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。     

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。     

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。     

冰核真菌研究进展

水在０℃以下仍未结冰的现象称为水的过冷却作用,小体积纯净水的过冷却温度可接近－４０℃。引起水从液态向固态转变的物质称冰核,不同种类冰核催化活性差异很大,非生物冰核可在－１０℃左右诱发小体积水结冰,而自然界中活性最强的冰核来自生物体,已报道某些细菌（如Pseudomonas syringae和Erwinia hericola的一些菌株）可产生在－１℃～－２℃下催化水结冰的冰核。研究者将一些可以产生在－５℃以上具有冰核活性冰核的生物称为冰核生物。自１９７４年Ｍａｋｉ［１］首次从赤杨树叶中分离到在－２℃～－５℃下诱发植物结冰发生霜冻的…     

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]…     

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     

X.ampelina TS206发酵制备冰核活性蛋白的研究

优化XanthomonasampelinaTS2 0 6的发酵制备条件可以提高冰核活性和生物量。通过摇瓶培养对该菌进行培养基成分和温度的筛选。实验结果表明 :( 1 )鱼蛋白胨和乳糖的浓度对冰核活性的提高产生显著影响程度 ,玉米浆影响程度较小 ;( 2 )生物量影响程度大小的顺序依次为鱼蛋白胨 >乳糖 >甘油 >玉米浆。优化的发酵培养基配方为 :酵母粉 1 0g L、大豆蛋白胨 1 0g L、硫酸镁 0 .5g L、蔗糖 2 0g L、鱼蛋白胨 1 5g L、乳糖 1g L、甘油 2 0g L、玉米浆 1 0g L ,pH 7.0。其较优的发酵温度为 1 8℃ ,发酵时间为4 8h ,能获得较高的冰核活性和生物量。     

昆虫低温生物学:Ⅱ.冰核物质(冰核蛋白)和昆虫的耐冻性

体系在低于熔点温度时才结冰的现象 ,叫过冷却 (supercooling)。体系开始结冰时的温度称为过冷却点 (supercooling point,SCP)。在适当的低温 ,体系内需存在一起始结冰的冰核 ,才能诱导冰晶产生 ,此物质称为冰核剂 (icenucleating agents,INA)。昆虫体内各腔室充满组织液 ,各腔室 (如消化系统和细胞内 )因所含 INA的冰核活性的不同 ,而使结冰的温度各异 ,所受低温伤害也不同。冰核常存在于昆虫血淋巴内 ,提高溶液的 SCP,降低其过冷却能力 ,引起胞外结冰。冰核物质的活性越高 ,SCP越高 ,虫体也能在较高的低温结冰。昆虫体内有不同性质…     

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。     

冰核真菌胞外冰核产生条件、成冰生物学特性及其分离纯化的研究

果糖和葡萄糖作碳源、硫酸铵作氮源、培养温度25℃以下,培养液初始pH5-7和少量通气都利于胞外冰核产生,最佳培养时间受培养温度和接种菌龄的制约,而低温处理,光照及加丝裂霉素C和HNPA对胞外冰核产生无明显影响。胞外冰核耐热（40℃处理后仍保持较高活性）,酸碱适应性强（pH2-13);温度50℃以上,蛋白酶K和高浓度脲处理可完全失活或严重破坏其冰核活性,表明蛋白和真菌胞外冰核的必需成分;胞外冰核溶液中盐浓度高于50mmol/L时,活性才受抑制。25％冰乙醇可有效沉淀真菌胞外冰核,同时又保持较高活性;分离纯化的初步研究显示真菌胞外冰核分子量很大,电荷组成和分布上不均一,所带净电荷为负,本研究加深了对真菌胞外冰核的认识,为进一步纯化奠定了基础。     

Phosphatidylinositol, a phospholipid of ice-nucleating bacteria.

