氧化葡糖杆菌1.637山梨糖还原酶基因sr功能初步研究

目的:研究山梨糖还原酶基因sr(B932_3022)在氧化葡糖杆菌1.637生长代谢中的作用。方法:构建sr基因同源敲除质粒p GX-srup-Gm-down,PCR扩增得到打靶片段,电击转化导入氧化葡糖杆菌1.637,通过庆大霉素抗性和PCR筛选sr基因重组敲除菌,分别以山梨醇和山梨糖为碳源考察野生菌与敲除菌生长和山梨糖代谢的差异。结果:抗性和PCR验证结果显示敲除了氧化葡糖杆菌1.637的sr基因;与野生菌相比,敲除菌生长出现滞后,以山梨醇为碳源时敲除菌山梨糖积累增多,而以山梨糖为碳源时山梨糖消耗减少。结论:氧化葡糖杆菌1.637的sr基因敲除影响菌株前期生长与山梨糖代谢。     

炭疽芽胞杆菌mntA基因缺失突变株的构建及鉴定

目的:通过同源重组的方法敲除炭疽芽胞杆菌减毒AP422株的mntA基因,使菌株进一步减毒,用于构建新的疫苗候选株。方法:利用PCR方法扩增mntA基因上下游同源臂后与温敏质粒连接,构建打靶载体,并转化炭疽芽胞杆菌减毒AP422株;利用抗生素和温度2种选择压力实现同源重组,敲除目标基因mntA,然后利用Cre-LoxP系统去除抗性筛选标记,得到无抗性标记的缺失突变株,并利用PCR和Western印迹等方法对重组菌进行系统鉴定,最后分析突变株的生物学性状。结果:敲除了AP422株的mntA基因,获得了无抗性标记的缺失突变株,突变株的生存竞争能力比原始菌株明显减弱。结论:突变株获得了进一步减毒,可用于构建新的疫苗候选株。     

鼠伤寒沙门菌spvBC基因编辑株的构建

利用λRed重组系统和pBAD原核表达载体构建鼠伤寒沙门菌spvBC质粒毒力基因修饰菌株,为深入探究沙门菌毒力基因spv的功能和致病机制及宿主抗感染免疫提供工具菌。以pKD4为模板,PCR扩增含spvBC同源臂的卡那霉素抗性基因以构建同源打靶片段,再将其电转入含有质粒pKD46的鼠伤寒沙门菌中进行同源重组,随后将质粒pCP20电转导入阳性转化子,消除卡那霉素抗性基因,PCR鉴定敲除株的构建。PCR扩增含酶切位点的spvBC基因片段,扩增产物与原核表达载体pBAD/gⅢ分别双酶切后连接构建pBAD-spvBC重组质粒,PCR筛选阳性菌落并测序鉴定。将构建成功的pBAD-spvBC重组质粒电转导入spvBC敲除株中,Western blot测定不同浓度L-阿拉伯糖诱导SpvB和SpvC蛋白表达情况。PCR结果表明鼠伤寒沙门菌spvBC基因敲除成功;PCR及测序结果表明pBAD-spvBC重组质粒构建成功,Western blot结果表明13 mmol/L L-阿拉伯糖可诱导SpvB和SpvC蛋白正常表达。λRed重组系统可用于沙门菌质粒上大片段基因的敲除,pBAD原核表达载体可用于沙门菌质粒上大片段基因的回补,丰富了细菌质粒的基因修饰和编辑策略。     

炭疽芽胞杆菌假想S-层蛋白SLP缺失突变体的构建

目的：构建炭疽芽胞杆菌假想S-层蛋白SLP缺失突变体,以进行后续SLP的功能研究,为炭疽芽孢杆菌重要基因功能的研究建立技术平台。方法：利用PCR技术,分别扩增得到目的基因的上游同源臂（slp-F）和下游同源臂（slp-R）,将抗性基因（S）和上、下游同源臂先后连入穿梭质粒pKSV7,构建打靶载体pKSV7-FSR,经去甲基化后,电转化入炭疽芽胞杆菌A16R,通过同源重组敲除slp基因,并通过DNA测序和Western blot实验验证;对野生株和突变株37℃时的生长曲线及生化反应进行比较研究。结果：分别从DNA水平和蛋白质水平证实slp基因被成功敲除;突变株对数期生长较快,衰退较慢,与野生株的生化反应差异不明显。结论：获得了炭疽芽胞杆菌假想S-层蛋白SLP缺失突变体。     

