冰核细菌及冰核基因的应用研究进展

引起水由液态变为固态的物质称为冰核或成核剂。冰核种类繁多,目前已发现4属23种或变种的细菌、4属11种或变种的真菌和1种病毒,它们都具成冰活性。细菌冰核是一类蛋白质,也称冰蛋白,由细菌冰核基因编码。作为生物冰核领域的研究重点,冰核细菌的研究已涉及到促冻杀虫、防霜冻、植物病害等多个领域;同时冰核细菌已成功地应用于人工降雪、制冷和高敏检测等方面,具有广阔的应用前景。主要对冰核细菌的应用研究现状和发展进行综述。     

冰核细菌生物学特性及其诱发植物霜冻机理与防霜应用

就国内外有关冰核细菌生物学特性及其诱发植物霜冻机理与防霜应用的研究进展作以概述。阐述了冰核细菌种类、分布、影响冰核活性的成冰因素,冰核活性等级划分、冰核细菌保存方法以及冰核细菌诱发植物霜冻机理;简介了冰核细菌分子生物学研究进展;药剂和生防菌能够防除植物上冰核细菌减轻或控制霜冻危害,并已取得成效,是防御植物霜冻的一条新途径。     

冰核细菌表达冰核蛋白特性的研究

选用１００２５Ａ和ＱＦ－９５－Ｆ１９两株分离自杨树的冰核活性细菌,探讨了两株菌不同生长阶段与它们冰核活性表达的特性。实验结果显示,冰核活性细菌在ＭＰＤＡ培养液中表达冰核蛋白的特性及活性与细菌浓度、菌龄以及培养的环境条件相关,两株菌在表达冰核活性时对培养基的营养组分没有表现出特殊的要求。同时还进一步阐明了不同生长温度冰核活性细菌对冰核蛋白表达的影响。     

冰核细菌表达冰核蛋白特性的研究*

选用１００２５Ａ和ＱＦ－９５－Ｆ１９两株分离自杨树的冰核活性细菌,探讨了两株菌不同生长阶段与它们冰核活性表达的特性。实验结果显示,冰核活性细菌在ＭＰＤＡ培养液中表达冰核蛋白的特性及活性与细菌浓度、菌龄以及培养的环境条件相关,两株菌在表达冰核活性时对培养基的营养组分没有表现出特殊的要求。同时还进一步阐明了不同生长温度冰核活性细菌对冰核蛋白表达的影响。     

冰核活性细菌(INAB)对菜青虫血淋巴免疫系统影响的研究

实验采用浸泡和喂食2种方法,研究了冰核活性细菌(INAB)在不同处理时间对菜青虫Pierisrapae血淋巴免疫系统的影响。结果表明,采用水浴法可使菜青虫血淋巴免疫力被破坏,而喂食法效果则不明显;同时结果还表明冰核活性细菌主要是在低温条件下通过体表侵入菜青虫血淋巴而影响其免疫力。     

冰核活性细菌基因的研究进展及其应用

冰核活性细菌是一类可以诱导过冷水结冰而导致霜冻灾害的细菌,它已成为一种重要的生物资源获得广泛的研究与开发,在基础理论和应用研究方面取得了较大进展。近些年来,许多国家的学科研究者对于冰核基因的研究与开发开展了大量的工作。自1985年克隆出了第一个冰核基因后,目前已经从冰核细菌中克隆了8个冰蛋白基因并完成了测序。冰核活性细菌及基因资源具有广阔的研究开发的价值,且具有良好的应用前景。所以,对于冰核活性基因的结构特点及近年来的研究现状有个总体了解是很有必要的。本文主要介绍了冰核活性基因及其应用方面的研究进展情况。     

冰核微生物中冰核基因重复序列PCR分析

在本试验的条件下,Pseudomonoas svringae inaZ基因产生的蛋白的主要重复基元的编码区：5’－GCCGGTTATGGCAGCACGCTGACC－3’序列既在冰核真菌、细菌中存在,也在非冰核真菌、细菌中存在,即冰核细菌、真菌和非冰核细菌、真菌都有扩增产物,并且产物呈多态性,同一个种不同菌株间也呈多态性,说明该引物不适合用于鉴定真菌、细菌中冰核基因是否存在,也不能用于区分冰核真菌和非冰核真菌以及分区冰核细菌和非冰核细菌,更不能根据其扩增片段的量和大小说明冰核真菌、细菌冰核活性的强弱。     

