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。     

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。     

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(Erwinia ananas 110)冰核基因克隆和序列分析

从作者自行分离的冰核细菌（Erwinia ananas110)中克隆到我国第1个细菌冰核基因,并完成其序列测定和分析,所克隆基因编码区全长3921bp,编码1306aa,氨基酸序列明显分为3个区即N-端（161aa),C-端（41aa)的单一序列区和中部的高度重复序列R区（1104aa)。以16氨基酸为重复单元的R区占整个编码序列的84.5%。序列分析表明我们所克隆的基因为一个新冰核基因,将其命名为iceA。该基因已在GenBank上登录,登录号为：AF387802。     

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。     

菌种保存方法对冰核细菌冰核活性的影响

本文测定了5种保存方法及3种保存温度,对4株代表3个属4个种(变种)的冰核细菌冰棱活性的影响。结果表明,不同保存方法对冰核细菌的存活力和冰核活性存在着不同程度的影响,保存温度越高,对冰核活性的影响越大,不同菌种对保存的敏感性不同。所适合的保存方法也不同。真空冷冻干燥和灭菌水于-20℃冷冻保存对各菌株的存活力和冰核活性的影响都较小,适用于一般冰棱细菌的长期保存。而10%甘油冷冻保存则不适用于冰核细菌的保存。     

玉米鼠耳病及其媒介昆虫二点叶蝉消长动态和玉米受害损失测定

对四川省南充市仪陇县二点时蝉及其所传播的玉米鼠耳病的田间消长规律以及玉米受害后的产量损失状况进行了研究。结果表明：温度低于16℃时,玉米不发病;28℃和31℃时玉米鼠耳病的潜伏期较短,分别为4．85和4．64d。在高、中、低3个海拔高度上,玉米鼠耳病的田间消长动态受二点叶蝉种群发生动态的影响,并于5月下旬至6月上旬达到最大病情指数,分别为5．75、10．97和9．09。玉米的生长和产量均受到玉米鼠耳病发生程度的影响,随着该病病级的上升,玉米株高、地面直径、穗长、穗粗、穗粒数、穗粒重及千粒重均相应下降。通过二元线性回归方程对玉米单穗籽粒重损失率作通径分析,表明在玉米的产量构成中．千粒重下降是导致产量损失的主要因子。     

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。     

细菌冰核基因的应用研究

细菌冰核基因的应用研究已成为生物冰核领域的研究热点,研究涉及细菌细胞表面展示、促冻杀虫、报告基因、病原微生物高敏检测、作物抗寒育种等多个领域,显示良好应用前景。通过对国外在该方面的研究现状的综述,和我们在冰核基因促冻杀虫研究方面重要进展的介绍,对今后我们拟开展的这一研究工作进行了展望。     

The role of ice nucleation active bacteria in supercooling of citrus tissues

The frost sensitivity of Citrus sinensis in relation to the presence of biogenic ice nuclei was studied. In commercially managed citrus groves the ice nucleation active (INA) bacterium Pseudomonas syringae reached 6 × 10⁴ colony forming units (CFU) leaf⁻¹, a population sufficiently high to catalyze ice formation. However, a transient loss of bacterial nucleation activity was noticeable at subzero field temperatures, followed by resumption as temperatures rose. This loss was apparently due to a temporary transition of INA to ice nucleation inactive (INI) bacteria. Field application of Bordeaux mixture, copper hydroxide, streptomycin, and 2-hydroxypropylmethanethiolsulfonate (HPMTS), resulted in reduction of INA bacterial populations to detectability (≤ 10² CFU leaf⁻¹) limits. However, the corresponding reduction in ice nucleation events in treated samples as compared to controls at nucleation temperature ≥−3°C was not as dramatic. It ranged from approximately 7% in samples treated with the bactericide HPMTS, to 35% in samples treated with chemicals possessing combined bactericidal - fungicidal action (coppers). Since a quantitative relationship exists between ice nucleation events on individual leaves and the INA bacterial populations harbored by these leaves, these results suggest the co-existence of a bacterial and a proteinaceous, yet non-bacterial ice nucleating source in citrus, both active at ≥−3°C.     

