应用多重PCR鉴定微生物肥料常用芽孢杆菌

[目的]枯草群芽孢杆菌中枯草芽孢杆菌(Bacillus subtilis)、解淀粉芽孢杆菌(B.amyloliq-wefaciens)、地衣芽孢杆菌(B.licheniformis)和短小芽孢杆菌(B.pumilus)是微生物肥料中常用菌种,用传统方法鉴定费时费力,有必要建立检测和鉴定这些芽孢杆菌的种特异性PCR方法.[方法]利用已登录的gyrA、rpoA和16s rRNA基因序列分别设计和筛选上述菌种的特异引物并建立多重PCR反应体系.[结果]以基因组DNA为模板,扩增芽孢杆菌、类芽孢杆菌和短芽孢杆菌3属15种的标准菌株(共33株),4个目标种分别产生了大小不同的唯一的产物,除个别种与短小芽孢杆菌引物有交叉反应外,其余参考菌株均为阴性.从23株枯草群菌株的基因组DNA扩增发现,PCR鉴定与常规鉴定结果一致.[结论]本文建立的多重PCR方法具有较好的特异性,可快速准确鉴定枯草群的4个种,在微生物肥料检测方面有良好的实用前景.     

多重PCR技术检测微生物肥料中巨大芽孢杆菌和蜡样群芽孢杆菌的研究与应用

巨大芽孢杆菌是微生物肥料生产中的常用菌种, 与之形态上相似的蜡样群芽孢杆菌(蜡样芽孢杆菌、苏云金芽孢杆菌、蕈状芽孢杆菌)则是产品中常见的污染菌, 传统方法区分两者费时费力, 有必要建立检测这两类芽孢杆菌的PCR方法。本文利用已登录的spoOA基因序列分别设计和筛选了上述两个种(群)的特异引物, 并建立了多重PCR检测技术。使用该方法对巨大芽孢杆菌、蜡样群芽孢杆菌和其他芽孢菌共3属13种24株标准菌株的基因组DNA进行扩增, 以检验其特异性。结果显示, 巨大芽孢杆菌、蜡样群芽孢杆菌基因组DNA分别产生大小不同的唯一产物, 其他芽孢杆菌均为阴性。该多重PCR检测方法的灵敏度经测定为105 CFU/mL。同时对10株待测菌株和8个微生物肥料产品进行检测, 其鉴定结果与常规鉴定结果一致。以上结果表明, 本文建立的多重PCR方法具有较高的特异性和灵敏度, 可快速、准确鉴定巨大芽孢杆菌和蜡样群芽孢杆菌, 在微生物肥料检测方面有良好的实用前景。     

一种快速分离检测产脂肽类生物表面活性剂枯草芽孢杆菌的方法

脂肽(Lipopeptide)是由枯草芽孢杆菌(Bacillus subtilis)等微生物产生的一类具有较强表面活性的生物表面活性剂.枯革杆菌磷酸泛酰巯基转移酶基因(afp)是枯草芽孢杆菌中参与脂肽代谢的功能性基因.采用sfp基因PCR对从环境中得到的一组产生表面活性剂的微生物进行筛选,结合Tricine-SDS-PAGE电泳对PCR结果呈阳性的菌蛛的代谢粗初提物进行检测,初步鉴定得到两株枯草芽孢杆菌.进一步利用16S rDNA序列的系统发育学分析确定这两种菌株为枯草芽孢杆菌,并利用TLC、HPLC鉴定其产物为脂肽类表面活性剂,从而建立了一套快速分离检测产生脂肽类生物表面活性剂的枯草芽孢杆菌方法.     

枯草芽孢杆菌群β-甘露聚糖酶基因克隆及同源性分析

本文对33株枯草芽孢杆菌群菌株进行β-甘露聚糖酶活性筛选,其中的32株具有β-甘露聚糖酶活性,只有1株无β-甘露聚糖酶活性.通过基因克隆测序的方法获得33株枯草芽孢杆菌群菌株β-甘露聚糖酶基因编码区全序列,对酶基因进行同源性分析并构建系统发育树;在β-甘露聚糖酶基因系统发育树中,33株枯草芽孢杆菌群菌株聚为3个分支,分别是枯草芽孢杆菌分支、地衣芽孢杆菌分支和解淀粉芽孢杆菌分支;枯草芽孢杆菌、地衣芽孢杆菌和解淀粉芽孢杆菌β-甘露聚糖酶基因种内同源性大于91%,而种间同源性为60%69%.     

