柔嫩艾美耳球虫孢子化卵囊cDNA文库的构建

用建立表达性文库的方法 ,构建了Eimeriatenella孢子化卵囊噬菌体ZAP表达性cDNA文库。首先用TRIzol试剂盒从E .tenella孢子化卵囊中提取总RNA ,再用Oligo(dT)₁₂纤维素柱从总RNA中分离mRNA ,以mRNA为模板 ,Oligo(dT)₁₈Linker Primer为引物 ,反转录合成cDNA第一链 ,再在DNA聚合酶Ⅰ作用下置换合成第二链cDNA。cDNA第二链合成后用PfuDNA聚合酶补平XhoⅠ位点 ,再与EcoRⅠAdapters连接 ,经XhoⅠ酶切后 ,凝胶电泳回收 500bp～4.0kp之间的cDNA片段 ,纯化后的双链cDNA与载体ZAPExpressvector连接。体外包装后得到E .tenella孢子化卵囊的cDNA表达性文库 ,经测定该文库的容量为 6×10⁶ ,扩增后文库的滴度为 1×10¹¹ Pfu mL ,经PCR测定 ,该文库的重组率为 96%。     

毛冠鹿大脑组织全长cDNA文库构建

运用SMART技术构建了毛冠鹿(Elaphodus cephalophus)大脑组织全长cDNA文库。提取大脑组织总RNA,Oligotex mRNA Kit纯化、获得poly(A) RNA,以CDSⅢ/3′PCR引物进行逆转录,LD-PCR扩增获得全长双链cDNA,经SfiⅠ酶切及柱层析分离后,500 bp以上的片段与载体λTripIEx2连接,体外包装得到cDNA文库。经鉴定原始文库滴度为5.1×105pfu/ml,扩增后文库滴度为1.5×109pfu/ml,重组率达到85%以上,插入片断平均长度约为1.0 kb,说明构建文库质量符合要求,可用于大脑特异表达基因的筛选。从该文库中克隆到了rig基因全长,包含5′和3′非编码区,从第43至477个核苷酸为一完整阅读框(ORF),此阅读框可编码一个145氨基酸的rig蛋白。     

麋鹿血液cDNA文库的构建

为分离出MHCⅠ、Ⅱ基因,利用TRIzol 试剂从麋鹿血液组织中提取总RNA,经mRNA 纯化试剂盒纯化后,根据Stratagene 公司的cDNA 文库构建系统构建了麋鹿血液组织的cDNA 文库。初始文库滴度为1.96 ×106 pfu/ ml,总库容为1.96 ×106 个独立克隆;cDNA 插入片段平均长度约为1.0 kb;重组率为95.6 % 。文库各项指标均达标准经典cDNA 文库的基本要求。利用本实验室设计的可扩增MHCⅡ类DQA 基因第二外显子的引物检测扩增后的文库,得到了特异性非常好的目标条带,说明本文所建文库可以用于麋鹿MHC 相关基因的分离。     

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.     

野蚕黑卵蜂寄主识别利它素的cDNA文库构建

根据家蚕性附腺中含有的利它素能诱导野蚕黑卵蜂寄生涂有该利它素的人造卵及能用家蚕卵繁育野蚕黑卵蜂的特性 ,用化蛹 9天的家蚕雌蛹性附腺分泌部的高丰度的mRNA构建了富含利它素基因的cDNA文库。构建成的未扩增文库的滴度为 3.2 9× 10 4 pfu μL ,重组率为 90 .0 5 % ,扩增后的文库总滴度为 1.5 6× 10 7pfu μL。用PCR方法对总文库和随机挑取的噬菌斑进行扩增 ,表明λZiplox载体中已含有大小为 0 .5～ 8.0kb的cDNA插入片段 ,并在 2 .5kb、1.1kb和 0 .75kb处cDNA插入片段相对集中。所有分析结果表明 ,采用特定的发育时期和特定的组织提取的mRNA构建而成的cDNA表达文库具有较高频率的利它素基因片段和合适的滴度 ,定向插入技术并结合λZiplox噬菌体的表达功能 ,使构建的cDNA文库具有表达外源DNA的功能 ,有利于进行利它素基因的免疫筛选。     

