拮抗细菌Enterobacter cloaeae B8的一株抗菌素抗性突变体(英文)     

徐平  陈卫良  朱伟光  李德葆 《浙江大学学报(农业与生命科学版)》1992,(4)

为了研究拮抗细菌Enterobacter cloacae B8在野外水稻叶面上的定殖情况及拮抗作用,由自然选择和Tn5诱变从B8产生了一株抗菌素抗性突变体BX8.BX8能在含利福平达400μg/ml或卡那霉素达300μg/ml的LB培养基中生长.实验表明该抗菌素抗性的产生没有降低BX8的拮抗活性.该抗性比较稳定,在无抗菌素培养基中培养.BX8至少分裂生长60代内抗性不变.  相似文献   

GFP标记的短小芽胞杆菌（Bacillus pumilus）转座突变株的构建初探   总被引：1，自引：0，他引：1  

沈新迁  胡晓璐  刘通 《上海交通大学学报(农业科学版)》2012,30(4):15-20

以水稻叶面附生菌短小芽孢杆菌(B.pumilus)为研究对象,通过构建pMOD-egfp-Tetr转座复合体,采用电击转化法对短小芽孢杆菌DX01菌株进行了Tn5转座插入诱变,获得了大量的转化子。对转化子进行了遗传稳定性分析和分子鉴定,并且在荧光显微镜下观察到了强烈的绿色荧光,研究结果表明外源DNA已经成功整合进细菌的基因组中,gfp基因在短小芽孢杆菌中实现了组成型表达。  相似文献   

热不对称PCR(TAIL-PCR)和Tn5转座子质粒拯救法快速分离水稻条斑病菌致病相关基因   总被引：2，自引：0，他引：2  

袁亮  李玉蓉  张希福  郭威  车亦舟  陈功友 《农业生物技术学报》2009,17(6)

构建植物病原菌突变体库是进行新基因发掘和功能基因组学研究的有效途径之一.为快速准确地鉴定Tn5的插入位点和相应的功能基因,研究采用热不对称PCR(TAIL-PCR)和Tn5转座子质粒拯救两种技术,鉴别了在筛选水稻条斑病菌(Xanthomonas oryzae pv.oryzicola)Tn5插入突变体库过程中获得的8个在水稻(Oryza sativa)上毒性减弱的突变体.结果显示,TAIL-PCR和Tn5质粒拯救结合使用,可有效地鉴别Tn5的插入位点和相应的功能基因.TAIL-PCR鉴别的5株突变体是Tn5分别插入在分泌系统结构蛋白F基因(gspF)、cAMP调控蛋白、膜镶嵌蛋白酶酶原、S-蛋硫氨酸脱羧酶亚基和gspJ基因上;Tn5质粒拯救法鉴别的3株突变体是Tn5分别插入在致病性蛋白、功能未知的保守蛋白和鞭毛特异性ATP合酶基因上.序列同源性分析发现,8株突变体Tn5插入的基因与基因组测序的BLS256菌株中的对应基因同一性达100%.这表明,TAIL-PCR和Tn5质粒拯救技术可有效地应用于植物病原菌Tn5突变体库的鉴别和目标基因的分离.  相似文献   

Temperature‐responsive genetic loci in pectinolytic plant pathogenic Dickeya solani          下载免费PDF全文