一株多功能芽孢杆菌的鉴定

【摘 要】 目的 从土壤筛选所得1株芽孢杆菌并对该菌进行鉴定.方法 通过表型特征、生理生化特性和16S rDNA序列同源性分析（Gen Bank登录号为: JN609382）。结果 形态学观察：菌落呈圆形,白色微黄,中央凸起,半透明,长时间培养容易形成褶皱;革兰染色为阳性;芽孢中生,周生鞭毛,细长且多。生理生化特征：参照《常见细菌手册》和《伯杰氏细菌鉴定手册》,其中接触酶反应、V-P测定、卵磷脂以及革兰染色呈阳性;能够利用D-葡萄糖、D-木糖、D-甘露醇产酸,并能使淀粉水解;不能利用丙酸盐,不能形成吲哚,不能水解酪氨酸,生理生化试验结果显示该芽孢杆菌与枯草芽孢杆菌的特征一致。分子生物学鉴定：在Gen Bank上经过BLAT分析,其与枯草芽孢杆菌的相似性最高（99％）。结论 鉴定该菌为枯草芽孢杆菌。     

玉米根系内生细菌种群及动态分析

2000-2002年,先后对辽宁省14个玉米主栽品种进行了根系内主要细菌种群分析．结果表明．玉米内生细菌的主要种群为芽孢杆菌属(Bucillus spp．),此外还包括肠杆菌属、沙雷氏杆菌属、假单胞菌属、黄单胞菌属和棍状杆菌属．其中Bacillus分布最广,已鉴定出8个种,包括枯草芽孢杆菌、巨大芽孢杆菌、蜡状芽孢杆菌、地衣芽孢杆菌、炭疽芽孢杆菌、蕈状芽孢杆菌、短小芽孢杆菌、环状芽孢杆菌．Bacillusspp．总量占根系内生细菌总量比苗期和成株期分别为75．5％和76．6％．内生细菌在不同玉米品种和不同生育期之间存在程度不同的差异．研究发现,品种的遗传背景与其内生细菌的种类和数量显著相关．     

一株拮抗多重耐药菌的芽孢杆菌的初步研究

目的 对一株具有拮抗多重耐药菌的芽孢杆菌的特性进行研究.方法 采用菌落法测试多株芽孢杆菌对18种不同来源致病菌的抑制效应,并对其中一株可拮抗多重耐药菌的芽孢杆菌(KC株)进行鉴定和电泳分析.结果 该芽孢杆菌(KC株)经生理生化以及16S rDNA鉴定为枯草芽孢杆菌;KC株对人源、鱼源、畜禽源和鸡粪源的多重耐药菌有不同程度的拮抗效应;SDS-PAGE分析提示,35 kD左右的分泌蛋白可能参与了KC株的抑菌活性.结论 具有拮抗多重耐药菌的芽孢杆菌为枯草芽孢杆菌,且该菌分泌的35 kD蛋白可能参与抑菌活性.     

芽孢杆菌TS02特异RAPD标记的SCAR标记转化和验证

利用自主分离的蜡状芽孢杆菌菌株TS02, 采用RAPD方法对TS02及其同源性相近的5株芽孢杆菌(地衣芽孢杆菌、枯草芽孢杆菌、凝结芽孢杆菌、巨大芽孢杆菌、短小芽孢杆菌) 进行了RAPD条带特异性分析, 从TS02基因组中筛选获得了一个533 bp的特异RAPD标记TSR1。TSR1克隆、测序后, 根据其序列设计出一对特异引物P1/P2进行扩增, 结果只在TS02中扩增得到目的片段, 而其余对照菌株扩增为阴性, 从而证明试验得到了在种水平上对该菌种进行准确鉴定的特异SCAR标记。     

芽孢杆菌TS02特异RAPD标记的SCAR标记转化和验证

利用自主分离的蜡状芽孢杆菌菌株TS02,采用RAPD 方法对TS02及其同源性相近的5株芽孢杆菌(地衣芽孢杆菌、枯草芽孢杆菌、凝结芽孢杆菌、巨大芽孢杆菌、短小芽孢杆菌)进行了RAPD条带特异性分析,从TS02基因组中筛选获得了一个533 bp的特异RAPD标记TSR1.TSR1克隆、测序后,根据其序列设计出一对特异引物P1/P2进行扩增,结果只在TS02中扩增得到目的片段,而其余对照菌株扩增为阴性,从而证明试验得到了在种水平上对该菌种进行准确鉴定的特异SCAR标记.     

拮抗菌株Kc-t99的鉴定及其抑菌活性研究

通过传统分类与分子生物学技术相结合的方法对从京郊菜园土壤中分离筛选的拮抗菌株Kc-t99进行鉴定,并采用生物测定的方法评价其抑菌活性.结果表明,菌株Kc-t99的形态学特征和生理生化特性与枯草芽孢杆菌基本一致,其16S rDNA序列与GenBank中已鉴定的枯草芽孢杆菌的16S rDNA序列同源性高达98.06%,据此初步确定其为枯草芽孢杆菌.抑菌试验表明该菌株对供试的5种病原真菌和4种细菌均具抑菌活性,其中对甘蓝枯萎病菌、黄瓜角斑病菌和桃褐腐病菌抑制作用显著.     

