沙冬青高活性抗冻蛋白的发现

关于植物的抗冻作用机制已有许多论述［１—３］,然而低温植物中是否存在高活性的抗冻蛋白？抗冻作用的主要作用机制是什么？为研究这些问题,我们以沙冬青（Ａｍｍｏｐｉｐｔａｎ－ｔｈｕｓｍｏｎｇｏｌｉｃｕｓ）为材料,分离和部分纯化了植物抗冻蛋白。经测定,其分子量包括４５．７ｋＤ和８１．２ｋＤ等,在５０ｍｇ／ｍｌ的浓度下,其熔点约为－１５℃,所以它的抗冻活性比已经发现的鱼类［４—６］或黑麦抗冻蛋白（在６０ｍｇ／ｍｌ的浓度时冰点为－１．１℃）［２］活性高得多。在１００—２００倍的相差显微镜下可观察到其冰晶呈多…     

高寒山区牧草根中丙二醛、渗透调节物、多胺季节动态与抗冻力关系研究

温带地区的高山多年生草本植物可在－３０℃组织结冰状况下生存,然而人们并不了解其抗冻的生理机理。本研究目的拟通过测定自然生境下生长的４种高寒山区禾本科牧草（无芒雀麦（Ｂｒｏｍｕｓ ｉｎｅｒｍｉｓ Ｌｅｙｓｓ．）、花雀麦（Ｂｒｏｍｕｓ　 ｓｉｎｅｎｓｉｓ　 Ｋｅｎｇ．）、垂穗披碱草（Ｅｌｙｍｕｓ ｎｕｔａｎｓ Ｇｒｉｓｅｂ．）、草地早熟禾（Ｐｏａ ｓｐｈｙｏｎｄｙｌｏｄｅｓ　 Ｔｒｉｎ．）根中渗透调节物、膜脂过氧化产物、多胺含量季节变化,以了解他们与牧草抗冻的关系。结果表明,在晚秋（９月１～１５日）牧草根中ＭＤＡ含量增高,尔后下降,冬季保持恒定。总碳水化合物（ＴＮＣ）,可溶性糖、脯氨酸、可溶性蛋白质含量随晚秋气温下降而增加,在１１月达到最高,尔后下降,且持续到翌年春季。随晚秋气温下降从９月到１１月根中多胺含量迅速增加,４种牧草平均增加１８０％,其中亚精胺（Ｓｐｄ）占多胺含量的５３％。在１１月牧草根中多胺几乎完全消失。上述物质在晚秋入冬增加正好与牧草抗冻锻炼时间相吻合,因而是植物抗冻适应的重要生理响应和植物越冬的低温保护物质。他们在降低细胞冰点、防止细胞结冰引起的膜机械伤害,抑制膜脂过氧化保护膜稳定性方面具有重要作用。     

热电偶湿度计和冰点渗透计测定PEG6000渗透势的比较

在植物抗旱生理研究中,常用聚乙二醇（PEG）水溶液作为渗透胁迫的条件。而PEG6000对植物产生的胁迫作用要比相对分子质量低的PEG效果更好[4]。因此,测定不同浓度PEG6000溶液的渗透势,对于正确设置植物渗透胁迫条件是很重要的。本文用热电偶湿度计和冰点渗透计两种方法,比较测定了不同浓度PEG6000溶液的渗透势。材料与方法将PEG6000配制成一系列不同浓度的水溶液：100、200、300、400‘500g·（100ml水）－’。用PEG溶液湿润滤纸圆片,放入热电偶湿度计测定小室中。小室壁涂上凡土林,以减少壁对水分的吸收。热电偶探头滴加标准…     

昆虫抗冻蛋白的结构与生物学特性研究

抗冻蛋白(antifreeze proteins AFPs)是一类抑制冰晶生长的蛋白质,它能以非依数性形式降低溶液的冰点而对其熔点影响甚微,因而也被称作热滞蛋白。近几年来对于昆虫抗冻蛋白的研究取得了较快的发展,已有20多种昆虫抗冻蛋白被分离纯化。就昆虫抗冻蛋白的结构特征、生物学特性以及在农业、医学和食品工业等方面的应用进行介绍。     

过冷却和昆虫的耐寒性

过冷却（超冷,supercooling）现象,由Fahrenheit（1842）发现,即为在一定的条件下,当温度低于水的冰点时,水仍能保持液体状态的现象。1过却点及测定方法Beclune6Cw（1901）利用电热测温,首次发现动物在结冰时的过冷却现象。过冷却点是指在逐步冷却过程中自发地结冰产生时的温度。过冷却的能力是指液体的熔点与过冷却点倍CPsllpereoolingpoint）间的差异,通常用过冷却点来表示。当自发的结冰发生时,通过测定潜热的消政。能够准确地测定SCP。特别是陆生节肢动物的超冷极限,可以方便地以过冷却点的形式测到。过冷却点测定最常用的…     

neo Trichosanthin及其突变体Y70A neo Trichosanthin的晶体结构研究

天花粉蛋白（Ｔｒｉｃｈｏｓａｎｔｈｉｎ,ＴＣＳ）的一个亚型,ｎｅｏＴｒｉｃｈｏｓａｎｔｈｉｎ（ｎ－ＴＣＳ）,及其突变体Ｙ７０Ａｎ－ＴＣＳ被克隆和表达为重组蛋白。用悬滴汽相扩散法得到ｎ－ＴＣＳ和Ｙ７０Ａｎ－ＴＣＳ的晶体。在ＭａｒＲｅｓｅａｒｃｈ面探测器系统上分别收集了０．２０ｎｍ和０．２０５ｎｍ分辨率的Ｘ射线衍射数据。用同晶差值傅立叶法解析了结构。最后晶体学Ｒ因子分别为０．１８３和０．１８４。键长的     

