芽孢杆菌对桉树幼苗的促生效果及其ACC脱氨酶活性的研究

【目的】筛选出能显著促进桉树幼苗生长的芽孢杆菌菌株,探究酶活性与桉树幼苗生长的相关性,初步揭示芽孢杆菌对桉树幼苗的促生机制。【方法】以分离自广东广州、阳江桉树林地土壤的32个芽孢杆菌菌株为研究对象,测定桉树幼苗接种盆栽试验以及菌株ACC脱氨酶活性与幼苗N、P养分。【结果】接种菌株2306、2403、2301能够显著促进桉树幼苗高生长和生物量积累,尤以菌株2306的促生效果最佳,其苗高、生物量分别比对照增加53.1%和190.2%。【结论】芽孢杆菌的ACC脱氨酶活性与桉树幼苗高生长相关极显著,与生物量相关显著;而且上述3个菌株均能提高桉树幼苗的N、P含量。研究结果将进一步丰富桉树促生菌资源,促进桉树微生物肥料的开发。     

具ACC脱氨酶活性大豆根瘤内生菌的筛选、抗性及促生作用北大核心CSCD

【目的】从大豆根瘤中筛选具ACC(1-氨基环丙烷-1-羧基)脱氨酶活性的内生细菌,对活性菌株的抗盐碱性、系统分类地位以及代表菌株的促生长作用进行研究,为发掘和应用抗逆、促生优良菌种资源提供理论基础。【方法】以ACC作为唯一氮源测定菌株产ACC脱氨酶特性,采用标准曲线法测定α-丁酮酸含量,比色法定量测定ACC脱氨酶活力,固体平板筛选法对活性菌株进行抗性分析,通过菌体形态及生理生化特性测定、16S rRNA基因序列同源性分析鉴定菌株分类地位,采用盆栽试验验证代表菌株的促生作用。【结果】从河南省13个市(地区)36个点采集的大豆根瘤中筛选出8株ACC脱氨酶内生细菌,其中菌株DD132的酶活性最高(15.712 U/mg)。筛选菌株可耐受4%–6%NaCl,其中菌株DD165、DD132可耐受9%NaCl盐浓度。在pH 11时5株(DD14、DD132、DD67、DD141、DD131)生长良好,说明这些菌株有较强耐碱性。8株产ACC脱氨酶菌株分属于4属,即芽孢杆菌属(Bacillus)、肠杆菌属(Enterobacter)、寡养单胞菌属(Stenotrophomonas)和泛菌属(Pantoea)。接种试验表明内生菌DD132对小麦幼苗生长具有明显促生长作用。【结论】大豆根瘤内具ACC脱氨酶高活性菌株有较强耐盐碱性,其中菌株DD132对小麦幼苗生长有明显促生长作用。为发掘和应用抗逆、促生的优良菌种资源提供理论基础。     

一株产1-氨基环丙烷-1-羧酸脱氨酶的氢氧化细菌的分离鉴定及酶活力测定

[目的]以结瘤豆科植物紫花苜蓿根际土壤为研究材料,筛选具有ACC脱氨酶活力的氢氧化细菌,探索氢氧化细菌植物促生作用机制.[方法]利用持续通H2 的气体循环培养体系、矿质盐固体培养基,分离、培养氢氧化细菌,观察菌株形态并测定生理生化特征;16S rDNA序列分析法构建系统发育树;采用薄层层析法筛选ACC脱氨酶阳性菌株,茚三酮显色法测定ACC脱氨酶活力.[结果]分离的37株细菌中有8株菌氧化氢和自养生长能力较强,初步确定为氢氧化细菌,从中筛选出1株ACC脱氨酶阳性菌株WMQ-7.菌株WMQ-7的形态特征、生理生化特征与恶臭假单胞菌(Pseudomonas putida)的特征基本一致;16s rDNA序列(GenBank登录号为EU807744)在系统发育树中与恶臭假单胞菌同属一个类群,序列同源性99%.鉴定菌株WMQ-7为恶臭假单胞菌,其ACE脱氨酶活力为0.671 U/μg[结论]采用气体循环培养体系分离氢氧化细菌,克服了传统配气法的局限.ACC脱氨酶阳性菌株的筛选,为深入研究氢氧化细菌作为植物根际促生菌的菌株特性和促生机制提供理论依据.     