Erwinia herbicola冰核活性蛋白的分离、电泳分析鉴定

对Erwinia herbicola(A25)菌株的冰核活性蛋白的分离纯化及电泳进行研究。主要方法①按双温培养的方法,获得了较高的生物量积累和强冰核活性的诱导表达。②实验采用渗透压冲击法破碎细菌细胞,破碎率达98．67％。③通过差速离心法获取不同冰核活性蛋白组分,测定各组分的冰核活性和SDS－PAGE电泳图谱分析。建立了冰核活性因子的高冰核活性与离心力之间的关系;利用SDS－PAGE还建立了具冰核活性蛋白的分子量的大小与冰核活性蛋白组分之间的关系。证实了具高冰核活性蛋白质最小结构单位约为26．0kD。     

一种新型的报告基因—冰核基因

冰核基因 (ＩｃｅＮｕｃｌｅａｔｉｏｎＡｃｔｉｖｅｇｅｎｅ ,ＩＮＡｇｅｎｅ)是从冰核细菌中克隆的编码冰核蛋白的基因 ,它编码的冰核蛋白具有很强的冰核活性。当ｉｎａ基因与目的基因一起转入生物体后 ,可以通过冰核活性的测定来检测目的基因是否表达。冰核基因与通常的报告基因有着根本意义上的不同 ,它对信号的检测不是由于酶的催化反应 ,而是一种物理过程 (水的液 固状态的改变 )。ｉｎａ基因克服了其它报告基因的一些缺点 ,扩大了报告基因应用范围[1] 。现在 ,已经有很多研究植物 细菌相互作用的实验室应用ｉｎａ基因作为报告基因[4]…     

聚乙烯醇-海藻酸盐共固定化冰核活性细菌——黄单胞菌TS206的研究

冰核活性细菌固定化在食品冷冻浓缩中的应用具有重要意义,冰核活性和抗渗漏能力是衡量其性能的两个重要技术指标。研究采用PVA和海藻酸盐作为固定化载体,通过两者优良性能的互补而建立对冰核活性细菌Xanthomonas ampelina TS206的共固定化技术。结果表明,细胞投入量对冰核活性有较大影响,基础固定化条件对固定化技术指标的综合评分影响程度大小的顺序依次为：海藻酸钠浓度＞硼酸浓度＞PVA浓度＞CaCl2浓度,各因素的较优水平是：海藻酸钠浓度1%,硼酸浓度5%,PVA浓度8%,CaCl2浓度1.1%;研究还发现冰核活性与固定化凝胶珠的添加量正相关,与固化时间相关性较小,渗漏量受固定化凝胶珠的添加量和固化时间影响不显著。     

冰核真菌的冰核活性及其种类鉴定*

作者从1993年起至今,从已分离和搜集到的500多株真菌中筛选出在-5℃具有冰核活性的真菌6株,它们-5℃的冻滴率高低顺序为F9502（100%）=F9501（100%）＞F9401（96％）=AS3．494（96%）＞F9801（94%）＞F9802（92%）,结冰点高低顺序为F9502（-2．7℃）＞F9501（-3．4℃）＞F9401（-4.4℃）＞AS34594（-4.5℃）＞F9801（-4．7℃）＞F9402（-4．8℃）,以F9502菌株活性强而稳定。经鉴定确定：F9401和F9402菌株为FusartumSportFichioldesSherb．;AS3．4594菌株为FavenaceumSacc．;F9501和F9502菌株为F.gramlnearumSchwabe;F9801菌株为F．monilijormeSheldon。F．Sportrlchloides和F．graminearum作为冰核真菌未见报道。本结果为揭示冰核真菌与植物冻害关系及冰核真菌开发应用研究提供了菌种资源,有重要应用价值。     

冰核真菌的冰核活性及其种类鉴定

作者从1993年起至今,从已分离和搜集到的500多株真菌中筛选出在-5℃具有冰核活性的真菌6株,它们-5℃的冻滴率高低顺序为F9502（100%）=F9501（100%）＞F9401（96％）=AS3．494（96%）＞F9801（94%）＞F9802（92%）,结冰点高低顺序为F9502（-2．7℃）＞F9501（-3．4℃）＞F9401（-4.4℃）＞AS34594（-4.5℃）＞F9801（-4．7℃）＞F9402（-4．8℃）,以F9502菌株活性强而稳定。经鉴定确定：F9401和F9402菌株为FusartumSportFichioldesSherb．;AS3．4594菌株为FavenaceumSacc．;F9501和F9502菌株为F.gramlnearumSchwabe;F9801菌株为F．monilijormeSheldon。F．Sportrlchloides和F．graminearum作为冰核真菌未见报道。本结果为揭示冰核真菌与植物冻害关系及冰核真菌开发应用研究提供了菌种资源,有重要应用价值。     

