芸薹属查尔酮异构酶基因家族RNA干扰载体的构建

作为类黄酮途径的第二步关键酶,查尔酮异构酶(chalcone isomerase,CHI)催化四羟基查尔酮成为柚皮素,用于合成各种类黄酮物质,影响多种性状。芸薹属含有多种重要的油料、蔬菜和观赏植物。本研究从甘蓝型油菜克隆了芸薹属CHI基因家族的干扰片段BCHII(761bp),采用NcoⅠ+AatⅡ和BamHⅠ+XbaⅠ双酶切方案分别将其反义片段和正义片段亚克隆到植物RNA干扰(RNAi)平台载体pFGC5941M的启动子与间隔区、间隔区与终止子之间,形成了11674bp的RNAi载体pFGC5941M-BCHII(简称pBCHII),多种PCR检测表明2个片段的插入方向正确,重组载体完整,将其转化根癌农杆菌菌株LBA4404以后获得了工程菌株LBA4404-pBCHII-①。本研究结果将促进研究CHI与芸薹属异花授粉、植株色彩、黄籽和抗逆性等性状的关系和开展相关分子育种。     

芸蔓属查尔酮异构酶基因家族RNA干扰载体的构建

作为类黄酮途径的第二步关键酶,查尔酮异构酶(chalcone isomerase,CHI)催化四轻基查尔酮成为抽皮素,用于合成各种类黄酮物质,影响多种性状.芸蔓属含有多种重要的油料、蔬菜和观赏植物.本研究从甘蓝型油菜克隆了芸蔓属CHI基因家族的干扰片段BCHII(761 bp),采用NcoI+AatII和BarnHI+XbaI双酶切方案分别将其反义片段和正义片段亚克隆到植物RNA干扰(RNAi)平台载体pFGC5941M的启动子与间隔区、间隔区与终止子之间,形成了11674by的RNAi载体pFGC5941M-BCHII(简称pBCHID,多种PCR检测表明2个片段的插入方向正确,重组载体完整,将其转化根癌农杆菌菌株LBA4404以后获得了工程菌株LBA4404-pBCHII-.本研究结果将促进研究CHI与芸蔓属异花授粉、植株色彩、黄籽和抗逆性等性状的关系和开展相关分子育种.     

芸薹属类黄酮3'-羟化酶基因家族RNA干扰载体的构建

目的：构建芸薹属类黄酮3′羟化酶（F3′H）基因家族的RNAi载体。方法：采用PCR克隆了607bp的芸薹属F3’H基因家族的RNAi片段,将其反义片段、正义片段分别采用NcoⅠ＋AatⅡ、BamHⅠ＋XbaⅠ插入到改进型植物RNAi基础载体pF-GC5941M的启动子与间隔区、间隔区与终止子之间,形成11366bp的重组载体pFGC5941M-BF3′HI（简称为pBF3′HI）,复合PCR鉴定后转化根癌农杆菌,获得工程菌株。结果：载体构建成功。结论：构建了RNAi载体pBF3′HI,可用于芸薹属植物花色、种皮色泽、茎叶色彩等性状的机理研究和遗传修饰。     

芥菜型油菜黄籽基因分子标记的开发

该研究在前期对‘吴旗黄芥’黄籽性状遗传图谱研究的基础上,利用白菜及拟南芥基因组信息,在芸薹属白菜A基因组BAC克隆KBrH105I15上设计了5对SCAR引物,在拟南芥第3染色体黄籽基因的同源区域At3g14120与At3g29615附近设计了6对IP引物,11对引物分别扩增F2群体(‘吴旗黄芥’ב武功褐芥’)的1 212个单株,开发与‘吴旗黄芥’黄籽基因更近的分子标记。结果表明:来自于白菜A基因组BAC克隆KBrH105I15上的Y12(Y12为共显性标记),以及来自于拟南芥第3染色体同源区域At3g24180的IP-6表现与‘吴旗黄芥’黄籽基因紧密连锁,其遗传距离分别为0.2和0.1cM,较之前最近的标记距离分别缩短0.3和0.2cM。这2个标记的开发对开展‘吴旗黄芥’黄籽基因的克隆奠定了基础。     

小麦淀粉合酶基因III片段的克隆及反义和RNA干扰载体的构建

采用RT-PCR方法从小麦品种豫教2号的发育籽粒中克隆出淀粉合酶III基因（starch synthase III, SSIII）部分cDNA片段(509bp) (GenBank No. EF466009),同源性比较结果显示,它与GenBank 上已报道的SSIII基因有高度同源性。以pWM101质粒为基础,构建了由35S启动子调控的SSIII基因的反义表达载体pWM101SSIII;另外,还以pFGC5941质粒为基础,构建了SSIII基因的RNAi干扰载体pFGC5941SSIII,这些载体的构建为研究此基因的功能打下了很好的基础。     