诱发RNase A活性的最低水含量阈值的测定

通过ＲＮａｓｅＡ的水合等温线,确定ＲＮａｓｅＡ吸附水时所处的相对湿度及其水合值的关系;通过将酶与底物的水溶液在低温下混合,冰冻和冷干后的酶－底物复合物在不同相对湿度下与水相互作用（酶反应）两天,经０．０１ＮＮａＯＨ猝灭反应后用紫外差吸收光谱法测相对酶活;从而建立了含微量水的ＲＮａｓｅＡ活性的测定方法,并得到诱发ＲＮａｓｅＡ活性的最低水含量阈值约为０．１６克水／克酶,即当一个ＲＮａｓｅＡ分子周围有约１２２个水分子时ＲＮａｓｅＡ开始具有生物活性。     

从绿茶中浸提茶氨酸

茶氨酸,学名为N-乙基-γ-L-谷氨酰胺,化学式为C7H14O3N2,属于酰胺类化合物。主要存在于绿茶叶中。纯净的茶氨酸为白色针状晶体;具有焦糖香和类似于味精(谷氨酸钠)的鲜爽味;难溶于无水乙醇、乙醚和汽油等有机溶剂,极易溶于水;常压下,熔点为490～491K(同时发生分解反应)。茶氨酸在水溶液中能够发生弱电解质行为,其水溶液显示弱酸性：在6mol／LHCl溶液中还能够发生水解反应,生成L-谷氨酸[又称左     

一种用于优化PCR引物设计的短链DNA熔点分析方法

目前,PCR引物设计主要依赖于软件对引物熔点的模拟计算,而PCR退火条件的优化需进行不同条件下的扩增实验。为开发一种可高效、精确评价引物和确定退火条件的方法,本研究采用高分辨率熔解曲线（high resolution melting,HRM）测定技术直接分析短链DNA的熔点,用于引物优劣性的评价,并为退火条件的优化提供参考。本文用HRM法直接测定了非完全互补的双链DNA以及DNA发卡结构的熔点,结果显示：（1）与完全互补的双链DNA相比,较为稳定的单碱基错配A?G、G?G和T?G的熔点只降低2℃ ~ 3℃,部分双碱基错配的熔点只降低4℃ ~ 6℃,单碱基突出熔点只降低4℃~ 5℃。因此,如果采用的退火温度不当,部分错配的非目的模板可能会被扩增。（2）即使发卡结构的茎干区只有6 bp,当其环区碱基少于10 nt时,其熔点也可达到60℃以上。此外,环区的长度对发卡熔点也有较大影响。根据本研究结果发现,引物设计时应尽量避免模板引物结合区同其邻近的30 nt碱基有6 bp以上的互补部分。综上所述,本研究证明HRM熔点法是一种高效评价引物及确定退火温度的方法。     

天花粉蛋白小结构域N端同源片段的溶液构象：T18肽的圆二色性和荧光研究

应用圆二色性,内源荧光和疏水荧光探针法进一步研究Ｔｌ８肽的溶液构象及其相互转化。发现Ｔ１８肽在水溶液中为β折叠结构,且在高浓度（＞１ｍｇ／ｍｌ）时形成疏水聚合物;Ｌｙｓ１５和Ｉｌｅ３－Ｉｌｅ４是形成β折叠疏水簇的关键因素。并讨论了蛋白质肽链和溶剂环境对肽段二级结构的调制作用。     

Antifreeze proteins in higher plants

Overwintering plants produce antifreeze proteins (AFPs) having the ability to adsorb onto the surface of ice crystals and modify their growth. Recently, several AFPs have been isolated and characterized and five full-length AFP cDNAs have been cloned and characterized in higher plants. The derived amino acid sequences have shown low homology for identical residues. Theoretical and experimental models for structure of Lolium perenne AFP have been proposed. In addition, it was found that the hormone ethylene is involved in regulating antifreeze activity in response to cold. In this review, it is seen that the physiological and biochemical roles of AFPs may be important to protect the plant tissues from mechanical stress caused by ice formation.     

