大熊猫肠道抗生素耐药菌质粒的研究

对分离自四川省卧龙保护区大熊猫肠道的6株大肠杆菌的抗生素抗性与质粒的关系进行了研究,结果显示：6株大肠杆菌中都存在不同大小的质粒DNA;采用SDS高温法处理每株大肠杆菌后,有2类质粒在琼脂糖凝胶上的条带数减少,菌株的抗生素抗性水平也有所降低,有2株菌的某些抗生素抗性完全消失;将分离自6株大肠杆菌的质粒转化大肠杆菌JM109,获得1个氨苄青霉素抗性转化子.这些结果表明,大熊猫肠道中的大肠杆菌的抗生素抗性与它们含有的质粒有关.     

邻氯硝基苯降解菌OCNB-1的分离鉴定与降解质粒

以邻氯硝基苯为唯一碳源、氮源和能源,从某化工厂废水处理站活性污泥中分离出一株细菌OCNB-1, 经微生物自动测定仪WSVTK-R07.02和16S rDNA扩增测序初步鉴定为Pseudomonas putida;在培养基pH值为8.0、培养温度为32 ℃、摇床转速为120 r/min的条件下,考察了该菌株降解邻氯硝基苯的能力,发现菌株42 h内将起始浓度为1.07 mmol/L的该化合物降解了约80%;经质粒检测,在OCNB-1菌株中发现一条质粒条带,选用7种内切酶对质粒pOCNB-1进行单酶切,得出质粒大小约为32 kbp;抗生素抗性实验表明菌株对红霉素、氨苄青霉素、青霉素钠的抗性与质粒无关,对利福平、氯霉素的抗性与质粒有关;通过细菌接合,质粒转移到无降解邻氯硝基苯能力的Pseudomonas.stutzeri受体菌中,接合子Pseudomonas.stutzeri能利用邻氯硝基苯为唯一碳源、氮源和能源,证明质粒为降解质粒.     

RP_4质粒在兼性自养多能硫杆菌中的接合转移

本实验以大肠杆菌(Escherichia coli)HB101(RP_4)作为供体菌,以多能硫杆菌(Thiobacillus versutus)作为受体菌,通过接合的方法将RP_4质粒转移到了多能硫杆菌中.接合频率为1.9×10~(-5),RP_4质粒的三个抗性基因(Ap~R、Tc~R、Km~R)在多能硫杆菌中均得到了表达.再将RP_4质粒从多能硫杆菌反向转移到大肠杆菌HB101菌中,接合频率为3.3×10~(-1)～7.3×10~(-1),三个抗性基因也均得到了表达.实验证明,多能硫杆菌对于RP_4质粒既是一个受体菌,也是一个很好的供体菌.     

邻氯硝基苯降解菌Pseudomonas putida OCNB-1的分离、鉴定与降解质粒研究

摘要：以邻氯硝基苯为唯一碳源、氮源和能源从某化工厂废水处理站分离出一株细菌, 经微生物自动测定仪WSVTK-R07.02和16SrRNA扩增测序鉴定为Pseudomonas putida;在 培养基pH为8.0,培养温度32 ℃,摇床转速120 rpm 条件下考察了该菌株降解邻氯硝基苯的能力, 菌株42 h内将起始浓度为1.07 mM的对邻氯硝基苯降解近80%;经质粒检测,在菌株OCNB-1中发现一条质粒条带,选用7种内切酶对质粒pOCNB-1进行单酶切,得出质粒大小约为32 Kb;抗生素抗性实验表明菌株对红霉素、氨苄青霉素、青霉素钠的抗性与质粒无关,对利福平、氯霉素的抗性与质粒有关;通过细菌接合,质粒转移到无降解邻氯硝基苯能力的Pseudomonas.stutzeri受体菌中,接合子Pseudomonas.stutzeri能利用邻氯硝基苯为唯一碳源、氮源和能源,证明质粒为降解质粒。     

