Gateway○R技术构建交链孢菌JH505 cDNA文库

GatewayD○R技术构建cDNA文库,利用λ噬菌体的位点特异性重组,避免使用限制性内酶切割cDNA,能够解决常规方法构建cDNA文库的技术缺陷。首次应用GatewayD○R技术构建交链孢菌cDNA文库,经检测cDNA入门文库的滴度达到1×10.7cfu/mL,文库总容量为9×10.7cfu,平均插入片段为1510bp。通过LR重组把入门文库转换为表达文库,表达文库的滴度为1.58×10.6cfu／mL,文库总容量为6.32×10.6cfu,平均插入片段大小为1680bp。表达文库的构建为进一步克隆植物激活蛋白基因打下了基础。     

Gateway技术构建交链孢菌JH505 cDNA文库

Gateway(R)技术构建Cdna文库,利用λ噬菌体的位点特异性重组,避免使用限制性内酶切割Cdna,能够解决常规方法构建Cdna文库的技术缺陷.首次应用Gateway(R)技术构建交链孢菌Cdna文库,经检测Cdna入门文库的滴度达到1×107cfu/Ml,文库总容量为9×107cfu,平均插入片段为1510bp.通过LR重组把入门文库转换为表达文库,表达文库的滴度为1.58×106cfu/Ml,文库总容量为6.32×106cfu,平均插入片段大小为1680bp.表达文库的构建为进一步克隆植物激活蛋白基因打下了基础.     

鸡胚成纤维细胞cDNA表达文库的构建

鸡胚成纤维细胞(CEF)是研究鸡传染性法氏囊病病毒(IBDV)的主要细胞材料,而构建CEF的cDNA表达文库是筛选IBDV在CEF中的细胞受体,研究细胞嗜性的基础平台。采用Gateway技术构建CEF的表达文库,避免使用限制性内切酶切割cDNA,能够解决常规方法构建cDNA文库的技术缺陷。该技术将CEF的mRNA分离纯化后,以5′端生物素标记的Oligo(dT)primer为引物反转录后连接Adapter,层析柱纯化,通过BP重组反应构建cDNA入门文库,其平均滴度为1.1×106cfu/mL,文库总容量为1.2×107cfu,平均插入片段为2243bp,重组率为100%。通过LR重组反应将入门文库转换为表达文库,经测定平均滴度为5×105cfu/mL,文库总容量为5.5×106cfu,平均插入片段为2411bp,重组率为100%。结果表明,所构建的文库具有较高的重组率和较大的库容量,可作为较高质量的文库来研究IBDV的相关基因,为研究病毒受体和病毒入侵途径,进一步了解IBDV的致病机理奠定了基础。     

毛竹笋全长cDNA文库构建

Gateway技术构建cDNA文库,利用了λ噬菌体的位点特异性重组,避免使用限制性内酶切割cDNA,能够解决常规方法构建cDNA文库的技术缺陷。应用Gateway技术构建毛竹笋cDNA文库,经检测cDNA入门文库的滴度达到1.7×106cfu/mL,文库总容量为8.5×106cfu,平均插入片段在1.0 kb以上。毛竹笋文库的构建为进一步克隆毛竹纤维化分子机理打下了基础。     

草地螟中肠cDNA文库的构建及初步鉴定

以草地螟中肠为材料,分别采用、Oligotex mRNA kit、ZAP-cDNA(R)synthesis kit和ZAP-cDNA(R)Gigapack(R)Ⅲ Gold Cloning Kit提取草地螟幼虫中肠总KNA,分离纯化mRNA并构建了草地螟中肠cDNA表达文库.测得文库的原始滴度为3.73×106pfu/ml,蓝白斑测定重组率迭99.7%,插入片段平均长度约为1.8 kb.草地螟中肠cDNA表达文库的成功构建为利用文库筛选中肠靶标蛋白提供了材料来源,为研制、开发草地螟生防药剂的新靶标以及研究其作用的分子机制奠定了基础.     