Recent advances in the study of Kaposi's sarcoma-associated herpesvirus replication and pathogenesis

It has now been over twenty years since a novel herpesviral genome was identified in Kaposi's sarcoma biopsies. Since then, the cumulative research effort by molecular biologists, virologists, clinicians, and epidemiologists alike has led to the extensive characterization of this tumor virus, Kaposi's sarcoma-associated herpesvirus(KSHV; also known as human herpesvirus 8(HHV-8)), and its associated diseases. Here we review the current knowledge of KSHV biology and pathogenesis, with a particular emphasis on new and exciting advances in the field of epigenetics. We also discuss the development and practicality of various cell culture and animal model systems to study KSHV replication and pathogenesis.     

The structure, reproduction and taxonomy of Vidalia and Osmundaria (Rhodophyta, Rhodomelaceae)

Comprises species occurring mostly in subtidal habitats in tropical, subtropical and warm-temperate areas of the world. An analysis of the type species, V. spiralis (Sonder) Lamouroux ex J. Agardh, a species from Australia, establishes basic characters for distinguishing species in the genus. These characters are (1) branching patterns of thalli, (2) flat blades that may be spiralled on their axis, (3) width of the blade, (4) primary or secondary derivation of sterile and fertile branchlets and (5) position of sterile and fertile branchlets on the thalli. Application of the latter two characters provides an important basic method for separation of species into three major groups. Osmundaria , a genus known only in southern Australia, was studied in relation to Vidalia , and its separation from the Vidalia assemblage is not accepted. Species of Vidalia therefore are transferred to the older genus name, Osmundaria. Two new species, Osmundaria papenfussii and Osmundaria oliveae are described from Natal. Confusion in the usage of the epithet, Vidalia fimbriala Brown ex Turner has been clarified, and Vidalia gregaria Falkenberg, described as an epiphyte on Osmundaria pro/ifera Lamouroux, is revealed to be young branches of the host, Osmundaria prolifera.     

New or rare chromosome counts in Artemisia L. (Asteraceae, Anthemideae) and related genera from Kazakhstan

Fifteen chromosome counts of six Artemisia taxa and one species of each of the genera Brachanthemum, Hippolytia, Kaschgaria, Lepidolopsis and Turaniphytum are reported from Kazakhstan. Three of them are new reports, two are not consistent with previous counts and the remainder are confirmations of very scarce (one to four) earlier records. All the populations studied have the same basic chromosome number, x = 9, with ploidy levels ranging from 2x to 6x. Some correlations between ploidy level, morphological characters and distribution are noted.     

肝癌中HBV和HCV基因和抗原的分布及意义

采用原位分子杂交方法检测HCV RNA及HBV X基因;采用免疫组织化学方法研究HCV核心抗原,非结构区C33c抗原及HBxAg在肝细胞肝癌中的定位及分布.结果表明(1)HCV RNA、HBV X基因在肝细胞肝癌组织检出率分别为40%(55/136)和82%(112/136).HCV RNA定位于癌细胞的胞浆内,阳性细胞呈散在、灶状及弥漫分布三种形式;HBV X基因在肝癌细胞中的分布呈胞浆型、核型及核浆型,阳性细胞也呈上述三种分布形式;(2)HCV C33c抗原、核心抗原在肝细胞肝癌中的阳性率为81%(133/164)及86%(141/164).C33c抗原定位于癌细胞及肝细胞的胞浆内;核心抗原既定位于癌细胞核中,又可定位于胞浆中.C33c抗原阳性细胞以灶状分布为主;而核心抗原阳性细     

Selection with two alleles and complete dominance

For a plant selection model with frequency-independent viabilities, fertilities and selfing rates, it is shown that apart from global fixation, for certain parameter combinations a protected polymorphism and facultative fixation (either allele may become fixed according to initial frequencies) may both occur. Facultative fixation requires different selling rates for the dominant and recessive type. Protection of the polymorphism requires resource allocation for male and female function. In this connection the problem of purely genetically caused population extinction is discussed.
For general frequency dependence and regular segregation, the chances for establishment of a completely recessive gene are compared to those of a completely dominant gene. It is proven that the process of establishment of the recessive gene, despite a fitness advantage, may be considerably endangered by drift effects if random mating prevails. The recessive gene may reach the same effectivity in establishment as a dominant gene, only if the recessive homozygote mates exclusively with its own type during the period of establishment.