刺糖多孢菌电转化条件研究

以经理化诱变选育的多杀菌素高产菌株刺糖多孢菌(Saccharopolyspora spinosa)CB11为受体菌,将具有安普霉素(apramycin)抗性标记的整合型载体pSET152作为质粒供体,研究了制备刺糖多孢菌感受态细胞时菌体的生长阶段、质粒DNA浓度、电场强度等因素对电转化效率的影响,结果表明,在菌体培养4...     

链霉菌基因克隆载体及基因文库的构建

利用麦迪霉素产生菌中分离的质粒pSMY1(10．8kb),将pIJ30的硫链丝菌素抗性基因(tsr)克隆到psMY1上,获得了具有硫链丝菌素抗性选择标记质粒pSJ10。通过DNA体外缺失,片段播入、λ-COS片段的插入及与大肠杆菌质粒的重组等基因技术,获得了一系列psMY1衍生质粒,包括双标记质粒pSM3,大肠杆菌／链霉菌穿梭质粒pBMJ2和穿梭粘粒pNMJ1。通过对这些质粒的分析,确定了质粒psMY1的复制必需区为3．06kd的EcoRI—sphI片段。质粒pSJ10、pSM3、pNMJ1具有可选择标记,多个单一确切的可克降位点,有一定范围的宿主并能在链霉菌中稳定存在等特点,故可作为链霉菌基因克隆的载体。其中pNMJl(11．15kb)是大肠杆菌／链霉菌穿梭粘粒,能有效地运载28—38kb的外源DNA片段,并能在体外包装λ噬菌体外壳,转导大肠杆菌。利用载体pNMJl,通过λ-噬菌体蛋白体外包装,在大肠杆菌中建立了螺旋霉素产生菌的基因文库,其基因覆盖率可达90％。重组DNA分子可通过转化转移到链霉菌中。     

大肠杆菌-链霉菌穿梭载体的构建及应用

pIJ6021和pIJ4123是链霉菌的高拷贝表达载体,它们携带有受硫链丝菌素诱导的强启动子P_tipA。分别在它们的合适位点插入大肠杆菌质粒的复制子和在大肠杆菌中选择用的抗性标记基因(bla),得到了两个能在大肠杆菌和链霉菌中穿梭复制、并保持结构稳定的链霉菌表达载体：pHZ1271和pHZ1272。将透明颤菌(Vitreoscillia sp.)血红蛋白基因(vhb)克隆到pHZ1272中,用它转化变铅青链霉菌(Streptomyces lividans),经Western blotting分析和CO结合实验表明,在变铅青链霉菌中表达出了有生物活性的透明颤菌血红蛋白,从而证明所构建的pHZ1272载体具有在链霉菌中表达外源基因的功能。     

子囊霉素产生菌吸水链霉菌FIM260840基因转移系统的建立

目的:建立子囊霉素产生菌吸水链霉菌FIM260840的接合基因转移体系,以便基因敲除和外源基因表达等遗传操作。方法:以整合型质粒p SET152为出发质粒,通过接合转移构建并子囊霉素产生菌FIM260840的基因转移系统。结果:12.5μg/m L安普霉素可有效筛选接合子。经PCR验证,质粒成功整合到菌株FIM260840基因组DNA中,所获接合子的安普霉素抗性高达400μg/m L以上。接合子经多次传代后,导入的质粒p SET152仍稳定整合于接合子基因组DNA上。结论:建立了高效、简便的吸水链霉菌FIM260840的基因转移系统,为该菌的生物合成基因改造奠定了基础。     

阿扎霉素F产生菌链霉菌211726基因转移系统的建立

旨在建立阿扎霉素F产生菌链霉菌211726的基因转移系统,以便基因敲除和外源基因表达等遗传操作。以整合型质粒pSET152和pIB139为出发质粒,通过接合转移构建了阿扎霉素F产生菌链霉菌211726的基因转移系统。结果显示25μg/mL阿泊拉霉素可有效筛选接合子。经PCR验证,质粒成功整合到菌株链霉菌211726基因组中,接合子经多次传代后,导入的质粒pSET152和pIB139仍稳定整合于接合子基因组上。     

粤蓝链霉菌榴菌素生物合成基因orf20的功能

【目的】在大肠杆菌中,转录因子SoxR作为胞内氧化还原感应器,参与抗氧化胁迫的全局性调控。粤蓝链霉菌榴菌素生物合成基因簇内存在一个类soxR基因orf20,但其生理功能仍不清楚。【方法】将orf20基因在大肠杆菌中进行表达,分析携带重组质粒的大肠杆菌对百草枯抗性的变化。同时通过修改后的PCR-targeting方法构建粤蓝链霉菌orf20删除的突变株,分析突变株的表型变化和对百草枯抗性水平的变化。【结果】重组ORF20在羧基端含有组氨酸标签,并在大肠杆菌中获得可溶性表达,携带重组质粒pET28b-orf20的大肠杆菌对百草枯的抗性水平显著提高。粤蓝链霉菌orf20删除突变株仍具有产孢能力,生长特性没有改变,对百草枯的抗性水平也没有变化,但榴菌素的产量大幅提高,是野生株的3.3倍。【结论】在大肠杆菌中,orf20基因的编码产物能够被百草枯激活,替代SoxR参与抗氧化胁迫的调控。在粤蓝链霉菌中,orf20基因不参与抗氧化胁迫,而对榴菌素的产生有负调控效应。     