β－淀粉酶基因在黄单胞菌中的克隆及表达

广泛寄主范围载体ｐＫＴ２１０可在辅助质粒ｐＲＫ２０１３的协助下转移进入黄单胞菌（革兰氏阴性）中并能稳定存在；来源于枯草杆菌的β－淀粉酶基因与载体ｐＫＴ２１０形成重组质粒ｐＹＬ１，并以其转化大肠杆菌；通过三亲本接合将ｐＹＬ１引入带有利福平抗性的黄单胞菌ＮＫ０１－Ｒ中，通过抗性选择接合子，并测定接合子的淀粉酶水解能力及产胶能力．本文获得一株黄原胶产量比出发菌株提高２０％，且淀粉酶水解能力显著提高的工程菌株．     

Sanglifehrin A产生菌Streptomyces flaveolus DSM 9954遗传转化系统的建立

Sanglifehrin A的产生菌淡黄色链霉菌Streptomyces flaveolus DSM 9954,通过筛选,得到其菌丝培养基为TSB, 生孢培养基为ISP-4,抗性实验显示淡黄色链霉菌Streptomyces flaveolus DSM 9954对所选抗生素都较敏感. 利用原生质体转化和接合转移都成功地构建了两种遗传转移系统,并且从接合转移成功的链霉菌转化子中回收质粒pKC 1139,经EcoR Ⅰ单酶切电泳,验证了质粒确实通过接合转移从大肠杆菌转移至宿主链霉菌, 这为研究Sangl     

铜胁迫下菌株CL01促进紫云英生长的研究

从湖北某矿区植物根际土壤中筛选到了一株多种重金属抗性菌株CL01.根据形态学特征和16S rDNA序列比较了结果,鉴定其为Achromobacter xylosoxidans.当土壤所含铜浓度为75 mg/kg时,接种菌株CL01可以促进紫云英根延长,增加植株高度,提高紫云英的生物量.菌株CL01通过减少根际土壤可吸收的铜含量,减少了铜在紫云英根部的聚集,说明该细菌提高了植物的抗铜能力.     

β—淀粉酶基因在黄单胞菌中的克隆及表达

广泛寄主范围载体ｐＫＴ２１０可在辅助质粒ｐＲＫ２０１３的协助下转移进入黄单胞菌（革兰氏阴性）中并能稳定存在；来源于枯草杆菌的β－淀粉酶基因与载体ｐＫＴ２１０形成重组质粒ｐＹＬ１，并以其转化大肠杆菌；通过三亲本接合将ｐＹＬ１引入带有利福平抗性的黄单胞菌ＮＫ－０１－Ｒ中，通过抗性选择接合子，并测定接合子的淀粉酶水解能力及产胶能力。本文获得一株黄原胶产量比出发菌株提高２０％，且淀粉酶水解能力显著提高的工     

乳酸乳球菌nisin抗性基因的克隆及作为筛选标记的研究

在添加500U/mL nisin和溴甲酚紫的GM17选择性培养基上,分离到5株nisin抗性菌株。根据形态观察,生理生化特性和16S rDNA基因序列比对被鉴定为乳酸乳球菌。根据已报道nisin抗性基因设计引物,分别以这5株菌的染色体和质粒DNA为模板进行PCR扩增,结果从1株抗性菌株的染色体上得到目的基因产物。通过序列测定和同源性比对,证明为nisin抗性基因（nsr）。将nsr基因克隆到乳酸菌-大肠杆菌穿梭质粒pTRKH2上,重组质粒命名为pT-nsr。pT-nsr电转化乳酸乳球菌MG1614,获得的重组菌MG1614/pT-nsr在含有500U/mL nisin的培养基中生长情况良好。这表明nsr基因赋予宿主菌抗nisin特性,并且与红霉素抗性功能相同,因此nsr基因可以作为筛选标记用于食品级载体的构建。     

烟酸羟基化菌株睾酮丛毛单胞菌JA1抗性质粒的消除

睾酮丛毛单胞菌JA1可羟基化烟酸产生6-羟基烟酸,并且对多种抗生素具有抗性.碱变性法提取JA1细胞内的质粒, 检测到一个约19 kb大小的质粒.用0.002 5% SDS进行质粒消除实验,结果表明,质粒消除后菌株对卡那霉素敏感,但仍对氨苄青霉素、安普霉素和链霉素具有抗性,推测质粒携带卡那霉素的抗性基因.同时,质粒消除后,JA1菌株烟酸脱氢酶酶活性没有变化,推测烟酸脱氢酶的基因并不在质粒上.     