中华补血草内生与根际具ACC脱氨酶活性细菌的筛选及其生物多样性

【目的】获得江苏沿海滩涂盐生药用植物中华补血草内生及根际具有1-氨基环丙烷-1-羧酸(ACC)脱氨酶活性的细菌,研究其遗传多样性和潜在促生活性。【方法】从中华补血草和根际土壤分离筛选具有ACC脱氨酶活性的菌株,对其ACC脱氨酶活性定量检测,通过16S r RNA基因序列分析确定菌株系统发育地位。同时研究其固氮、溶磷、产植物生长素吲哚乙酸(IAA)及耐盐能力。【结果】分离筛选获得18株具有ACC脱氨酶活性的内生与根际细菌,定量检测发现其中有13株菌的ACC脱氨酶含量在20 nmolα-KA/(mg Pr·h)以上,有11株菌可以固氮,7株菌能够解磷,9株菌产生IAA。菌株的Na Cl盐耐受范围多数在0–13%之间。16S r RNA基因测序表明,活性菌株分属于7个属,多样性丰富,节杆菌属(Arthrobacter)为优势类群。其中菌株KLBMP 5180为节杆菌属的潜在新种。【结论】江苏沿海滩涂盐生药用植物中华补血草共生环境中具有丰富多样的具ACC脱氨酶活性的菌株,并存在潜在新物种资源,具有进一步研究价值。     

4株茶树根际促生菌菌株的鉴定及促生作用

【背景】根际促生菌可以促进植物生长、提高植物抗性。茶树根际具有特殊的根土微生物生境,可以获得具促生作用的有益微生物。【目的】探究4株茶树根际促生菌菌株的分类地位及促生作用,筛选优良的根际促生菌菌株。【方法】通过形态、生理生化特征、16S rRNA基因序列同源性比对鉴定4株茶树根际促生菌,采用钼锑抗比色法测定溶磷量,通过比色法测定ACC脱氨酶活性、CAS法测定产铁载体能力、Salkowski法测定产IAA (Indoleacetic acid)的能力进行促生作用研究,通过盆栽实验测试白菜、空心菜、苋菜及水稻的株高及鲜重以分析促生效应。【结果】鉴定KKS-6-N1为放射型土壤杆菌(Agrobacteriumradiobacter), KKS-7-N7为铜绿假单胞菌(Pseudomonas aeruginosa),GD3为Pseudomonashunanensis,GD12为弯曲芽孢杆菌(Bacillusflexus)。固氮菌株KKS-6-N1可产铁载体;固氮菌株KKS-7-N7具有解磷及产铁载体能力,分泌的IAA含量高达101.29mg/L;解钾菌株GD3具溶磷能力,分泌的ACC脱氨酶酶活为8.09μmol/(mg·h),相对铁载体含量为0.31;具固氮解钾性能的菌株GD12分泌的ACC脱氨酶活性为14.46μmol/(mg·h)。盆栽试验表明,4个菌株对白菜、空心菜、苋菜的株高和鲜重均有明显促进作用,尤以GD3效果更甚。【结论】茶树根际促生菌菌株Pseudomonas hunanensis GD3促生作用显著,具有开发成微生物菌肥的潜力。     

贵州喀斯特地区具ACC脱氨酶活性细菌的分离和鉴定

1-氨基环丙烷-1-羧酸(ACC)脱氨酶能够降解乙烯前体,从而有助于植物生长。具有ACC脱氨酶活性细菌在旱胁迫下具有植物促生作用。本研究从贵州地区选取典型喀斯特地貌区域159份土壤样品中分离并鉴定出具有ACC脱氨酶活性的细菌188株。利用16S r DNA测序分析将这些菌株归为14属63种,优势菌属为假单胞菌属和伯克氏菌属,分别有ACC脱氨酶活性的细菌18种和17种。对63种菌株的ACC脱氨酶活性定量检测,酶活最高的菌株是AL30ADF120(Cupriavidus oxalaticus),为3.639 U/mg。据我们所知,这是第一个有关喀斯特地区土壤中的具有ACC脱氨酶活性细菌名录,为将来研究ACC脱氨酶活性细菌的植物促生作用奠定了基础。     