R. Czajkowski  N. Kaczyńska  S. Jafra  M. Narajczyk  E. Lojkowska 《Plant pathology》2017,66(4):584-594

Fifty‐four Dickeya solani thermoregulated genes were identified using Tn5 transposon mutagenesis with an inducible gusA reporter system; 45 genes were up‐regulated at 37 °C, whereas nine were up‐regulated at 18 °C. The relative level of gene up‐regulation ranged from 2–1200 and 5–650 U/mg total proteins at 18 and 37 °C, respectively. Among the temperature‐regulated loci, genes coding for proteins involved in fundamental bacterial metabolism, membrane‐related proteins and pathogenicity‐corresponding factors and several hypothetical unknown proteins were found. The mutants were tested for their pathogenicity in planta and for features known to be important for D. solani virulence viz. production of pectinolytic enzymes, cellulases, proteases, siderophores and auxins as well as for motility and the ability to form a biofilm. Eight Tn5 mutants, four up‐regulated at high and four up‐regulated at low temperature, expressed visible phenotypes including the decreased ability to cause symptoms on potato tubers and chicory leaves, impairment in phospholipase production and/or deficiency in biofilm formation. The implications of environmental temperature on the ability of D. solani to cause disease symptoms in potato are discussed.  相似文献   

转座酶Tn3体外定向进化和酶活力提高方法的建立     

宋尚桥  马围围  李晓剑  曾素先  李鑫  严瑾  孙翠翠  黎宗强 《中国畜牧兽医》2020,47(4):984-991

试验旨在对转座酶Tn3的酶活力提高和进化进行初步探索。采用PCR扩增、限制性酶切、DNA连接、重组质粒的转化、易错PCR的优化及构建、D值的测定、酶切鉴定方法对转座酶Tn3进行体外定向进化研究。结果表明,用SacⅠ和XbaⅠ对重组质粒进行酶切,得到了450 bp的酶切产物;在含有卡那霉素的LB培养基培养菌体24 h,测量其D值,经过3轮重复性的研究,其D值和增殖能力从开始的0上升至0.18、0.42、0.60,说明Tn3活性有了明显的提高;将筛选得到的进化型重组质粒进行质粒抽提,用内切酶XhoⅠ和XbaⅠ进行双酶切鉴定,发现进化型重组质粒酶切产物条带相对较小;之后对进化型重组质粒进行基因测序分析,发现Tn3基因序列的多个位点发生了突变以及Tn3-Gal4靶向序列中间的CCR5-delta32基因被切除。说明成功完成了转座酶Tn3基因的克隆;确立了易错PCR的最优反应体系;选择卡那霉素作为筛选标记对进化型Tn3进行筛选,初步验证利用含有卡那霉素的LB培养基和对菌液D值的测定来进行筛选是可行的;Tn3基因序列突变和基因敲除,说明不论是在功能上还是在基因序列上Tn3都发生了改变,这种变化正是向着需要的方向进行的。  相似文献   

Pseudomonas for biological control of dutch elm disease. I. Labeling,detection and identification of pseudomonas isolates injected into elms; comparison of various methods     

R. J. Scheffer  D. M. Elgersma  Letty A. De Weger  G. A. Strobel 《European journal of plant pathology / European Foundation for Plant Pathology》1989,95(5):281-292

To understand the mechanisms involved in biological control of Dutch elm disease byPseudomonas, data were needed on the distribution of the introduced bacteria within elm and on the development of the bacterial population over a period of time.As traditional biochemical identification techniques are not suitable for distinguishment between individualPseudomonas isolates, three alternative approaches were compared.

1)	Chemotaxonomy, using lipopolysaccharide pattern, cell envelope protein pattern or DNA restriction fragment pattern. These techniques were reliable, but tedious.
2)	Labeling bacteria with a transposon (Tn903) or a plasmid construct (pMON5003) with a metabolic marker (Lac ZY, coding for -galactosidase and lactose permease) allowed for a reliable identification of reisolates. However, populations of transposon-labeled bacteria in elms declined much faster than populations of the unlabeled wild type. The plasmid carrying the metabolic marker disappeared from the bacterial populations over time. Apparently both the transposon and the plasmid were a disadvantage to the bacteria compared with the wild type parent strains.
3)	Immunoagglutination of representative reisolates with an antiserum against theP. fluorescens isolate in use proved to be specific and fast. For routine purposes the immunoagglutination test therefore was the best method of the various ones employed.

