肠道沙门菌O：4（B）菌群脉冲场凝胶电泳分子分型

为了明确2010年食源性疾病监测中分离的肠道沙门菌O：4（B）菌群的PFGE分子型别,探讨其多态性及其与分子流行病学关系,我们将分离获得的29株O：4（B）血清型沙门菌进一步鉴定培养,挑取单个菌落增菌,供试菌株基因组DNA用限制性内切酶xbaI消化酶切后进行脉冲场凝胶电泳分型,所得结果用Bionumerics5．1软件进行聚类分析。实验结果表明,根据电泳指纹图谱,可将29株肠道沙门菌O：4（B）菌群分为24个PFGE型别,菌株间的相似值在56.31％～100％之间,同一血清型别的沙门菌有多个PFGE型别。脉冲场凝胶电泳对O：4（B）群沙门菌有较高的分型能力,可有效的应用于食物中沙门菌溯源分析及分子流行病学研有。     

水稻联合固氮菌质粒的鉴定及固氮(nif)基因定位

应用改进的Casse法提取质粒,鉴定了供试菌株的质粒存在情况:高NH_4~+和N_2培养的Alcaligenes faecalis A_15,Enterobacter cloacae E26,E.cloacaeEnSs和klebsiella planticola DWUL2分别携有1—2个大质粒,分子量在30—200Md之间;K.planficola和Pseudomonas saccharophila含有小质粒;K.oxytocaNG13不携质粒。供试菌株中DNA与nif探针R1 nif DH和Ec nif B-Y均具有同源性。A.faecalis A15,E.cloacae E26和K.oxytoca NG13的nif基因位于染色体上,而E.cloacae EnSs的nif基因则位于一个较大质粒上。     

苏云金芽孢杆菌S1478—1分离株的特性鉴定及cry1Ac同源基因克隆

本研究对从海南岛尖峰岭热带雨林自然保护区的土壤样品中分离出的＆菌株S1478～1进行了特性鉴定,研究表明S1478—1分离株菌落形态和生长特征和励参照菌株HD73极其相似。16SrDNA序列分析表明,S1478—1分离株与其它＆thuringiensis、B．cereus和B.anthracis的16S rDNA序列相似性达到99％。分离株能产菱形伴胞晶体,SDS-PAGE蛋白电泳分析表明,菌株在生长后期,形成芽孢同时分泌130kD大小的晶体蛋白。生物测定表明S1478—1分离株对小菜蛾具有很高的毒杀活性,LC50值高达5．159×10^8cfu／mL。初步显示S1478—1分离株可作为防治鳞翅目害虫的生物农药菌株。利用PCR—RFLP方法鉴定S1478—1分离株含有cry1Ac同源基因,以PCR粘性端克隆方法扩增全长基因,序列测定表明该基因ORF为3537bp,编码1178个氨基酸,推定的编码蛋白分子量为133．3kD,与其它cry1Ac基因序列最高达到99％同源,因此,该基因可作为杀虫工程菌及培育转基因抗虫作物的候选基因。     

毒死蜱降解菌株Bacillus latersprorus DSP的降解特性及其功能定位

从土壤中分离到一株能有效降解毒死蜱的细菌DSP，该分离株鉴定为侧芽孢杆菌（Bacillus latersprorus）。在纯培养条件下测定了分离株DSP对毒死蜱的降解性能。在接种量为菌浓度OD415=0．2，pH7．0、25℃条件下，测得1、10mg L^-1毒死蜱的降解符合一级动力学特征，其降解半衰期分别为1．48d、5．00d，100mg L^-1毒死蜱对DSP菌有明显的抑制作用；分离株DSP在不同pH及温度下对毒死蜱的降解作用为pH7．0〉pH5．0〉pH9．0，35℃〉25℃〉15℃。该菌株含有一个20kb左右的质粒，通过吖啶橙与升温法对质粒消除实验证实，随着质粒的丢失，菌株利用毒死蜱的能力也丧失，用热击法和CaCl2法将菌株质粒转人大肠杆菌JM109和质粒消除处理的DSP^-菌中，随着质粒的获得，这些转化子获得了降解毒死蜱的能力。研究结果表明，Bacillus latersprorus DSP降解毒死蜱的功能和质粒有关。     

