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。     

苏云金杆菌在华北果园土壤中消长动态的研究

本文报道了产生核黄素的苏云金杆菌CH菌株在果园土壤中的消长动态。研究结果表明 ,将CH菌株制剂喷洒于苹果园土壤表面后 1个月内 ,CH菌株的菌数基本上维持在喷洒后的 10 5/ g水平 ,其后菌数急剧减少 ,2个月后从 10 5/ g的水平减少到 10 4 / g的水平 ,并长时间维持 10 4 / g这一水平 ;CH菌株制剂喷洒于果园土壤后对土壤中固有的Bacillus属细菌数的消长没有明显的影响 ,Bacillus属细菌数长时间维持在 10 6～ 10 5/ g之间 ,处于一种相对稳定的状态 ;在土壤透水性和灌水条件良好的土壤条件下 ,喷洒于土壤表面的苏云金杆菌可缓慢向深处移动。     

Ice‐‐bacteria as antagonists in biological frost protection

Investigated was the efficacy of 4 ice nucleation‐inactive (ice^‐) bacterial strains (A506, GSPB 1147, 1181, 2357) at 4 ice nucleation active (ice⁺) strains (553, 554, GSPB 1139, 2035) on the surface of 4 culture crops (corn, tomato, bush‐ and field bean). Examinated was the reduction of frost damage at the used culture crops by inoculation with ice⁺ and ice^‐ strains at 4 different variants (A‐D) under laboratory conditions. The ice nucleation activity was determined by the tube‐freezing‐assay. An effective reduction of ice⁺ bacteria was possible, when plant surfaces were preinoculated (3days) with ice^‐bacteria before ice⁺ bacterial strains colonized the surfaces of plants. A statistical comparison of mean values of obtained results showed, the ice crystallization (INT) by inoculation started at ‐3°C and in mean (MNT) below ‐5°C independent of the used ice⁺ and ice^‐ strains. Obtained differences were not significant. As untreated as colonized plants with antagonists started to freeze at ‐5 and ‐6°C and in average at ‐7°C. However, the preinoculation resulted in 1.38 K differences for the temperatures, at which 50% of leaves were frozen (PROZ 50) in favour of preinoculated plants. This difference in freeze temperatures by preinoculation with ice^‐ bacteria was discussed as a possible method of frost protection.     

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.     

我国冰核活性细菌的优势种类调查与研究

１９８６￣１９９４年,从国内１７个省、市、自治区的６８种植物上分离到了２５０株冰核活性细菌,经鉴定分别属于３个属的１７个种或致病变种。其中出现最多的是菠萝欧文氏菌,共１３３株,占总数的５３．２％,其次是丁香假单胞菌群,共７０株,占２８％,其它种类的冰核活性细菌共４７株,仅占１８．８％。因此,我国冰核活性细菌的优势菌种是Ｅ．ａｎａｎａｓ,其次是Ｐ．ｓｙｒｉｎｇａｅ ｐｖｓ。在低纬度的南方地区中Ｅ．ａ     

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 Use of High-resolution Infrared Thermography (HRIT) for the Study of Ice Nucleation and Ice Propagation in Plants

Freezing events that occur when plants are actively growing can be a lethal event, particularly if the plant has no freezing tolerance. Such frost events often have devastating effects on agricultural production and can also play an important role in shaping community structure in natural populations of plants, especially in alpine, sub-arctic, and arctic ecosystems. Therefore, a better understanding of the freezing process in plants can play an important role in the development of methods of frost protection and understanding mechanisms of freeze avoidance. Here, we describe a protocol to visualize the freezing process in plants using high-resolution infrared thermography (HRIT). The use of this technology allows one to determine the primary sites of ice formation in plants, how ice propagates, and the presence of ice barriers. Furthermore, it allows one to examine the role of extrinsic and intrinsic nucleators in determining the temperature at which plants freeze and evaluate the ability of various compounds to either affect the freezing process or increase freezing tolerance. The use of HRIT allows one to visualize the many adaptations that have evolved in plants, which directly or indirectly impact the freezing process and ultimately enables plants to survive frost events.     

丝裂霉素C对Erwinia herbicola表达及运转冰核活性蛋白方式的影响

利用添加丝裂霉素C(MMC)于不同培养基中,并在不同培养条件下对草生欧文氏菌10025A(E．herbicola 10025A)诱导培养,首次证实该菌表达冰核活性蛋白的活性及运转分泌冰核活性蛋白方式是不同。研究结果显示,MMC的加入可以提高细菌培养物的冰核活性,该现象可以解释为MMC对E．herbicola的作用能够激活细胞SOS应急系统的反应,诱导细胞合成部分参与修复DNA损伤的酶和蛋白因子,达到对E．herbicola诱导表达冰核蛋白通过高尔基体形成小液泡,向膜外分泌的方式,同时对E．herbicola细胞分裂后的形态也发生明显的影响。这对研究细胞在恶劣环境的生存机制以及获取该条件下的蛋白具有重要的意义,对低温生物的研究也将产生积极地影响。     

The freezing characteristics of cauliflower curd

Cultivar differences in frost resistance and the heritable nature of resistance were demonstrated using seedling cauliflower plants. Such cultivar differences were not however expressed in the curd. Selection for frost resistance in cauliflower should therefore use whole plant screening techniques. Curd material when frozen as isolated florets, supercooled over the range – 1°C to – 12°C and the mean freezing point of all curds tested was -6°C to -7.25°C (overall mean -6.44°C). Curd florets which supercooled but did not freeze were completely undamaged, whereas freezing always led to cell damage and death observed as water-soaking of the floret surface and measured using an electrical conductivity method. The large range of freezing points measured suggests a range of active ice nucleators either on or within the florets. When curds were frozen intact the ability of florets to supercool was severely restricted which was attributed to the seeding of freezing by the internal growth of ice crystals. A crop protection strategy needs to identify and control or modify warm temperature nucleators in cauliflower curd.     

Biosynthesis of Cephalosporins

Abstract

Certain aerobic, Gram-negative bacteria, including the epiphytic plant pathogen, Pseudomonas syringae, possess a membrane protein that enables them to nucleate crystallization in supercooled water. Currently, these ice-nucleating (IN) bacteria are being used in snow making and have potential applications in the production and texturing of frozen foods, and as a replacement of silver iodide in cloud seeding. A negative aspect of these IN bacteria is frost damage to plant surfaces. Thus, of the various types of biological ice nucleators, bacteria have been the subject of most research and also appear relevant to the anticipated practical uses. The intent of this review is to explain the identification and ecology of the ice-nucleating bacteria, as well as to discuss aspects of molecular biology related to ice nucleation and consider existing and potential applications of this unique phenomenon.