因的双价杀虫基因植物表达载体pGBI1214ABC.
    The effects of cultural time, lysis solution and lytic conditions on production and purity of insecticidal protein from Bacillus thuringiensis strain HD-1 were investigated to get high quality insecticidal protein as a antigen.
    研究了培养时间对苏云金杆菌(Bacillusthuringiensis)HD-1菌株孢晶混合物的产量、杀虫晶体蛋白裂解液的种类和处理温度与时间对杀虫晶体蛋白提取率及纯度的影响。
    The best lysis solution used to treat the complex of endospore and crystal protein is 50? mmol/L Na_2CO_3. There are lots of complex protein in insecticidal protein when 40? mmol/L NaOH lytic solution is used to lytic complex of endospore and crystal protein.
    采用50mmol/LNa2CO3裂解液处理孢晶混合物,可获得纯度高的130kD杀虫晶体蛋白,而用40mmol/LNaOH提取的杀虫晶体蛋白中含有较多杂蛋白。
    The results show that when the pure Bt insecticidal protein is assayed by a indirect ELISA, there are no significant differences in sensitivity.
    结果表明,在采用纯的杀虫晶体蛋白进行灵敏度检测时,2种酶标抗体的测定结果非常相近,无明显差别,灵敏度可达7·8~15·6ng。 但在检测植物样本时,不同酶标抗体则存在差异。
 

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