Improving DNA damage repair ability ofE. coli to UV-radiosensitivity by DNA-mediated gene transfer of low energy ion beam

Using calcium chloride method of transfer gene as control, a new technique of transferring gene by low energy ion beam has been applied to the study of improving DNA damage repair ability ofE. coli to UV-radiosensitivity. The genome DNA pieces ofDeinococcus radiodurans, as “foreign” genetic materials, were introduced into the UV-radiosensitive strains ofE. coli by implantation of 20 keV Ar⁺ at doses ranging from 1 × 10¹⁵ to 2 × 10¹⁵ ions/cm². Results show that the transfected strains present higher UV-radioresistance than that of un-transfected ones and start ones. The survival rate of transfected strains and their unscheduled DNA synthesis (UDS) ability is increased, indicating that the transfer gene is a success.     

Stable isotopic analysis on sus bones from the Wanfabozi site, Tonghua, Jilin

In order to study the sus diets and explore the possibility to distinguish domestic pig from wild boar through dietary analysis, stable carbon and nitrogen isotopes of 28 pig bones from archaeological site of Wanfabozi in Tonghua City, Jilin Province were analyzed. The δ^13C and δ^15N values of uncontaminated bones show that the overall pigs in the site were generally herbivores and ate mainly C3 plants. Significant difference of δ^15N values was observed between wild boar and domestic pigs, which may result from the higher consumption of animal protein in domestic pigs other than from that in wild boar, possibly from human leftover or waste,. The dietary difference between wild boar and domestic pigs has great potential to differentiate wild boar and domestic pigs in the early stage of pig domestication.     

Insights into the subunit interactions of the chloroplast ATP synthase

Subunit interactions of the chloroplast F0F1- ATP synthase were studied using the yeast two-hybrid system. The coding sequences of all the nine subunits of spinach chloroplast ATP synthase were cloned in two-hybrid vectors. The vectors were transformed into the yeast strains HF7c and SFY526 by various pairwise combinations, and the protein interactions were analyzed by measuring the yeast growth on minimal SD medium without serine, lucine and histidine. Interactions of γ Subunit with wild type or two truncated mutants of γ sununit, △εN21 and △εC45, which lose their abilities to inhibit the ATP hydrolysis, were also detected by in vitro and in vivo binding assay. The present results are largely accordant to the common structure model of F0F1-ATP synthase. Different from that in the E. Coli F0F1-ATP synthase, the δ subunit of chloroplast ATP syn- thase could interact with β,γ,ε and all the CF0 subunits in the two-hybrid system. These results suggested that though the chloroplast ATP synthase shares the similar structure and composition of subunits with the enzyme from E. Coli, it may be different in the subunit interactions and con- formational change during catalysis between these two sources of ATP synthase. Based on the present results and our knowledge of structure model of E. Coli ATP synthase, a deduced structure model of chloroplast ATP synthase was proposed.     

Cloning and analysis of a cDNA encoding acetohydroxy acid isomeroreductase from G2 pea

Using cDNA representational difference analysis (cDNA RDA) method, we have successfully isolated a gene fragment whose expression was specifically induced by external GA₃ application. Screening a G2 pea cDNA library using this fragment as a probe, we obtained a 2036 bp full-length cDNA. It contains a 1746 bp open reading frame and encodes a protein of 581 amino acids with a theoretical molecular weight of 64 ku. It shares high-level sequence identity withAAIR genes from other plant species. This cDNA was cloned into expression vector and recombinantE. coli DH5α cells with remarkable AAIR enzyme activity were obtained.     

多重耐药UPEC细菌毒力基因分布特征及进化分型分析

本研究对临床尿路感染病例病原菌耐药情况、毒力基因分布及其进化分型特征,以期为耐药性尿路感染病原防控提供参考。通过对收集的尿样进行细菌分离培养,结合革兰氏染色镜检、16S rDNA对细菌进行鉴定,利用K-B纸片扩散法对细菌进行药敏试验,并通过PCR扩增进行毒力基因及系统发育和进化分型分析。本研究成功检测出20株尿路致病性大肠杆菌（Uropathogenic Escherichia coli, UPEC）,药敏试验显示多数菌株具有多重耐药特征,该批UPEC对青霉素、红霉素、万古霉素耐药率达100%;对羧苄西林、氨苄西林、阿奇霉素耐药率达90%以上;对四环素、诺氟沙星、链霉素、耐药率达80%以上;对环丙沙星、庆大霉素、卡那霉素、氟氧沙星、头孢曲松耐药率达70%以上;对头孢噻肟、呋喃妥因、氯霉素耐药率在35%以上;对阿莫西林与多粘菌素B耐药率低,分别仅为10%与5%。六个毒力基因（iutA、fimH、hly、fyuA、tsh、traT）检出率分别为85%、95%、85%、75%、25%、10%。其系统发育分型中,归属B2群的细菌最多,占比40%(n=8);D群次之,占比35%(n=7);其次为...     