Fostriecin产生菌Streptomyces pulveraceus遗传转化体系的建立与优化

【目的】建立并优化链霉菌Fostriecin产生菌Streptomyces pulveraceus的遗传转化系统。【方法】以整合型质粒pSET152为出发质粒,通过供体菌E.coli ET12567/pUZ8002与受体菌Streptomyces pulveraceus进行接合转移。【结果】确定了链霉菌Streptomyces pulveraceus的最佳接合转移条件:培养基为终浓度含15%甘氨酸的MS培养基;孢子热激条件为50°C 10 min;阿伯拉霉素覆盖的时间为18 h,终浓度为20 mg/L。同时,把组成型启动子ermE+与绿色荧光蛋白基因(gfp)克隆到pSET152载体上,通过接合转移整合到该链霉菌中,gfp获得表达。【结论】建立Fostriecin产生菌的遗传转化系统,并发现甘氨酸能显著提高链霉菌的接合转移效率。     

铜绿假单胞菌全局调控因子RsmA的缺失影响2个吩嗪基因簇的表达

[目的]为了研究铜绿假单胞菌全局调控因子RsmA对两个吩嗪(Phenazine)合成基因簇phz1和phz2的调控方式与机制.[方法]采用基因缺失和抗性基因(gentamycin resistance cassette,aacC1)插入相结合的策略构建了rsmA基因缺失突变株PA-RG ;通过构建互补表达载体和过表达载体,进一步确认RsmA对绿脓菌素的调控作用 ;采用电转化方法将构建的翻译融合表达载体pMEZ1(phz1'-'lacZ)和pMEZ2(phz2'-'lacZ)分别导入铜绿假单胞菌突变株PA-RG和野生株PAO1,采用Miller法测定融合β-半乳糖苷酶活性.[结果]在GA培养基中,互补分析和过表达分析表明,RsmA抑制绿脓菌素的合成.此外,pMEZ1在突变株PA-RG中的表达增强,为野生株的2-3倍 ;而pMEZ2在突变株PA-RG中的表达降低,野生株是突变株的2倍.[结论]由此初步判定,铜绿假单胞菌全局调控因子RsmA对两个不同吩嗪合成基因簇的调控作用具有特异性,在一定程度上RsmA负调控phz1,正调控phz2.     

利用异源正调控基因acyB2构建埃莎霉素Ⅰ高产菌株

必特螺旋霉素(bitespiramycin,BT)是以异戊酰螺旋霉素(简称为埃莎霉素)Ⅰ、Ⅱ和Ⅲ为主要成分的多组分抗生素。通过阻断3-O-酰基转移酶基因(sspA),获得了只产埃莎霉素Ⅰ的WSJ-2菌株,但其发酵产物中含有大量螺旋霉素,而埃莎霉素Ⅰ含量较低。为提高4″-异戊酰基转移酶基因(ist)在WSJ-2中的表达水平,从而提高埃莎霉素Ⅰ的产量,首先构建了含有ist基因和其正调控基因acyB2连锁片段的重组质粒pSET152-ia,然后将其导入到埃莎霉素Ⅰ产生菌WSJ-2中,通过具有阿普拉霉素(apramycin)抗性标记的链霉菌整合型载体pSET152整合到WSJ-2的染色体上,获得新的埃莎霉素Ⅰ产生菌WSJ-IA。在不同发酵时间定量检测ist的表达水平,WSJ-IA中ist的表达量要明显高于WSJ-2。WSJ-IA高产菌株的发酵单位从(280±20)μg/ml提高至(1160±108)μg/ml,较原始菌株WSJ-2提高了314%,而且在WSJ-IA发酵产物中埃莎霉素Ⅰ与螺旋霉素Ⅰ含量的比值是WSJ-2的2.4倍左右,说明在引入acyB2基因的同时提高了菌株的发酵单位和埃莎霉素Ⅰ的产量。     

黑暗链霉菌DNA同源重组系统的构建

以黑暗链霉菌Tt-49基因组为模板,利用PCR方法,扩增安普霉素生物合成关键基因aprF-G的上、下游序列,作为同源交换臂,并将红霉素抗性基因筛选标记及其启动子插入两交换臂之间,以温敏型质粒pKC1139为基础,构建用于阻断黑暗链霉菌Tt-49安普霉素生物合成的重组质粒pFD8.该质粒通过E.coil ET12567/pUZ8002去甲基化修饰后,经接舍转移进入黑暗链霉菌Tt-49,利用红霉素抗性筛选得到3株阳性转化子,分别命名为Tt-49 AG1、Tt-49 AG2和Tt-49 AG3.通过PCR鉴定,证明pFD8已插入黑暗链霉菌Tt-49基因组的目标位点.以亲株作对照,对3株工程菌进行红霉素抗性能力考察,发现3株工程菌的抗红霉素能力均高迭1 000 μg/mL以上.     

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.