Inhibition of bacterial ice nucleation by polyglycerol polymers

The simple linear polymer polyglycerol (PGL) was found to apparently bind and inhibit the ice nucleating activity of proteins from the ice nucleating bacterium Pseudomonas syringae. PGL of molecular mass 750 Da was added to a solution consisting of 1 ppm freeze-dried P. syringae 31A in water. Differential ice nucleator spectra were determined by measuring the distribution of freezing temperatures in a population of 98 drops of 1 microL volume. The mean freezing temperature was lowered from -6.8 degrees C (control) to -8.0,-9.4,-12.5, and -13.4 degrees C for 0.001, 0.01, 0.1, and 1% w/w PGL concentrations, respectively (SE < 0.2 degrees C). PGL was found to be an ineffective inhibitor of seven defined organic ice nucleating agents, whereas the general ice nucleation inhibitor polyvinyl alcohol (PVA) was found to be effective against five of the seven. The activity of PGL therefore seems to be specific against bacterial ice nucleating protein. PGL alone was an ineffective inhibitor of ice nucleation in small volumes of environmental or laboratory water samples, suggesting that the numerical majority of ice nucleating contaminants in nature may be of nonbacterial origin. However, PGL was more effective than PVA at suppressing initial ice nucleation events in large volumes, suggesting a ubiquitous sparse background of bacterial ice nucleating proteins with high nucleation efficiency. The combination of PGL and PVA was particularly effective for reducing ice formation in solutions used for cryopreservation by vitrification.     

Cold requirement for maximal activity of the bacterial ice nucleation protein INAZ in transgenic plants

The bacterial ice nucleation gene inaZ confers production of ice nuclei when transferred into transgenic plants. Conditioning of the transformed plant tissue at temperatures near 0°C greatly increased the ice nucleation activity in plants, and maximum ice nucleation activity was achieved only after low-temperature conditioning for about 48 h. Although the transgenic plants contain similar amounts of inaZ mRNA at both normal and low temperatures, low temperatures are required for accumulation of INAZ protein. We propose that the stability of the INAZ protein and thus ice nucleation activity in the transgenic plants is enhanced by low-temperature conditioning.     

A kinetic model describing cell growth and production of highly active, recombinant ice nucleation protein in Escherichia coli

A structured kinetic model, which describes the production of the recombinant ice nucleation protein in different conditions, was applied. The model parameters were estimated based on the variation of the specific growth rate and the intracellular product concentration during cultivation. The equations employed relate the cellular plasmid content or plasmid copy number with the cloned-gene expression; these correlations were successfully tested on the experimental data. The optimal nutrient conditions for the growth of Escherichia coli expressing the inaZ gene of Pseudomonas syringae were determined for the production of active ice nucleation protein. The kinetics of the cultures expressing the inaZ gene were studied in a bioreactor at different growth temperatures and nutrient conditions.     

大豆和玉米冠层光合有效辐射各分量日变化

通过实测大豆和玉米冠层光合有效辐射各分量并计算其反射率、透射率,分析了各分量日变化规律及其影响因素。结果表明:光合有效辐射分量(FPAR)在一天中均接近于常数,特别是在8:00—11:00和14:00—16:00相对稳定;晴天大豆冠层入射光合有效辐射变化曲线较阴天平滑,反射率和透射率曲线没有阴天平滑;由于云层的吸收和散射作用,阴天中光合有效辐射(PAR)最大值的出现时间比晴天晚1h左右;植被冠层空间异质性对光合有效辐射各分量影响较大,不同作物类型的各分量之间有较大差异;大豆冠层空间异质性较玉米小,其光合有效辐射各个分量曲线较平滑;线性光量子传感器与太阳入射方向垂直投影线成30°时,冠层入射光合有效辐射平均偏离度值最小,为0.657%。     

Effect of ice nucleation by droplet of immobilized silver iodide on freezing of rabbit and bovine embryos

Silver iodide was immobilized by applying the insoluble reaction between sodium alginate and calcium chloride. The immobilized silver iodide was immersed into a freezing solution in order to trigger ice nucleation. Temperature change during cooling and postthaw in vitro development of embryos were examined in order to evaluate the effectiveness of the immobilized silver iodide (AgI alginate-gel droplet) on embryo development. Samples containing the AgI alginate-gel droplets released the latent heat of fusion at a higher subzero temperature than samples without the AgI alginate-gel droplets. When the AgI alginate-gel droplet was added to the freezing solution of rabbit and bovine embryos, they were successfully preserved in liquid nitrogen.