耐碳青霉烯酶肺炎克雷伯菌的分离鉴定及生物学特性分析

以临床尿路感染患者尿液为研究对象,通过分离培养及16s rDNA进行肺炎克雷伯菌的分离鉴定;采用Kirby-Bauer纸片法进行药敏实验,并对分离株进行碳青霉烯酶表型筛选;通过PCR检测常见的碳青霉烯酶耐药基因、荚膜血清型和毒力基因分布情况;对分离的肺炎克雷伯菌株进行了生物被膜形成能力及其对小鼠致病力的分析。本研究从采取的86例尿液标本中分离检出11株耐碳青霉烯酶肺炎克雷伯菌,分离率为12.79%。耐碳青霉烯酶基因PCR检测结果显示,blaNDM、blaVIM、blaIMP和blaKPC基因在分离株中均呈现不同程度的分布。耐药性分析发现11株肺炎克雷伯菌对氨苄西林耐药率为90.91%,对头孢噻肟耐药率为63.64%,对链霉素、庆大霉素、氯霉素和诺氟沙星的耐药率较低。耐碳青霉烯类肺炎克雷伯菌荚膜分型结果显示,分离的11株细菌中10株均为强毒力型菌株（90.9%）,其中K57血清型4株（36.4%）,K1血清型1株（9.1%）,K2血清型1株（9.1%）,K5血清型1株（9.1%）,K20血清型3株（27.3%）,提示该批分离株具有较强的致病力。此外,毒力因子分布结果显示,其毒力因子rmpA（54.5%）、Aerobactin F（54.5%）在菌株中分布较为广泛。生物被膜形成能力检测及小鼠致病性试验结果显示,9株分离株均有较强的生物被膜形成能力,菌株致病力可能与荚膜血清型及生物被膜形成能力相关。综上所述,临床分离的致尿路感染病原肺炎克雷伯菌呈现多重耐药特征,强毒力菌株以K57荚膜型为主,K1、K2均有分布,提示对尿路感染病原应加强耐药监测,合理使用抗菌药物,有效防控多重耐药和强毒力菌株的感染与流行。     

养殖环境病原菌以及超级细菌气溶胶的发生与传播的监测

为了观察和评估养殖环境耐药细菌气溶胶的形成与扩散,采用Andersen-6级空气样品收集器在养鸡场鸡舍环境舍内、外(到下风方向800 m)收集空气样品;与此同时,采集鸡只粪便样品。对样品分离的大肠杆菌(E.coli)进行药物敏感性测试。结果显示,各个地点分离的E.coli对利福平和青霉素完全耐药,但他们对妥布霉素、庆大霉素敏感;有不同数量的菌株对氟哌酸、链霉素、头孢哌酮、氯霉素、复合磺胺和四环素有不同程度的耐药性。采用多重PCR方法,分别对从鸡、猪、牛舍(共21个)及其环境中分离到的480株大肠杆菌,进行了5种毒素基因即STa,STb,LTa,Stx1和Stx2/Stx2e的检测。结果表明,分离株都携带有一定数量的毒素基因,很多携带2种或2种以上。通过对11个动物舍(鸡、猪、牛、兔)空气和粪便样品分离的426株肠球菌对四环素类(TetM)、氨基糖甙类抗生素及万古霉素VanA和VanB主要耐药基因的检测表明,14.55%的细菌对β-内酰胺酶耐药;分离株都存在不同程度的对四环素类抗生素的耐药性;共检测出31株肠球菌携带vanA和vanB耐药基因;绝大多数肠球菌携带氨基糖苷修饰酶(AME)基因的一种或几种。     