The nature of the phospholipids of the various bacteria that have ice nucleation activity in supercooled water has been determined. The seven bacteria studied included Pseudomonas syringae, Erwinia herbicola, three Escherichia coli K-12 strains that are phenotypically Ice+ because they contain plasmids with different amounts of either P. syringae or E. herbicola cloned DNA, and two E. coli K-12 strains without cloned ice gene DNA. All five Ice+ bacterial strains contained small amounts (0.1 to 1.0% of the total phospholipids) of phosphatidylinositol (PI), a phospholipid not previously detected in E. coli, Pseudomonas, or Erwinia species. The Ice- E. coli strains also contained trace level of PI that amounted to 2 to 30% of the level found in the Ice+ E. coli strains. Extracts of Ice+ strains contained low but measurable activities of PI synthase, while the activities in Ice- strains amounted to only 8 to 12% or less of that found in extracts of Ice+ bacteria. The functioning of the ice gene apparently increased both the PI synthase activity and the PI content of Ice+ strains from low endogenous levels. The relative ice nucleation activity at -4 degrees C or above (class A nucleation activity) of all Ice+ strains was found to be proportional to their PI content. The addition of myo-inositol (5 x 10(-4) M) to synthetic culture media increased the class A nucleation activity of both Ice+ E. coli strains and P. syringae up to sevenfold but had no stimulating effect on ice nucleation at lower temperatures (class B and class C nucleation activities). If these cells after fusion with PI vesicles were incubated with an energy source, the class A nucleation activity increased 70-fold over that present before fusion. These results indicate that PI plays an important role in ice nucleation at warm temperatures and is a likely precursor or component of the class A structure.     

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。     

Release of cell-free ice nuclei by Erwinia herbicola.

Several ice-nucleating bacterial strains, including Erwinia herbicola, Pseudomonas fluorescens, and Pseudomonas syringae isolates, were examined for their ability to shed ice nuclei into the growth medium. Only E. herbicola isolates shed cell-free ice nuclei active at -2 to -10 degrees C. These cell-free nuclei exhibited a freezing spectrum similar to that of ice nuclei found on whole cells, both above and below -5 degrees C. Partially purified cell-free nuclei were examined by density gradient centrifugation, chemical and enzymatic probes, and electron microscopy. Ice-nucleating activity in these cell-free preparations was associated with outer membrane vesicles shed by cells and was sensitive to protein-modifying reagents.     

Immunological characterization of ice nucleation proteins from Pseudomonas syringae, Pseudomonas fluorescens, and Erwinia herbicola.

Antibodies were raised against the InaW protein, the product of the ice nucleation gene of Pseudomonas fluorescens MS1650, after protein isolation from an Escherichia coli clone. On Western blots (immunoblots), these antibodies recognized InaW protein and InaZ protein (the ice nucleation gene product of Pseudomonas syringae S203), produced by both E. coli clones and the source organisms. The InaZ protein appeared in P. syringae S203 during stationary phase; its appearance was correlated with the appearance of the ice nucleation-active phenotype. In contrast, the InaW protein occurred at relatively constant levels throughout the growth phases of P. fluorescens MS1650; the ice nucleation activity was also constant. Western analyses of membrane preparations of P. syringae PS31 and Erwinia herbicola MS3000 with this antibody revealed proteins which were synthesized with development of the nucleating phenotype. In these species the presence or absence of the nucleating phenotype was controlled by manipulation of culture conditions. In all nucleation-positive cultures examined, cross-reacting low-molecular-weight bands were observed; these bands appeared to be products of proteolytic degradation of ice nucleation proteins. The proteolysis pattern of InaZ protein seen on Western blots showed a periodic pattern of fragment sizes, suggesting a highly repetitive site for protease action. A periodic primary structure is predicted by the DNA sequence of the inaZ gene.     

