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。     

芸薹属原花青素跨膜转运蛋白TT12、AHA10基因家族RNA干扰载体的构建

拟南芥TT12和AHA10基因分别编码MATE类型和H+泵类型的跨膜转运蛋白,介导原花青素单体向液泡的转运,对种皮色素的积累起重要作用。甘蓝型油菜是重要的油料作物,但缺乏黄籽基因型,现有黄籽材料表型不稳定,黄籽性状的分子机理不清,缺乏相关的分子育种。克隆了芸薹属TT12和AHA10基因家族的RNA干扰载体片段BTT12I和BA-HA10I,以基于pFGC5941而改造的植物RNA干扰基础载体pFGC5941M为骨架,构建了相应的RNAi载体pFGC5941M-BTT12I(简称pBTT12I)和pFGC5941M-BAHA10I(简称pBAHA10I),通过分子鉴定后转化农杆菌,获得工程菌株,有利于通过基因沉默技术研究TT12和AHA10基因在芸薹属种皮色素跨膜转运中的作用和机理,探索黄籽性状的分子育种。     

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.     

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。     

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。     

携带大片段BIBAC克隆在不同农杆菌中的遗传稳定性研究

大片段克隆在农杆菌中的稳定性是利用可转化大片段载体进行农杆菌介导遗传转化的关键问题.选用插入片段分子质量分别为150kb和50kb的BIBAC克隆,测定了在3种农杆菌AGL-1,EHA105和LBA4404中的遗传稳定性.从第1、3、5次继代培养的农杆菌中抽提的质粒及酶切结果显示,由于农杆菌背景质粒的干扰,难以判断质粒的降解与否.将农杆菌质粒再转化到大肠杆菌宿主中,发现来自农杆菌AGL-1和EHA105的质粒出现了明显的降解,片段变小,而来自农杆菌LBA4404的质粒没有变化.结果表明,大片段BIBAC质粒在不同农杆菌菌株中的稳定性不同,在农杆菌LBA4404中比较稳定,适合用于遗传转化.     

植物查尔酮异构酶研究进展

黄酮类化合物属于多酚类次生代谢物,具有广泛的药用价值。查尔酮异构酶（CHI）是黄酮类代谢途径中的一个关键酶,催化分子内环化反应,使双环的查尔酮转化为有生物学活性的三环（2S）-黄烷酮。植物体内的CHI活性与类黄酮物质的合成有着密切联系,CHI转基因研究对于提高植物类黄酮含量有重要意义。简要概述了查尔酮异构酶的结构特点、催化反应机理以及CHI转基因的研究进展。     

水稻OsNCED3基因的RNAi载体构建

水稻OsNCED3基因是水稻抗逆过程中重要的基因之一.以水稻中花10号幼苗为材料,提取基因组DNA.设计引物扩增区段cDNA并引入相应的酶切位点,以基因组DNA作为模板,进行RNAi-OsNCED3顺式和反式目的片段的PCR扩增.将PCR产物连接到pMD19-T载体上,经酶切和PCR检测后进行测序.测序结果表明:RNAi-OsNCED3顺式和反式目的片段均已正确的连接到pMD19-T载体上.然后将RNAi-OsNCED3顺式和反式目的片段通过酶切和连接,连接到含有发夹结构的质粒pFGC5941上.PCR及双酶切结果显示,构建的pFGC5941-OsNCED3即RNAi-OsNCED3载体结构完整.     

分蘖洋葱查尔酮异构酶基因的克隆及表达载体的构建

旨在得到分蘖洋葱查尔酮异构酶基因,探明该基因的功能。克隆查尔酮异构酶基因并构建了其超表达和干扰载体,以期得到用于研究查尔酮异构酶基因功能的表达载体。从分蘖洋葱叶片中克隆得到查尔酮异构酶基因cDNA全长743 bp,GenBank登录号为KJ489062。结果显示,该基因633 bp的开放读码框编码210个氨基酸的多肽序列。将PCR扩增克隆得到的分蘖洋葱查尔酮异构酶基因片段连接到干扰载体pCAMBIA1 301和超表达载体SOL2095中,成功构建了35S启动子控制的植物表达双元载体pCAMBIA1301-CHI和SOL2095-CHI。     

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