川西北牦牛源产肠毒素大肠杆菌的分子流行病学调查

为调查川西北地区牦牛源产肠毒素大肠杆菌的流行情况,对采自川西北甘孜州和阿坝州的26份腹泻牦牛粪便样品进行大肠杆菌分离,分别以分离的大肠杆菌DNA和牦牛腹泻粪便样本提取的总DNA为模板,采用PCR方法扩增产肠毒素大肠杆菌黏附因子K99+菌毛基因片段,分析产肠毒素大肠杆菌在牦牛腹泻中存在情况,同时比较两种DNA提取方法对产肠毒素大肠杆菌PCR检出率的差异;以分离自外表健康牦牛粪便的105株大肠杆菌DNA为模板,采用PCR方法扩增产肠毒素大肠杆菌黏附因子K99+菌毛基因片段,比较牦牛源产肠毒素大肠杆菌在外表健康牦牛和腹泻牦牛中的携带情况.结果:从26份牦牛腹泻粪便样本中共鉴定出84个大肠杆菌菌落携带K99菌毛基因,这些菌落分别来自所有26份牦牛腹泻样品,因此,牦牛腹泻样本100%分离并检测到携带K99+菌毛基因的产肠毒素大肠杆菌;而粪便总DNA检测结果为K99+菌毛基因阳性率84.62%(22/26),粪便中提取的总DNA模板K99+菌毛基因PCR检出率略低于分离鉴定的细菌DNA检出率;105株外表健康牦牛粪便样本分离株中检测到携带K99+菌毛基因的产肠毒素大肠杆菌34株,检出率为32.38%(34/105),产肠毒素大肠杆菌在牦牛腹泻样本中的检出率显著高于外表健康牦牛粪便的检出率(P0.001).结果表明,产肠毒素大肠杆菌在牦牛粪便中存在广泛,是引起牦牛腹泻的一种重要病原菌;以粪便中提取的总DNA为模板,可快速检测牦牛粪便中产肠毒素大肠杆菌的存在.     

产丁醇大肠杆菌工程菌的构建

【目的】为了在大肠杆菌(Escherichia coli)中导入改良的丁醇合成途径,使非生产菌株大肠杆菌具备产丁醇的能力。【方法】克隆大肠杆菌乙酰转移酶基因atoB和丙酮丁醇梭菌(Clostridium acetobutylicum)丁醇合成途径关键酶基因(crt、hbd、adhE),构建多顺反子表达质粒pSE380-atoB-adhE-crt-hbd;克隆齿垢密螺旋体(Treponema denticola)反式烯酰辅酶A还原酶基因ter,构建表达质粒pSTV29-ter,并将双质粒导入到大肠杆菌。【结果】构建的工程菌能半厌氧发酵产微量丁醇,产量为0.08g/L。【结论】大肠杆菌中的丁醇合成途径导入成功,构建了产丁醇的大肠杆菌工程菌。     

Desulfurization metabolite of Rhodococcus erythropolis LSSE8-1 and its related desulfurizational gene fragments

Rhodococcus erythropolis LSSE8-1 is a newly isolated biodesulfurizaion strain from the soil of Chishui gas field, Guizhou Province, China. The analysis of its metabolism product shows that the strain is a kind of biocatalyst able to oxidize dibenzothiophene (DBT) to 2-hydroxydi-phenyl (HBP), and therefore the sulfur in DBT is selectively removed. By using DBTO2 (dibenzothiophene 5,5-dioxide) as substrate, both DBT and HBP are found in the culture,which shows that the reaction from DBT to DBTO2 is reversible in the cell. While using 0.5 mmol/L DBT as control,0.01-0.4 mmol/L DBTO2 shows poisonous effect to the cell,which will explain why there is no DBTO2 accumulation in the process of biodesulfnrization. After treatment by lysozme,the plasmid DNA of the strain is isolated by alkaline method to be used as the template of PCR reaction. Three dsz gene fragments of 1.3, 1.0 and 1.2 kb respectively were amplified.Each fragment is ligate with PGEM-T vector, and cloned into E. coil DH5a. The clone DNA is sequenced and the result shows that dsz related genes are highly conservative. The identities of dszA and dszB with respect to IGTS8 are 100%,and the identity of dszC with that of IGTS8 is 99%.