冰核细菌在我国北方玉米上的消长动态规律

研究证明,菠萝欧文氏菌(Erwinia ananas)为我国北方玉米上优势冰核细菌种类,占总体INA细菌95 %以上。采用定量定性和定期取样分离方法,首次研究INA细菌在玉米上的消长动态规律。结果表明:玉米不同生长发育阶段是影响INA细菌在玉米上数量分布和消长动态变化的重要因素,以抽雄至成熟期间分布INA细菌数量最多,高达10 7～10 8CFU/ g,比拔节至抽雄期高出2～3个数量级,比苗期至拔节期高出4～5个数量级;同时还指出,玉米不同播期,对INA细菌数量分布影响显著,差异很大,其中INA细菌分布数量消长变化,以正常播种(1.9×10 7CFU / g) >中期播种(7.9×10 5CFU/ g) >晚期播种(5 .0×10 4 CFU/ g) ;研究指出,处于抽雄至成熟期间的玉米上分布的INA细菌数量最多,因此期间(8月上旬至9月下旬) ,气温逐渐降低,昼夜温差大,田间结露多,加上玉米处于成熟阶段,抗INA细菌能力弱,这些因素有利于低温(5～2 0℃范围内生长)型INA细菌生长繁殖,故使INA细菌分布数量最多     

Cloning and expression of bacterial ice nucleation genes in Escherichia coli.

Epiphytic populations of Pseudomonas syringae and Erwinia herbicola are important sources of ice nuclei that incite frost damage in agricultural crop plants. We have cloned and characterized DNA segments carrying the genes (ice) responsible for the ice-nucleating ability of these bacteria. The ice region spanned 3.5 to 4.0 kilobases and was continuous over this region in P. syringae Cit7R1. The cloned fragments imparted ice-nucleating activity in Escherichia coli. Substantial increases in the nucleating activity of both E. coli and P. syringae were obtained by subcloning the DNA fragments on multicopy plasmid vectors. Southern blot analysis showed substantial homology between the ice regions of P. syringae and E. herbicola, although individual restriction sites within the ice regions differed between the two species.     

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 .     

Genetic Transfer of Episomic Elements Among Erwinia Species and Other Enterobacteria: F′lac+

The episomic element F'lac(+) was transferred, probably by conjugation, from Escherichia coli to Lac(-) strains of Erwinia herbicola, Erwinia amylovora, and Erwinia chrysanthemi (but not to several other Erwinia spp. In preliminary trials). The lac genes in the exconjugants of the Erwinia spp. showed varying degrees of stability depending on the strain (stable in E. herbicola strains Y46 and Y74 and E. amylovora strain EA178, but markedly unstable in E. chrysanthemi strain EC16). The lac genes and the sex factor (F) were eliminated from the exconjugants by treatment with acridine orange, thus suggesting that both lac and F are not integrated in the Erwinia exconjugants. All of the tested Lac(+) exconjugants of E. herbicola strains Y46 and Y74 and E. amylovora strain EA178, but not of E. chrysanthemi strain EC 16, were sensitive to the F-specific phage M13. The heterogenotes (which harbored F'lac(+)) of E. herbicola strains Y46 and Y74, E. amylovora strain EA178, and E. chrysanthemi strain EC16 were able to transfer lac genes by conjugation to strains of E. herbicola, E. amylovora, E. chrysanthemi, Escherichia coli, and Shigella dysenteriae. The frequency of such transfer from Lac(+) exconjugants of Erwinia spp. was comparable to that achieved by using E. coli F'lac(+) as donors, thus indicating the stability, expression, and restriction-and-modification properties of the sex factor (F) in Erwinia spp.     

一株新的溶磷草生欧文氏菌的分离、鉴定及其溶磷效果的初步研究

从湖南、湖北、云南等地磷矿开采场的土壤样品中筛选到一株溶磷能力较强的菌株P21,结合生理生化指标和16S rDNA序列分析鉴定其属于草生欧文氏菌菠萝变种(Erwinia herbicola var.ananas).该菌能溶解磷酸三钙、羟基磷灰石、磷酸铁、磷酸锌,其中对磷酸三钙和羟基磷灰石的每升液体培养基溶磷量(P2O5)分别高达1206.20mg、529.67mg.溶磷菌草生欧文氏菌菠萝变种P21对产地不同的8种磷矿石溶解能力不同,对云南晋宁和昆阳、四川雅安、江苏锦屏等地磷矿石有较强的溶解能力,每升液体培养基溶磷量分别为96.64mg、78.46mg、67.07mg、65.24mg,对其它产地的磷矿石溶解能力较差.实验表明,培养液的pH下降与溶磷菌P21的溶磷量无直接关系.