产荧光假单胞菌分离鉴定及其质粒DNA复制区的克隆

从国内多个地区的65份土样中分离纯化了产荧光假单胞菌株146株,其中菌株DSC3和DKXC1含有质粒,细胞形态,菌落特征的观察和检测证明DKXC1菌株为恶臭假单胞菌（Pseudomonas putida),以其为出发菌株,克隆了该菌质粒DNA复制子1.7kb片段,序列分析结果表明该DNA片段由3个ORF组成,分别编码311、130、115个氨基酸;它与已知的Pf-E.coli穿梭质粒pUCP19的复制区同源性只有49%,该复制子DNA序列已在GenBank登记,登记号为AF273219,这是国内分离克隆的第一个Pseudomonas质粒DNA复制子,以此复制子构建了4.7kb的E.coli-Pf穿梭载体pYQSPE,该质粒在荧光假单胞菌P303中能够稳定复制和遗传。     

西北部分地区鸡眼草根瘤菌的多样性及系统发育分析

对分离自西北部分地区鸡眼草(Kummerowia striata)的53株根瘤菌,进行唯一碳源和氮源利用、抗生素、染料和苯酚抗性、耐盐性等98项生理生化性状测定,并结合16S rDNA PCR-RFLP分析,对供试菌株进行了遗传多样性研究.结果表明,不同地理来源,甚至来源于同一地区的不同菌株在碳、氮源利用、耐盐性、对苯酚抗性程度等方面存在着一定差异,12.5%的菌株能耐受300 μg/mL氯霉素,58.9%的菌株能耐受2%NaCI,38%的菌株能耐受700 mg/L苯酚.通过16S rDNA PCR-RFLP分析,53株供试菌株分别属于慢生根瘤菌属(Bradyrhizobium)、中慢生根瘤菌属(Mesorhizobium)、根瘤菌属(Rhizobium)、中华根瘤菌属(Sinorhizobium)和土壤杆菌属(Agrobacterium),说明分离自鸡眼草的根瘤菌在种及属的水平上具有非常丰富的遗传多样性.     

马桑弗兰克氏菌的生物学特性的多样性

报道了分离自我国尼泊尔马桑根瘤的２０株内生放线菌的系统的生物学特性。这些菌株在形态上具弗兰克氏菌属的典型特性，即丝状菌丝体上有孢囊和泡囊。少数菌株还有串珠状生殖菌丝。但它们在培养特征，生理特性，细胞化学组分，拮抗性，细胞可溶性蛋白和脂酶同功酶电泳图谱，质粒类型和限制酶切图谱彼此均有较大差异，几乎没有两个菌株在所有检测指标的结果上是相似的。表明马桑弗兰克氏菌在生物学特性上有明显的多样性。     

黄河三角洲贝壳堤放线菌多样性及抑菌活性

研究旨在揭示黄河三角洲贝壳堤放线菌多样性,以便从中寻找新的具有生物防治功能的微生物资源。采用3种分离培养基,利用稀释平板涂布法对贝壳堤土壤样品进行分离;通过形态特征、生理生化实验及16S rDNA基因测序对放线菌进行了分类鉴定。结果表明分离到的174株放线菌分属于10个科,13个属,其中链霉菌属(42%)和拟诺卡氏菌(11%)为优势菌属。16S rDNA基因分析表明,61%的菌株与已知菌的相似性都在99%以下,其中菌株BK-17的16S rDNA序列与同源性最近的菌株相似率仅为95.2%。以2株细菌和5株植物病原真菌作为指示菌,进行了抑菌活性测定,有94株至少对1种指示菌有抑菌作用,有8株对7种指示菌都有很强的拮抗作用。以上研究结果表明黄河三角洲贝壳堤土壤中存在丰富的放线菌资源,存在很多潜在的新菌种和活性菌株,有进一步研究和开发的价值。     