小麦淀粉合酶基因Ⅲ片段的克隆及反义和RNA干扰载体的构建

采用RT-PCR方法从小麦品种豫教2号的发育籽粒中克隆出淀粉合酶Ⅲ基因(starch synthase Ⅲ,SSⅢ)部分cDNA片段(509bp) (GenBank No.EF466009),同源性比较结果显示,它与GenBank 上已报道的SSⅢ基因有高度同源性.以pWM101质粒为基础,构建了由35S启动子调控的SSⅢ基因的反义表达载体pWM101SSⅢA;另外,还以pFGC5941质粒为基础,构建了SSⅢ基因的RNAi干扰载体pFGC5941SSⅢsa,这些载体的构建为研究此基因的功能打下了很好的基础.     

pFGC5941的改造及拟南芥NAC1和SIP1双基因反义表达载体的构建和遗传转化

拟南芥中的SIP1基因编码的蛋白与拟南芥盐胁迫应答中的关键蛋白SOS2存在互作关系,而NAC1为拟南芥中介导生长素信号促进其侧根发生的蛋白。本研究中我们将SIP1基因和NAC1正义基因以及SIP1基因和NAC1反义基因分别整合到一个经改造的具有2个35S启动子的可用于双基因表达的载体pFGC5941S中,构建了两个双基因表达载体pFGC5941S SIP1 NAC1 sense和pFGC5941S SIP1 NAC1 anti。并将这两个载体通过农杆菌介导的方法转化到野生型拟南芥中,共获得15株转基因植株。对这些转基因植株进行盐胁迫实验发现,在含75mmol·L-1 NaCl的MS培养基上,相比于野生型,pFGC5941S SIP1 NAC1 sense转基因植株主根增长,侧根数量明显增多,而pFGC5941S SIP1 NAC1 anti转基因植株长势与野生型苗相似。由此我们推测可能只有当SIP1和NAC1同时过表达时,才会促进盐胁迫下拟南芥侧根的发育。     

pFGC5941的改造及拟南芥NAC1和SIP1双基因反义表达载体的构建和遗传转化

拟南芥中的SIP1基因编码的蛋白与拟南芥盐胁迫应答中的关键蛋白SOS2存在互作关系,而NACl为拟南芥中介导生长素信号促进其侧根发生的蛋白。本研究中我们将SIPl基因和NAC1正义基因以及SIP1基因和NAC1反义基因分别整合到一个经改造的具有2个35S启动子的可用于双基因表达的载体pF—GC5941S中,构建了两个双基因表达载体pFGC5941S-SIP1-NAC1-sense和pFGC5941S-SIP1-NACl-anti。并将这两个载体通过农杆菌介导的方法转化到野生型拟南芥中,共获得15株转基因植株。对这些转基因植株进行盐胁迫实验发现,在含75mnlol·L-1 NaCl的MS培养基上,相比于野生型,pFGC5941S-SIP1-NAC1-sense转基因植株主根增长,侧根数量明显增多,而pFGC5941S-SIP1-NAC1-anti转基因植株长势与野生型苗相似。由此我们推测可能只有当SIP1和NAC1同时过表达时,才会促进盐胁迫下拟南芥侧根的发育。     

油菜BnCr4基因RNAi载体构建及植株转化

该实验构建了含甘蓝型油菜黄化相关基因BnCr4特异片段反向重复结构的RNA干扰(RNAi)载体pFGC5941-Cr4,通过根癌农杆菌介导转化油菜,获得47株抗Basta的抗性再生油菜植株,其中10株经PCR鉴定为阳性转基因植株.随机选取3株经鉴定的转基因阳性油菜植株进行半定量RT-PCR分析,结果显示,相对于非转基因的野生型油菜,3株转基因植株中BnCr4基因的表达量分别降低了78.5%、8.5%、11.8%,表明该干扰载体转入油菜能特异引起植株BnCr4基因表达量下降.     

甘蓝型油菜Fad2与Fae1基因双干扰RNAi载体的构建

将来源于甘蓝型油菜XY15的Fad2与Fae1基因编码区,长度分别为349bp和426bp的两个保守序列片段连接成807bp的大片段。然后分别以正反向插入到pFGC5941干扰载体查耳酮合成酶内含子的两端,构建成RNAi载体。以期在菜籽中转录后能有效抑制Fad2与Fae1两个基因的表达。所构建的RNAi载体最终序列全长3kb,经限制性内切酶消化、PCR扩增验证和序列测定,证明目标序列与GenBank数据库中的碱基序列一致,并且为正反向插入到pFGC5941载体上。通过农杆菌介导的油菜转化,已获得油菜抗性再生植株144个。     

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