云南植物上冰核活性细菌鉴定

从云南植物上分离到９２株冰核活性细菌,并进行了鉴定。其中菠萝欧文氏菌６１株,占６６．３％;草生欧文氏菌２株,占２．２％;丁香假单胞菌２１株,占２２．８％;黄瓜角斑病菌２株,占２．２％;菜豆荚斑假单胞菌６株,占６．５％。云南省冰核活性细菌的优势种类是菠萝欧文氏菌,其次是丁香假单胞菌类。     

Perturbation of bacterial ice nucleation activity by a grass antifreeze protein

Certain plant-associating bacteria produce ice nucleation proteins (INPs) which allow the crystallization of water at high subzero temperatures. Many of these microbes are considered plant pathogens since the formed ice can damage tissues, allowing access to nutrients. Intriguingly, certain plants that host these bacteria synthesize antifreeze proteins (AFPs). Once freezing has occurred, plant AFPs likely function to inhibit the growth of large damaging ice crystals. However, we postulated that such AFPs might also serve as defensive mechanisms against bacterial-mediated ice nucleation. Recombinant AFP derived from the perennial ryegrass Lolium perenne (LpAFP) was combined with INP preparations originating from the grass epiphyte, Pseudomonas syringae. The presence of INPs had no effect on AFP activity, including thermal hysteresis and ice recrystallization inhibition. Strikingly, the ice nucleation point of the INP was depressed up to 1.9 °C in the presence of LpAFP, but a recombinant fish AFP did not lower the INP-imposed freezing point. Assays with mutant LpAFPs and the visualization of bacterially-displayed fluorescent plant AFP suggest that INP and LpAFP can interact. Thus, we postulate that in addition to controlling ice growth, plant AFPs may also function as a defensive strategy against the damaging effects of ice-nucleating bacteria.     

Molecular cloning of an ice nucleation gene from Erwinia ananas and its expression in Escherichia coli

Abstract An approximately 7 kbp genomic DNA fragment was cloned from an ice nucleation-active (ina) strain of Erwinia ananas and defined as to its restriction enzyme site. When the DNA fragment was introduced into E. coli MM294, a potent ice nucleation activity was expressed. Both 0.7 kbp truncation from the 5'-end and 1.7 kbp truncation from the 3'-end were also effective in expressing the ice nucleation activity in E. coli . Therefore, the resulting DNA fragment of approximately 5 kbp was considered to be an ina gene and named ina A. It existed as a unique gene in this strain of E. ananas . No corresponding ina gene existed in an ice nucleation-inactive strain of E. milletiae .     

大麦黄矮病毒的冰核活性与作物霜冻的关系

对大麦黄矮病毒(Barley Yellow Dwarf Virus,BYDV)的冰核活性,以及它的侵染与作物霜冻的关系进行了研究.利用人工摸拟霜箱,测试了接种BYDV的小麦等作物植株的霜冻温度,并用ELISA法检测了供试植株的病毒含量.结果表明,接种样品与对照相比,感病品种的霜冻温度升高1—2℃以上,抗病品种的霜冻温度变化不大.离体叶片测定结果表明,“中7902”的叶片中,病毒含量与霜冻温度成正相关,说明BYDV可以起到异源冰核(heterogeneous ice nuclei)的作用,它的侵染能影响作物的抗霜冻能力.用“Vali小液滴冻结法”检测提纯的BYDV,证明BYDV具冰核活性,从而首次发现病毒也能起到生物冰核的作用.     

A first test of the hypothesis of biogenic magnetite-based heterogeneous ice-crystal nucleation in cryopreservation

An outstanding biophysical puzzle is focused on the apparent ability of weak, extremely low-frequency oscillating magnetic fields to enhance cryopreservation of many biological tissues. A recent theory holds that these weak magnetic fields could be inhibiting ice-crystal nucleation on the nanocrystals of biological magnetite (Fe₃O₄, an inverse cubic spinel) that are present in many plant and animal tissues by causing them to oscillate. In this theory, magnetically-induced mechanical oscillations disrupt the ability of water molecules to nucleate on the surface of the magnetite nanocrystals. However, the ability of the magnetite crystal lattice to serve as a template for heterogeneous ice crystal nucleation is as yet unknown, particularly for particles in the 10–100 nm size range. Here we report that the addition of trace-amounts of finely-dispersed magnetite into ultrapure water samples reduces strongly the incidence of supercooling, as measured in experiments conducted using a controlled freezing apparatus with multiple thermocouples. SQUID magnetometry was used to quantify nanogram levels of magnetite in the water samples. We also report a relationship between the volume change of ice, and the degree of supercooling, that may indicate lower degassing during the crystallization of supercooled water. In addition to supporting the role of ice-crystal nucleation by biogenic magnetite in many tissues, magnetite nanocrystals could provide inexpensive, non-toxic, and non-pathogenic ice nucleating agents needed in a variety of industrial processes, as well as influencing the dynamics of ice crystal nucleation in many natural environments.