铁锰结核土壤锰氧化细菌多样性及新菌属分析

尽管细菌的锰氧化作用被认为是自然界中氧化锰矿物形成的主要成因,但目前国内外对陆地土壤环境中锰氧化细菌的种群组成与多样性方面的研究甚少。本研究对采集于山东崅峪一处含铁锰结核的棕壤进行了可培养锰氧化细菌分离、活性测定与多样性调查,结果发现表层土壤(A层:0～20 cm)的可培养锰氧化细菌是最丰富的,但是高锰氧化活性的细菌主要分布在心土层(B层:20～40 cm)和底土层(C层:>75 cm)。通过对具有高锰氧化活性的分离菌株16S rRNA基因的扩增、测序和序列BLAST比对分析,发现了7个此前未见报道的具有锰氧化活性的新菌属。此外,对5株具有高锰氧化活性的分离菌株和土壤样品的进行了16S rRNA基因V3产物的变性梯度凝胶电泳(DGGE)分析,结果显示此5株高锰氧化菌株并非都是土壤中的高丰度细菌。     

苏云金芽孢杆菌基因组研究概况

迄今为止,全球已有2个Bt株菌株完成了全基因组测序,1个Bt菌株正在拼接中,15个Bt菌株正在进行测序中。已有22个晚质粒完成了全序列测定。助是作为生物农药使用最广泛的微生物菌株,也是最为成功地将其杀虫晶体蛋白基因应用于植物转基因的微生物。在基因组进化、新基因发现、基因表达调控等方面一直是科学家研究的热点,并取得了相当多的成果。本文概述了苏云金芽孢杆菌基因组测序现状、基因组特征及比较基因学等方面的研究进展。     

Persistence of plasmid RP4 in Pseudomonas putida and loss of its expression of antibiotic resistance in a groundwater microcosm

We examined the stability of plasmid RP4 and its expression of antibiotic-resistance genes in suspended and sorbed Pseudomonas putida in aquifer microcosms. Test tubes containing different proportions of sterilized aquifer soil and groundwater were inoculated with bacteria and incubated for up to 26 d. Serial dilutions were made to agar plates with or without antibiotics, to quantify the functional stability of the plasmid. The structural integrity of RP4 was examined by plasmid extraction, digestion with restriction enzymes, and agarose gel electrophoresis. The plasmid-borne resistance gene expression disappeared in 80-90% of the cells during day 1 of incubation in aquifer soil and then remained at that frequency throughout the experiment. The RP4 plasmid was present in cells without antibiotic-resistance gene expression, indicating that the observed loss of plasmid-encoded activity was most likely due to a reduction in expression of the resistance genes. The increased growth rate in groundwater amended with glucose and phosphate had no significant influence on plasmid loss or antibiotic-resistance expression, suggesting that plasmid loss and antibiotic-resistance expression were independent of the growth rate. Most of the reduction of resistance gene expression was associated with the presence of soil particles, and 70% of the resistance expression was retained in bacteria incubated for 1 d in groundwater alone. Bacteria sorbed to the soil particles had a lower frequency of expression of resistance genes than suspended bacteria, but the difference was not caused by sorbed inorganic or organic chemicals. Resistance gene expression was partly recovered in suspended bacteria after in vitro exposure to the antibiotics and after first isolating on agar without antibiotics and then replica plating to agar containing the antibiotics.     