一株人参内生1-氨基环丙烷-1-1羧酸(ACC)脱氨酶活性细菌的筛选、鉴定及其对宿主生长的影响

【目的】从人参内生细菌中获得具有1-氨基环丙烷1-羧酸(ACC)脱氨酶活性的菌株,并进行促生效果的验证。【方法】结合初筛和复筛的方法筛选具有ACC脱氨酶活性的人参内生菌株;采用Ashby培养基和固氮酶基因验证其固氮潜能;菌碟法及钼锑抗比色法测定其解磷能力;CAS方法检测产生铁载体能力;通过室内及田间试验测定菌株对人参生长的促进作用。通过形态学、生理生化测定及16S rRNA序列分析明确菌株的分类地位。【结果】从120株人参内生菌中获得了一株具有较高ACC脱氨酶活性的菌株JJ8-3,其酶活性为α-酮丁酸6.7μmol/(mg·h);且具有解磷特性、固氮潜能和产生铁载体能力;能明显促进人参种子及根部的生长;经鉴定菌株JJ8-3为荧光假单胞菌(Pseudomonas fluorescens)。【结论】获得了一株具有ACC脱氨酶活性的人参内生细菌,将为其在促进植物生长中的应用和研究奠定基础。     

具有ACC脱氨酶活性的绞股蓝内生细菌的分离鉴定及其抑菌促生作用

采用富集筛选法从绞股蓝根中筛选得到6株具有ACC脱氨活性的细菌,其中菌株JDG-6、JDG-7、JDG-14、JDG-16、JDG-23均具有较强的分泌铁载体能力,但菌株JDG-32没有产铁载体能力。抑菌试验结果显示,菌株JDG-6、JDG-7、JDG-14和JDG16对一种或多种供试菌有抑菌作用,其中菌株JDG-14能抑制大肠埃希菌、藤黄八叠球菌和白色念球菌的生长。促生试验表明,菌株JDG-6、JDG-7和JDG-14均能促进水稻幼苗根的伸长,其中菌株JDG-14的促生作用最为明显,与对照组相比水稻幼苗的根长和根鲜重分别增长了26%和21%。通过形态特征观察、生理生化试验以及16S rDNA序列分析,菌株JDG-6、JDG-7、JDG-16和JDG-23属于假单胞杆菌属（Pseudomonas）,菌株JDG-14为木糖葡萄球菌（Staphylococcus xylosus）,而菌株JDG-32为枯草芽胞杆菌（Bacillus subtilis）。菌株JDG-6、JDG-7和JDG-14均具有ACC脱氨酶活性和抑菌活性的促生菌,具有农业应用潜力。     

氢氧化细菌SDW-16的分离鉴定及促生特性

利用气体循环培养体系从沙打旺根际土壤分离得到1株氢氧化细菌SDW-16(GenBank登录号:KF835389),16S rDNA序列分析和生理生化特征鉴定其为荧光假单胞菌(Pseudomonas fluorescens)。菌株对植物促生机制的初步研究表明菌株SDW-16除具有铁载体分泌能力外,还具有产IAA和ACC脱氨酶活性,其中产IAA量为(21.62±0.30)μg/mL,ACC脱氨酶活力高达(8 372.17±805.43) nmol/(mg·h)。菌株SDW-16具有多项促生能力且均高于其他菌株,说明菌株SDW-16有较高的促生特性,同时也初步证明了氢氧化细菌的促生机制。     

海州香薷（Elsholtzia splendens）根际铜抗性细菌的筛选及生物多样性

摘要：【目的】重金属耐性植物海州香薷根际铜抗性细菌的筛选及生物多样性研究将有助于了解微生物－超富集植物相互关系和植物修复机理、开发微生物－香薷重金属修复新技术。【方法】采用稀释平板涂布法从海州香薷根际筛选铜抗性菌株,测定菌株溶磷和产生吲哚乙酸、铁载体、1-氨基环丙烷-1-羧酸（ACC）脱氨酶的特性,采用16S rDNA限制性酶切多态性分析（amplified rDNA restriction analysis, ARDRA）研究铜抗性细菌的遗传多样性,根据16S rDNA相似性对产ACC脱氨酶的菌株进行了     

番茄果实ACC合成酶的性质

测定了经4步纯化、比活性达20000U／mg蛋白质以上的番茄果实伤诱导ACC合成酶的一些酶学性质。酶反应最适PH值为9．5;酶在pH8．0下最稳定,pH7．5－10短时间处理不会使酶发生不可逆变性;酶在pH8．0和9．5的Km值分别为23和4Dμmol／L;根据酶反应不同时间的产物累积量,得出反应速度随时间的变化符合函数关系式Vt＝V0e－kt,并根据此式求出酶的半寿期为107min。光照对酶活性有抑制作用。酶的DTNB化学修饰结果表明,在酶活性中心的PLP结合部位很可能有半胱氨酸残基存在。     

Expression of the ACC synthase and ACC oxidase coding genes after self-pollination and incongruous pollination of tobacco pistils