北京棒杆菌芳香族氨基酸转运蛋白基因敲除对L-色氨酸积累的影响

[目的]为了减少北京棒杆菌PD-67(Corynebacterium pekinense PD-67)从细胞外吸收色氨酸,降低细胞内色氨酸库的浓度,从而使色氨酸的反馈控制作用减弱,增加胞外L-色氨酸的积累量,构建北京棒杆菌PD-67的芳香族氨基酸转运蛋白基因aroP敲除的菌株,研究aroP基因敲除对菌株L-色氨酸积累的影响.并进一步研究在aroP敲除菌株中表达邻氨基苯甲酸合成酶(AS)基因对L-色氨酸积累的影响.[方法]运用PCR技术扩增aroP基因,与整合质粒连接后,用限制性内切酶法构建带有内部片段缺失的aroP基因的敲除载体.利用同源重组技术,敲除北京棒杆菌PD-67的aroP基因,构建菌株PD-67 ΔaroP,并用带有aroP基因的表达载体对PD-67ΔaroP进行互补验证.采用PCR技术扩增AS基因,与表达载体连接构建重组质粒.将重组质粒转入菌株PD-67ΔaroP,构建工程菌株PD-67 ΔaroP/pXAS.通过摇瓶发酵研究PD-67 AaroP和PD-67 ΔaroP/pXAS的发酵特性.[结果]经PCR验证获得了aroP基因缺陷的菌株.摇瓶发酵结果表明,与出发菌株相比,PD-67ΔaroP的L-色氨酸的积累量提高了65％.酶活分析结果表明,AS基因在菌株PD-67 △aroP中得到表达.AS基因表达使工程菌单位菌体产酸率提高了25.6％.[结论]北京棒杆菌PD-67中芳香族氨基酸转运蛋白基因arop的敲除能够提高胞外L-色氨酸的积累量.在arop基因敲除菌中表达AS基因,可以进一步提高工程菌的产酸率.     

Red/ET重组在基因打靶载体快速构建中的应用

通过合理应用Red/ET重组技术实现基因打靶载体的快速构建。在Red/ET重组介导下,首先从基因组DNA中将靶基因片段亚克隆至打靶质粒载体中,随后将两端带有50 bp同源臂的抗性筛选基因插入并替换靶基因上的目标序列,如此两步操作即可完成一个传统型基因敲除打靶载体的构建;结合Cre-loxP系统,在传统型基因敲除打靶载体的基础上,经过再一轮的Red/ET重组就能够成功实现条件性基因敲除打靶载体的构建。整个实验过程不需要PCR扩增长、短臂序列,也不涉及酶切、连接反应,因此,不仅省时、省力,而且所构建的基因打靶载体序列准确,无突变。此实验方法的建立为加速后基因组时代的基因功能研究提供了一条捷径。     

地衣芽胞杆菌FLP/FRT基因编辑系统的构建及验证

地衣芽胞杆菌有效的基因编辑工具有限,为了拓展和丰富其基因编辑手段,在地衣芽胞杆菌中构建一个抗性标记可重复使用的FLP/FRT基因编辑系统,并通过敲除α-淀粉酶基因amyL、蛋白酶基因aprE及敲入外源透明颤菌血红蛋白基因vgb对该系统进行初步验证。首先以温敏质粒pNZT1为载体分别构建amyL和aprE基因敲除质粒pNZTT-AFKF和pNZTT-EFKF,两个敲除质粒各自包含针对目标基因的同源臂、抗性基因及同向的FRT位点;将敲除质粒转化地衣芽胞杆菌并经过两次同源交换过程实现目标基因的敲除;最后导入一个FLP重组酶表达质粒通过FLP/FRT系统的重组作用介导抗性基因的回收。为进一步验证本系统的实用性及编辑效率,构建了包含透明颤菌血红蛋白编码基因vgb表达盒及基因组丙酮酸甲酸裂解酶编码基因pflB敲除盒的重组质粒pNZTK-PFTF-vgb,并以此进行外源基因vgb在基因组上的定向敲入。结果显示,成功敲除amyL及aprE并回收了抗性标记卡那霉素基因,敲除后淀粉酶活和蛋白酶活分别减少95.3%和80.4%;vgb基因成功整合入基因组pflB位点并回收了抗性标记四环素基因,并利用荧光定量PCR技术检测到vgb的整合表达。文中首次建立了一个适用于地衣芽胞杆菌的、抗性标记可重复使用的FLP/FRT基因编辑系统,并进行了基因敲除及基因敲入验证,为地衣芽胞杆菌遗传改造提供了良好的方法参考。     