质粒制备绝对定量PCR标准曲线方法的建立

用质粒制备绝对定量PCR标准曲线存在着作为标准品的双链环状质粒分子与待测样品单链线性cDNA分子结构不一致的问题.为了消除二者之间的差异,确保绝对定量PCR测定方法的准确性,我们在用质粒制备标准曲线和测定未知样品中特定基因mRNA分子拷贝数时进行了两个改进:1)用酶切的方法使环状质粒线性化,用线性质粒制备标准曲线;2)将未知样品cDNA扩增出一条正义链,再与作为标准曲线的质粒标准品同时开始荧光定量PCR循环.实验结果表明,1)具有相同拷贝数的环状和线性质粒在一起进行定量PCR测定时,环状质粒所得的Ct值大于线性质粒所得的Ct值(P＜0.05);2)本研究测定的cDNA样品中先扩增一次测出的起始拷贝数均大于未先扩增一次的测定结果.样品起始拷贝数两次测定结果的差异分析表明5个样品中有3个样品的测定差异达到了显著水平(P＜0.05).应用该方法改进的鸡GATA5基因绝对定量PCR的标准曲线能够准确地反应目的产物扩增.经改进的质粒制备绝对定量PCR标准曲线的方法更为简便易行,并提高了测定的准确性.     

苏云金杆菌天门变种晶体蛋白基因的克隆和表达

用改进的Birnboim法检测了苏云金杆菌9个变种的质粒组成,其中我国分离到的天门变种有12条质粒带,用Southern杂交法证明,天门变种的δ-内毒素基因位于1个约60MD的质粒上。该质粒经BglⅡ酶切后与Bam HI酶切的pBR322载体连接,转化大肠杆菌HB101。通过菌落原位放射免疫反应和Southern杂交方法,筛选带有δ-内毒素基因并能在大肠杆菌中表达的转化体。对玉米螟幼虫进行生物测定表明,阳性克隆pTCC65及pTCC18有较强的抑制幼虫生长的作用。     

链霉菌(Streptomyces TE66 ) MalQ基因克隆与原核融合表达

用PCR方法获得Streptomyces TE66 MalQ基因，将该基因插入原核表达载体pTrc-CKS中，对质粒所含外源片段双向测序，并经BLAST比较分析，发现其与GenBank中报道的Streptomyces coelicolor MalQ基因的同源性高达97％。重组质粒转化E.coli Top 10F’，经IPTG诱导后以融合蛋白形式表达，SDS-PAGE电泳显示MalQ基因与CKS表达的融合蛋白在目标位置约106kDa处有明显条带。经薄层层析分析粗酶液酶处理的麦芽三糖溶液，证实粗酶液具有麦芽糖转糖基活性。     

链霉菌(Streptomyces ST66)MalQ基因克隆与原核融合表达

利用PCR方法获得Streptomyces ST66 MalQ基因,将该基因插入原核表达载体pTrc-CKS中,对质粒所含外源片段双向测序,并经BLAST比较分析,发现其与GenBank中报道的Streptomyces coelicolor MalQ基因的同源性高达97%。重组质粒pTrc-MalQ转化大肠杆菌(Escherichia coli)Top10F',经IPTG诱导后以融合蛋白形式表达,SDS-PAGE电泳显示MalQ基因与CKS(CTP:CMP-3-deoxy-D-manno-octulosonate cytidylyltransferase or CMP-KDO synthetase)基因表达的融合蛋白在目标位置约106kD处有明显条带。经薄层层析分析粗酶液处理的麦芽三糖溶液,证实粗酶液具有麦芽糖转糖基活性。     

Capillary electrophoresis is essential for microsatellite marker based detection and quantification of adulteration of Basmati rice (Oryza sativa)

Microsatellite markers are employed for genotyping of Basmati varieties and assaying purity of market samples. However, employment of diverse electrophoresis techniques across laboratories has resulted in inconsistent allele sizes, creating doubts about the suitability of the assay. This study evaluated agarose gel electrophoresis, slab gel electrophoresis, and capillary electrophoresis techniques for their efficiency in the detection and quantification of adulteration in Basmati samples. Comparative analysis across 8 microsatellite loci in 12 rice varieties demonstrated that the capillary electrophoresis method showed less error (+/-0.73 bp) in the estimation of allele sizes compared to slab gel (+/-1.59 bp) and agarose gel (+/-8.03 bp) electrophoretic methods. Capillary electrophoresis showed greater reproducibility (<0.5 bp deviation) compared to slab gel (1 bp) and agarose (>3 bp) based methods. Capillary electrophoresis was significantly superior in quantification of the adulterant, with a mean error of +/-3.91% in comparison to slab gel (+/-6.09%). Lack of accuracy and consistency of the slab gel and agarose electrophoretic methods warrants the employment of capillary electrophoresis for Basmati rice purity assays.     