  相似文献   

短小芽孢杆菌转座突变株的GFP标记及在水稻上的定殖   总被引：4，自引：0，他引：4  

沈新迁  刘通  胡晓璐  顾振芳  陈云鹏 《中国农业科学》2012,45(24):5024-5031

【目的】研究GFP标记的短小芽孢杆菌在水稻植株上的定殖规律,为其生物防治应用提供科学依据。【方法】从短小芽孢杆菌GFP标记的转座突变株库中筛选强表达GFP且遗传稳定的突变株,用作水稻植株的定殖示踪。【结果】采用荧光酶标仪、流式细胞术检测及荧光显微镜观察等手段,对1 467株Tn5-egfp随机插入突变株的GFP表达作了定量和定性分析,最终获得8株荧光表达较强的突变株,并对突变株作了T-DNA插入拷贝数鉴定。在开放和封闭系统中,标记菌在水稻幼苗根际土壤中均可以存活15 d以上,且前者标记菌的数量下降相对更慢。【结论】短小芽孢杆菌在根毛区及侧根分生处的数量最多,并在根表面形成菌膜。该菌可通过水稻幼苗根部的伤口或幼嫩根毛等部位侵入根系,主要分布在皮层细胞及其间隙,在植物的维管束内亦可观察到荧光标记菌。研究结果表明该菌在水稻幼苗根际土壤中有着良好的定殖能力。  相似文献   

短小芽孢杆菌(Bacillus pumilus)DX01转座突变株的构建及转化体系的优化   总被引：1，自引：0，他引：1  

胡晓璐  沈新迁  傅科鹤  顾振芳  陈云鹏 《上海交通大学学报(农业科学版)》2011,29(1):68-74

Tn5转座子及其衍生载体被广泛应用于革兰氏阴性菌的基因功能研究,但尚未见有利用Tn5转座子导入革兰氏阳性菌短小芽孢杆菌(Bacillus pumilus)DX01菌株的报道.本研究通过构建Tn5转座载体,对短小芽孢杆菌DX01菌株进行了Tn5转座插入诱变,获得了大量的突变株.同时,考察了感受态细胞培养浓度、电击电压、电...  相似文献   

Molecular Monitoring of hrpB-disrupted Mutant of Ralstonia solanacearum in Tomato Plants     

Yoshinori MATSUDA  Hideyoshi TOYODA  Yasunari KATO  Koji KAKUTANI  Takayuki NAKANISHI  Miki BINGO  Teruo NONOMURA  Seiji OUCHI 《Journal of General Plant Pathology》2000,66(1):59-63

A nonpathogenic mutant of Ralstonia solanacearum was produced by the insertion of transposon Tn4431. The mutagenized gene was then cloned from a genomic DNA library by the gene tagging method, using the labeled lux operon located on Tn4431 of pUCD623 as a hybridization probe. From nucleotide sequence analysis of the transposon-inserted genomic clone, the hrpB gene was shown to be disrupted by the inserted transposon. Tomato plants were inoculated with the hrpB-disrupted mutant bacteria, for which multiplication and translocation were then monitored using the colony hybridization method. In addition, the original pathogenic bacteria in which the lux operon had been functionally ligated with the genomic promoter were also used for inoculation and traced by their bioluminescence. Multiplication of the hrpB-disrupted mutant was suppressed initially in the invaded root tissues and then in upper hypocotyl after translocation, suggesting that the pathogenic strain of R. solanacearum overcomes at least two steps of host responses expressed in root and hypocotyl tissues. Thus, our approach for molecular monitoring of the bacteria enabled us to precisely analyze the infection behavior of the pathogenic bacteria in planta. Received 16 April 1999/ Accepted in revised form 10 August 1999  相似文献   

以淀粉酶为标记选择水稻白叶枯病菌毒性基因突变体     

何晨阳  董晓艳 《南京农业大学学报》1992,15(3):30-34

  相似文献