In tobacco, as in other species, ethylene is produced in response to pollination. Although tobacco is a self-compatible species, it displays unilateral incongruity with other Nicotianaplants. Incongruous pollination also results in ethylene production, but this production differs depending on the pollen used and is related to the extent to which pollen tubes grow in the tobacco style. In the investigation reported here we followed the expression of the ACC synthase- and ACC oxidase-coding genes upon pollination of tobacco pistils and compared self-pollination with incongruous pollination. The pattern of expression of these genes also correlated with pollen-tube growth, although wounding alone cannot explain the results obtained. We also examined the expression of these genes upon pollination of immature tobacco pistils, in which different pollen tubes grew indistinctly inside the tobacco style and reached the ovary at the same rate. In this situation no significant differences in gene expression could be observed between the different pollinations. Ethephon, a substance that produces ethylene, could, in some cases, minimize the arrest of incongruous pollen tubes inside the style.     

Perspectives of bacterial ACC deaminase in phytoremediation

Phytoremediation of contaminated soil and water environments is regulated and coordinated by the plant root system, yet root growth is often inhibited by pollutant-induced stress. Prolific root growth could maximize rates of hyperaccumulation of inorganic contaminants or rhizodegradation of organic pollutants, and thus accelerate phytoremediation. Accelerated ethylene production in response to stress induced by contaminants is known to inhibit root growth and is considered as a major limitation in improving phytoremediation efficiency. Recent work shows that bacterial 1-aminocyclopropane-1-carboxylate (ACC) deaminase regulates ethylene levels in plants by metabolizing its precursor ACC into alpha-ketobutyric acid and ammonia. Plants inoculated with ACC deaminase bacteria or transgenic plants that express bacterial ACC deaminase genes can regulate their ethylene levels and consequently contribute to a more extensive root system. Such proliferation of roots in contaminated soil can lead to enhanced uptake of heavy metals or rhizodegradation of xenobiotics.     

RFLP analysis of 1-aminocyclopropane-1-carboxylate synthase ACC2 and ACC4 genes from Polish cultivars of tomato

An important trait of tomato is the rate of fruit ripening, strongly dependent on ethylene production. The ripening-related ethylene synthesis in tomato is controlled mainly by 1-aminocyclopropane-1-carboxylate synthase LE-ACS2 and LE-ACS4 isoenzymes (Rottmann et al., 1991, J. Mol. Biol. 222: 937; Lincoln et al., 1993, J. Biol. Chem. 268: 19422; Barry et al., 2000, Plant Physiol. 123: 979). In spite of numerous reports on the LE-ACS2 and LE-ACS4 gene expression, only ones considered the genomic organisation each of these genes (Rottmann et al., 1991; Lincoln et al., 1993) reported one copy of each of these genes in tomato cv VF36. In this article we suggest that the genomic organisation of LE-ACS2 and LE-ACSS4 genes may depend on tomato cultivars and may differ from that described by the above authors. The results of Southern analyses of genomic DNAs from 17-day old seedlings (cultivars Jaga, Halicz, Betalux, New Yorker) imply that the genomic organisation of LE-ACS2 and LE-ACS4 genes in Polish cultivars differs from that reported for cv VF36.     

1-aminocyclopropane-1-carboxylate (ACC) deaminase induced by ACC synthesized and accumulated in Penicillium citrinum intracellular spaces

We have already described how 1-aminocyclopropane-1-carboxylic acid (ACC), which is a precursor of the plant hormone ethylene, is synthesized in Penicillium citrinum through the same reaction by the catalysis of ACC synthase [EC 4.4.1.14] as in higher plants. In addition, ACC deaminase [EC 4.1.99.4], which degrades ACC to 2-oxobutyrate and ammonia, was also purified from this strain. To study control of induction of ACC deaminase in this organism, we have isolated and analyzed the cDNA of P. citrinum ACC deaminase and studied the expression of ACC deaminase mRNA in P. citrinum cells. By the analysis of peptides from the digests of the purified and modified ACC deaminase with lysylendopeptidase, 70 % of its amino acid sequences were obtained. These amino acid sequences were used to identify a cDNA, consisting of 1,233 bp with an open reading frame of 1,080 bp encoding ACC deaminase with 360 amino acids. The deduced amino acids from the cDNA are identical by 52% and 45% to those of enzymes of Pseudomonas sp. ACP and Hansenula saturnus. Through Northern blot analysis, we found that the mRNA of ACC deaminase was expressed in P. citrinum cells grown in a medium containing 0.05% L-methionine. These findings suggest that ACC synthesized by ACC synthase and accumulated in P. citrinum intracellular spaces can induce the ACC deaminase that degrades the ACC.