Are molecular weights of proteins determined by superose 12 column chromatography correct?

Our research on several proteins indicates that accurate molecular weights cannot be determined by Superose 12 column chromatography. In support of this statement, we present data on molecular weights of purified red kidney bean alpha-amylase inhibitor (RKB alphaAI) and white kidney bean alpha-amylase inhibitor (WKB alphaAI) to document this problem. The molecular weight of purified RKB alphaAI determined by Sephadex G-100 gel filtration, polyacrylamide gel electrophoresis, Superose 12 gel filtration and cDNA were 49.0, 51.0, 22.9, and 49.805 kDa (not glycosylated), respectively. The molecular weights of WKB alphaAI by several methods were as follows: Sephadex G-100 gel filtration, 51.0 kDa; Superose 12 gel filtration in 0.2 M NaCl buffer, 23.1 kDa; polyacrylamide gel electrophoresis (PAGE), 51.0 kDa; sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), 45.0 kDa; multiangle laser light scattering (MALLS), 49.940 kDa; laser-assisted time-of-flight mass spectrometry (LATOFMS), 56.714 kDa; and cDNA sequence (with 12.2% carbohydrate), 55.9 kDa. The data indicate there is ionic interaction between proteins and the matrix of Superose 12 in low ionic strength buffers and hydrophobic interaction at higher ionic strength buffers. Researchers should be cautious when using Superose 12 columns for molecular weight determinations.     

PCV2衣壳蛋白羧基端基因在T4噬菌体表面的展示

用PCR扩增猪圆环病毒II型广东分离株的衣壳蛋白羧基端基因,将PCR产物连接到重组型质粒pR上,转化DH5α细胞并筛选阳性克隆;重组质粒经PCR鉴定并测序后,转化E2菌,将重组E2菌与缺陷型噬菌体T4-Z1同源重组后,得到重组噬菌体,SDS-PAGE和Westernbloting分析,表明衣壳蛋白基因在噬菌体表面正确展示,表达的融合蛋白的分子量约为25Ku。     

珍珠黄杨叶片的蛋白质提取方法探讨

蛋白质样品制备是双向电泳的核心。为了找到一种适合提取珍珠黄杨叶片蛋白质的方法,本文以该树种扦插苗的叶片为材料,用TCA_丙酮沉淀法、Tris-饱和酚法和2-D Clean-Up Kit提取蛋白质,并进行双向凝胶电泳,采用银染法进行检测。结果表明,TCA-丙酮沉淀法得到的样品图谱背景模糊、拖尾;Tris-饱和酚法得到的样品图谱清晰,蛋白点饱满,无纵向或横向拖尾,但有蛋白点丢失;2-DClean-Up Kit提取的蛋白质样品得到了较好双向电泳图谱。     

The Identification of Finnish Oat Cultivars (Avena sativa L.) by the Use of SDS-PAGE of the Avenins in Homogeneous and Gradient

Abstract

The Finnish oat cultivars were identified by homogeneous and gradient SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis on the basis of 7–14 avenin bands. REM (relative electrophoretic mobility) values of the avenins among the Finnish oat cultivars were determined by gradient SDS-PAGE to calculate the PH% (pattern homology percentage). Most of the cultivars (58%) had a PH% of over 75%, which indicates quite a. high degree of similarity between the cultivars. Homogeneous SDS-PAGE was used in addition to gradient SDS-PAGE to compare the electrophoregrams of the cultivars by the gel separation systems. Resolution of the avenins was better by homogeneous than by gradient SDS-PAGE. Ten oat cultivars out of the 28 tested could be identified individually in homogeneous SDS-PAGE, as opposed to three which were identifiable by gradient SDS-PAGE.