柔嫩艾美耳球虫孢子发育阶段虫体抑制性消减文库的构建

为了分离鉴定柔嫩艾美耳球虫（Eimeria tenella）孢子发育阶段虫体的差异表达基因,分别以柔嫩艾美耳球虫未孢子化卵囊和孢子化卵囊为驱动组、子孢子为实验组,或未孢子化卵囊为驱动组、孢子化卵囊为实验组,利用抑制性消减杂交（SSH）技术,构建了2个子孢子cDNA消减文库和1个孢子化卵囊cDNA消减文库。随机从3个cDNA消减文库中分别挑取50个克隆,经PCR鉴定2个子孢子cDNA消减文库的重组率都为96%,孢子化卵囊cDNA消减文库的重组率为98%。从每个文库中随机挑取50个克隆测序,并进行同源性比较分析,结果显示：从孢子化卵囊cDNA消减文库中获得了13个单一有效序列,其中8个EST与已知蛋白同源性很高;从2个子孢子cDNA消减文库中共获得了40个单一有效序列,其中9个EST与已知蛋白同源,其余可能为柔嫩艾美耳球虫的新基因。这些结果为分离柔嫩艾美耳球虫新功能基因和进一步探索防治球虫病的方法提供了理论基础。     

柔嫩艾美耳球虫孢子发育阶段虫体抑制性消减文库的构建

为了分离鉴定柔嫩艾美耳球虫(Eimeria tenella)孢子发育阶段虫体的差异表达基因,分别以柔嫩艾美耳球虫未孢子化卵囊和孢子化卵囊为驱动组、子孢子为实验组,或未孢子化卵囊为驱动组、孢子化卵囊为实验组,利用抑制性消减杂交(SSH)技术,构建了2个子孢子cDNA消减文库和1个孢子化卵囊cDNA消减文库。随机从3个cDNA消减文库中分别挑取50个克隆,经PCR鉴定2个子孢子cDNA消减文库的重组率都为96%,孢子化卵囊cDNA消减文库的重组率为98%。从每个文库中随机挑取50个克隆测序,并进行同源性比较分析,结果显示:从孢子化卵囊cDNA消减文库中获得了13个单一有效序列,其中8个EST与已知蛋白同源性很高;从2个子孢子cDNA消减文库中共获得了40个单一有效序列,其中9个EST与已知蛋白同源,其余可能为柔嫩艾美耳球虫的新基因。这些结果为分离柔嫩艾美耳球虫新功能基因和进一步探索防治球虫病的方法提供了理论基础。     

中华大蟾蜍精巢组织全长cDNA文库的构建及泛素延伸蛋白基因Bg-ubi52的获得

运用SMART技术构建了中华大蟾蜍(Bufo bufo gargarizans)精巢全长cDNA文库。提取中华大蟾蜍精巢总RNA,用Clontech公司SMARTTM cDNA文库构建试剂盒反转录合成第一链cDNA,LD-PCR扩增获得全长cDNA双链;经SfiⅠ酶切、层析柱分离后,500bp以上的片段与λTriplEx2载体连接并包装,建成原始文库。经鉴定,原始文库滴度为2.21×106pfu/ml,重组率为91%;文库扩增后的滴度为2.94×109pfu/ml,重组率为93.7%。插入片段大小分布于0.4~2.0kb之间,平均长度约为1.0kb,说明已构建文库质量较高,为进一步筛选、克隆精巢特异表达基因奠定了基础。从该文库中克隆到了泛素延伸蛋白基因,全长561bp,包含完整的5′和3′非编码区,编码128个氨基酸,即泛素的76个氨基酸后融合了52个氨基酸的核糖体L40蛋白。     

Gateway(R)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1×107cfumL,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。     

铁皮石斛cDNA文库的构建及分析

首次报道铁皮石斛cDNA文库的构建及其分析.以Trizol一步法从4年生的铁皮石斛叶片中提取总RNA,分离纯化mRNA,LD-PCR法反转录合成双链cDNA,以λTripEX2为载体,构建铁皮石斛cDNA文库.原始文库的滴度为4.3×105 pfu/mL,总克隆数为3.1×105,重组率为97.6%,扩增后文库总滴度为6.8×109pfu/mL,对随机挑取的噬菌斑进行PCR鉴定,结果插入片段多分布在0.5～2.0kb之间,绝大部分在1.2～1.7kb左右,文库质量鉴定结果表明,构建的铁皮石斛cDNA文库具有较好的库容量、较高的重组率以及较大的插入片段.cDNA文库的构建为铁皮石斛药用成分相关功能基因的研究奠定了基础.     

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