SPF鸡肝脏细胞cDNA文库的构建及鉴定

旨在构建SPF鸡肝脏组织细胞的cDNA文库并且对文库质量进行鉴定。用TRIzol方法从SPF鸡肝脏组织细胞中提取总RNA,用紫外分光光度仪测定其含量后,应用SMART技术经过LD-PCR合成双链cDNA。利用CHROMA SPIN-400纯化柱纯化得到双链cDNA,并与线性pGADT7-Rec共转化进酵母Y187感受态,以同源重组的方式在酵母细胞内构建鸡肝脏酵母双杂交cDNA表达文库。结果显示,成功构建含有1.70×10~7个重组子的SPF鸡肝脏细胞cDNA文库,插入片段多数在0.4~2.0 kb之间,重组率达100%,重组子中平均插入的片段长度为1.0 kb,文库滴度为1.30×10~7 cfu/mL。结果表明,该文库达到了高质量文库所应具备的条件,为进一步筛选此文库与ARV相互作用的宿主蛋白以及研究其功能提供了数据依据。     

巨型艾美耳球虫孢子化卵囊cDNA文库的构建

为构建巨型艾美耳球虫(Eimeria maxima)孢子倾卵囊cDNA表灰文库,从E.maxima孢子化卵囊中提取总RNA,以总RNA为模板、λTriplex2TM为栽体,利用SMARTTM cDNA文库构建试剂盒构建全长cDNA表达文库.经测定构建的E.maxima cDNA表达文库的原始文库容量为1×106 pfu/ml,扩增后的文库滴度为5×1010pfu/ml,重组率为95%,插入片断主要集中在0.5～1kb之间.根据已知序列设计引物,能从文库中扩增出编码E.maxima免疫球蛋白重链结合蛋白的基因序列片段.结果 表明所构建的E.maxima cDNA表达文库质量良好,为克隆、筛选E.maxima的功能性基因奠定了基础.     

两个水稻品种(系)酵母双杂交cDNA文库的构建和比较分析

"武育粳3号"和"KT95-418"为两个遗传背景基本一致而对水稻条纹叶枯病表现为明显抗性差异的粳稻(Oryza sativa L.ssp. japonica)品种(系).利用SMART技术合成双链cDNA后,通过SfiⅠ酶切位点将cDNA片段定向插入到改造的载体NpGADT7中,构建了两品种(系)水稻的酵母双杂交cDNA文库.检测结果表明:所构建的两个文库库容量均大于1.0×106 cfu;初始文库滴度分别为1.0×1010 cfu/mL和5.0×1010 cfu/mL,扩增文库滴度均为1.0×1011 cfu/mL;两文库重组率均大于95 %;"武育粳3号"文库cDNA插入片段长度集中分布在700 bp～800 bp之间,"KT95-418"集中分布在750 bp～1 000 bp之间;小规模测序结果表明两文库中全长基因的比例均超过60 %.两品种(系)水稻酵母双杂交cDNA文库的构建为筛选分离抗病相关的变异基因及开展寄主与水稻条纹病毒(Rice stripe virus, RSV)互作的研究奠定了基础.     

Investigation of the mechanism of temperature sensitivity of the electroreceptors of ampullae of lorenzini

In experiments on Black Sea skates (Raja clavata), the potential of the receptor epithelium of the ampullae of Lorenzini and spike activity of single nerve fibers connected to them were investigated during electrical and temperature stimulation. Usually the potential within the canal was between 0 and –2 mV, and the input resistance of the ampulla 250–400 k. Heating of the region of the receptor epithelium was accompanied by a negative wave of potential, an increase in input resistance, and inhibition of spike activity. With worsening of the animal's condition the transepithelial potential became positive (up to +10 mV) but the input resistance of the ampulla during stimulation with a positive current was nonlinear in some cases: a regenerative spike of positive polarity appeared in the channel. During heating, the spike response was sometimes reversed in sign. It is suggested that fluctuations of the transepithelial potential and spike responses to temperature stimulation reflect changes in the potential difference on the basal membrane of the receptor cells, which is described by a relationship of the Nernst's or Goldman's equation type.I. P. Pavlov Institute of Physiology, Academy of Sciences of the USSR, Leningrad. I. M. Sechenov, Institute of Evolutionary Physiology and Biochemistry, Academy of Sciences of the USSR, Leningrad. Pacific Institute of Oceanology, Far Eastern Scientific Center, Academy of Sciences of the USSR, Vladivostok. Translated from Neirofiziologiya, Vol. 12, No. 1, pp. 67–74, January–February, 1980.     

Principles of organization and evolution of systems of regulation of functions

Evolution of living organisms is closely connected with evolution of structure of the system of regulations and its mechanisms. The functional ground of regulations is chemical signalization. As early as in unicellular organisms there is a set of signal mechanisms providing their life activity and orientation in space and time. Subsequent evolution of ways of chemical signalization followed the way of development of delivery pathways of chemical signal and development of mechanisms of its regulation. The mechanism of chemical regulation of the signal interaction is discussed by the example of the specialized system of transduction of signal from neuron to neuron, of effect of hormone on the epithelial cell and modulation of this effect. These mechanisms are considered as the most important ways of the fine and precise adaptation of chemical signalization underlying functioning of physiological systems and organs of the living organism