抗冻蛋白研究进展

抗冻蛋白是一类具有热滞效应、冰晶形态效应和重结晶抑制效应的蛋白质。简单介绍了各种抗冻蛋白的生化特征、作用机制及其应用研究 ,并对抗冻蛋白的基因和基因工程研究作了较为系统的综述。     

Antifreeze glycoproteins

Antifreeze glycoproteins (AFGPs) are a novel class of biologically significant compounds that possess the ability to inhibit the growth of ice both in vitro and in vivo. Any organic compound that possesses the ability to inhibit the growth of ice has many potential medical, industrial, and commercial applications. In an effort to elucidate the molecular mechanism of action, various spectroscopic and physical techniques have been used to investigate the solution conformations of these glycoproteins. This review examines the characterization of AFGPs and potential biological applications relating to stabilization of lipid membranes and vitrification adjuvants.     

抗冻蛋白结构基因片段的克隆

为了研究和开发利用抗冻蛋白或多肽,木文通过聚合酶链式反应（ＰＣＲ）合成了抗冻蛋白１１０ｂｐ的结构基因片段,然后将该基因片段克隆到大肠杆菌质粒载体Ｐ（Ｂｌｕｅｓｃｒｉｐｔ）Ⅱｋｓ＋／－上,获得了重组质粒,经酶切证明,获得的重组质粒中含有抗冻蛋白结构基因片段,以供该基因在大肠杆菌或酵母中表达抗冻蛋白。     

植物抗冻基因研究进展

低温是作物生长一大限制因素,许多作物不能在低温下生长或者容易受到冻害,所以通过基因工程的方法,转入抗冻基因,是扩大作物生长区域,稳定作物产量的有效办法。目前通过对植物抗冻基因在抗冻机理、研究技术、应用等方面的研究,已经取得了一定的成果。综述抗冻基因的来源、抗冻机理、研究技术,并做适度的展望。     

昆虫抗冻蛋白的研究进展

抗冻蛋白是一类与冰晶有亲合力,能够与冰晶结合并抑制冰晶生长的蛋白或糖蛋白。自20世纪60年代以来,研究人员已经分别从鱼类、昆虫、植物、真菌和细菌中发现多种抗冻蛋白。其中已知鱼类抗冻蛋白有5种,也是研究最详细的。但是,近几年来发现昆虫抗冻蛋白活性普遍比较高,因此受到研究人员重视,研究取得了较快的发展。主要讨论昆虫抗冻蛋白的结构特点、抗冻活性、作用机制和应用,并分析目前的研究现状提出一些待解决的问题,以期望对昆虫抗冻蛋白的研究进行比较系统化的整理。     

Isolation and Identification of Antifreeze Protein with High Activity in Ammopiptanthus mongolicus

The authors have isolated and partial purified antifreeze protein antifreeze protein (AFP) produced endogenously in Arnrnopiptanthus rnongolicus. The results show that the partial purified AFP ranged in size including 45.7 kD, 81.2 kD, and so on. At 50 mg/mL protein concentration, the temperature of melting point is –15 ℃, and its freezing point is even lower. Therefore, the AFP activity exhibited by the Arnmopiptanthus mongolicus is higher than that observed for AFP found in polar fishes or in winter rye. In addition, by a phase contrast light photomicroscope, the author have observed the morphology of individual ice crystals formed in the solution, including squares, rectangles, cones, hexagons. The morphology of these ice crystals are similar to those of ice crystals observed in polar fishes and in cold-acclimation winter rye.     

Antifreeze proteins in Alaskan insects and spiders

Prior to this study, antifreeze proteins (AFPs) had not been identified in terrestrial arthropods from the Arctic or anywhere in Alaska. The hemolymph of 75 species of insects and six spiders from interior and arctic Alaska were screened for thermal hysteresis (a difference between the freezing and melting points), characteristic of the presence of AFPs. Eighteen species of insects and three spiders were shown to have AFPs. Ten of the insects with AFPs were beetles including the first species from the families Chrysomelidae, Pythidae, Silphidae and Carabidae. In addition, the first Neuropteran to have AFPs was identified, the lacewing Hemerobius simulans together with the second and third Diptera (the first Tipulids) and the second and third Hemiptera, the stinkbug Elasmostethus interstinctus (the first Pentatomid), and the water strider Limnoporus dissortis (the first Gerrid). Prior to this study, 33 species of insects and three spiders had been reported to have AFPs. Most AFP-producing terrestrial arthropods are freeze avoiding, and the AFPs function to prevent freezing. However, some of the AFP- producing insects identified in this study are known to be freeze tolerant (able to survive freezing) to very low temperatures (-40 to -70 degrees C).     

Purification and Identification of Antifreeze Proteins in Ammopiptanthus mongolicus

The conventional protein chromatography technique was adopted to purify the antifreeze proteins (AFPs) from the leaves of Ammopiptanthus mongolicus ( Maxim. ) Cheng f. Two bands on native PAGE gel showed thermal hysteresis activity, one was band Bi, whose thermal hysteresis was 0.46 cE at 8 g/L, which showed two bands (67 kD, 21 kD) on SDS-PAGE gel; the other was B3, whose thermal hysteresis was 0.45 cE at 10 g/L, and it contained only a single protein (39.8 kD). Both B1 and B3 are not glycoproteins, because neither do they interact with Shift-reagent, nor show ultraviolet characteristics of a typical glycoprotein.