利用细菌人工染色体同源重组对小鼠基因进行条件型敲除

目的:探索通过细菌人工染色体(BAC)同源重组系统构建条件基因敲除载体的高效率方法,提高条件基因敲除小鼠(Flox小鼠)的构建效率。方法:利用作者自己构建的噬菌体重组酶系统,通过BAC同源重组进行条件型基因敲除载体构建工作。首先通过亚克隆构建了一系列载体含有同源臂的靶向质粒,线性化后,打靶片段经电穿孔法转入大肠杆菌内,与相应的BAC同源重组,再经过三步同源重组和一步位点特异性重组,构建小鼠条件型基因敲除载体。结果:高效率构建了小鼠基因的最终条件基因敲除载体。结论:通过BAC同源重组高效构建条件基因敲除载体,为条件基因敲除载体的构建提供了全新思路,并为FLox小鼠的建立,及相应基因在发育、生理、致病机制等方面的功能研究奠定了基础。     

λRed重组系统用于沙门菌质粒毒力基因spvC敲除株的构建

[目的]利用λRed重组系统敲除沙门菌质粒毒力基因spvC。[方法]首先以质粒p KD4为模板,扩增得到两侧含spvC同源臂、中间为卡那霉素抗性基因的线性DNA片段。再将此线性片段电转入具重组功能的感受态沙门菌菌株,发生重组后,卡那霉素平板筛选阳性转化子。最后利用表达FLP重组酶的质粒p CP20,将FRT位点之间的卡那霉素抗性基因消除,用PCR鉴定。Western Blot检测野生沙门菌和spvC敲除株感染的He La细胞ERK磷酸化水平。[结果]沙门菌质粒毒力基因spvC敲除株构建成功,spvC敲除株感染的He La细胞内ERK磷酸化水平升高。[结论]成功构建沙门菌质粒毒力基因spvC敲除株,验证了spvC基因的功能。     

RED同源重组法敲除发光光状杆菌晶体蛋白基因cipA和cipB

RED同源重组法敲除发光光状杆菌晶体蛋白基因cip和cipB,为发光光状杆菌基因敲除提供方法.采用TOPO克隆方法将基因cipA和cipB克隆到质粒pTA上,获得质粒pTA-cipA和pTA-cipB;采用Red/ET方法分别将Gentd基因和Aprd基因分别插入pTA-cipA和pTA-cipB的cipA和cipB基因中,并替换cipA和cipB基因部分序列,获得pTA-GentaIncipA和pTA-ApraIncipB;pTA-GentaIncipA通过BamH Ⅰ酶切,从0.7%琼脂糖胶上回收获得GentaIncipA,将GentaIncipA导入带有质粒pASK-αβγA的P luminescens subsp.Akhurstii中,通过的RED同源重组敲除发光光状杆菌晶体蛋白cipA:pTA-ApraInciB用EcoR Ⅰ酶切,从0.7%琼脂糖胶上回收获得ApraIncipB,将ApraIncipB导入带有质粒pASK-αβγA的P.luminescens subsp.Akhurstii中,通过的RED同源重组敲除发光光状杆菌晶体蛋白cipB;42℃下热激去除突变菌中的质粒pASK-αβγA.克隆PCR和SDS-PAGE电泳结果表明发光光状杆菌晶体蛋白基因cipA和cipB均被敲除.采用RED同源重组法成功地敲除了发光光状杆菌晶体蛋白基因cipA和cipB.     

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.