sRNA (istR)在肠炎沙门氏菌抗活性氮氧中的作用分析

sRNA(Small non-coding RNA,sRNA)为新近发现的基因表达调控分子,转录后水平调控靶基因表达,在细菌毒力、应激及对外界环境感应方面起调控作用。沙门氏菌是一种重要的人畜共患病病原菌,可引起人类食物中毒、败血症及伤寒等。以肠炎型沙门氏菌为模型,研究sRNA(istR)在肠炎沙门氏菌抗活性氮氧中的作用,为沙门氏菌的防治提供新方向。参考已报道的沙门氏菌全基因组及istR序列,设计引物,PCR扩增istR突变用基因片段,运用Red同源重组系统对肠炎沙门氏菌(SE2472)的istR基因进行定点敲除,构建敲除菌株(SE2472△istR),比较野生株和敲除株对活性氮氧的敏感性;构建回复表达质粒pHDB3-istR,将其转入istR敲除株构建回复株SE2472△istR-comp,以回复表达istR,分析istR表达对沙门氏菌istR敲除株抵抗活性氮氧的回复作用。活性氮抑菌结果表明,SE2472在pH 5.0、NaNO2浓度为20 mmol/L的LB液体培养基中培养3 h,存活率为20.40%;培养6 h,存活率降至0.05%。同等培养条件下,SE2472△istR的存活率分别为0.70%和0,SE2472△istR-comp生长情况与SE2472类似,存活率分别为21.40%和0.08%。同时用H2O2分析istR在沙门氏菌抗活性氧中的作用,活性氧抑菌结果表明,SE2472和SE2472△istR两者对H2O2的抑菌作用无明显差异。综合上述结果,推测istR在沙门氏菌抗活性氮中起着调控作用,在抗活性氧作用中没有调控作用。     

肠炎沙门菌肽脯氨酰顺反异构酶SlyD的基因敲除、表达及酶学特征

【目的】以肠炎沙门菌肽脯氨酰顺反异构酶SlyD为对象,构建基因缺失株及表达纯化该蛋白,为研究其在肠炎沙门菌致病性与应激等方面的作用奠定基础。【方法】参考Gen Bank登录的肠炎沙门菌基因组序列设计用于slyD基因敲除及原核表达的特异引物,运用自杀质粒介导的同源重组技术对肠炎沙门菌C50041 slyD基因进行敲除,构建C50041ΔslyD缺失株;原核表达SlyD蛋白,通过α-糜蛋白酶耦联法对其PPIase活性进行测定;利用生物信息学相关软件,分析SlyD蛋白的氨基酸序列及功能域。【结果】PCR鉴定与测序结果证明成功构建了肠炎沙门菌C50041ΔslyD缺失株,其生长特性与野生株基本一致;SDS-PAGE及PPIase活性分析表明,获得了具有生物活性的可溶性SlyD蛋白;生物信息学分析显示SlyD蛋白由FKBP样肽脯氨酰顺反异构酶结构域、分子伴侣功能域和金属结合区域3个功能区域组成。【结论】成功获得了肠炎沙门菌C50041ΔslyD缺失株和具有PPIase活性的重组SlyD蛋白。     

crgA调控三孢布拉霉合成类胡萝卜素

【目的】探究crgA基因在三孢布拉霉合成类胡萝卜素过程中的调控作用。【方法】克隆三孢布拉霉crgA基因并利用split-marker策略敲除该基因;在表型特征、关键酶基因转录水平、类胡萝卜素合成水平等方面将基因敲除株与野生株进行比较分析。【结果】与野生型菌株相比,crgA基因敲除菌产孢能力明显下降,而类胡萝卜素合成途径中的关键酶基因转录水平明显提高,在发酵120h后β-胡萝卜素的积累量提高了31.2%。将crgA基因重新导入到敲除菌后,该菌的性状恢复至野生型。【结论】crgA基因调控三孢布拉霉的生长和产孢能力,并通过调控类胡萝卜素关键酶基因表达来调控类胡萝卜素的合成,是一个负调控因子。     

鼠伤寒沙门菌rtsB基因缺失株的构建及其生物学特性

【背景】鼠伤寒沙门菌(Salmonella typhimurium)是一种重要的人畜共患病原菌,严重危害养殖业及人类健康。调控蛋白在病原菌的生存及感染过程中发挥重要作用。【目的】构建鼠伤寒沙门菌调控基因rtsB缺失株和互补株,分析调控蛋白RstB对鼠伤寒沙门菌生物学特性和致病性的影响。【方法】利用Red同源重组的方法构建鼠伤寒沙门菌SAT52的rtsB基因缺失株,并利用互补质粒构建互补株。然后比较分析野生株SAT52、缺失株?rtsB和互补株C?rtsB的生长特性、运动性、生物被膜形成能力、黏附入侵能力、胞内存活能力及致病性的差异。【结果】缺失rtsB基因不影响SAT52的生长速度,但导致运动能力增强,生物被膜形成能力减弱。细胞感染试验结果表明,rtsB基因有助于鼠伤寒沙门菌对Hela细胞的黏附入侵及RAW264.7细胞内的存活。动物试验结果表明rtsB基因缺失显著降低鼠伤寒沙门菌的致病力。【结论】rtsB基因在鼠伤寒沙门菌感染过程中发挥重要作用,可为阐释鼠伤寒沙门菌的致病机制提供参考。     

华癸根瘤菌7653R hfq基因突变株的构建及其生物学特性

【目的】研究hfq基因在Mesorhizobium huakuii 7653R抵抗外界不利环境和共生固氮中的功能特性。【方法】利用pK19mob同源重组方法构建7653R hfq基因的插入失活突变株7653RΔhfq,并构建互补菌株7653RΔhfq-C,对hfq在压力胁迫和共生固氮中的功能特性进行研究。【结果】与野生型7653R相比,突变株7653RΔhfq的生长速率降低,热激处理后致死率升高;hfq突变影响了7653R中部分sRNA的表达;在4.5%乙醇和50 mmol H_2O_2生长胁迫下,突变株适应性明显较野生型差。另外,接种突变株的紫云英结瘤能力和固氮酶活性都明显降低。【结论】hfq基因作为重要的转录后调控因子,在7653R抵御外界胁迫环境和与宿主紫云英的共生固氮中发挥了重要作用。     

绿针假单胞菌GP72中aurI/aurR系统调控功能

【背景】绿针假单胞菌(Pseudomonas chlororaphis) GP72是一株可生产吩嗪类抗生素吩嗪-1-羧酸(PCA)和2-羟基吩嗪(2-OH-PHZ)的生防根际促生菌。基因组比对发现GP72菌中存在aurI/aurR双元调控系统。【目的】研究该系统对GP72中吩嗪类物质的调控作用。【方法】将aurI基因在大肠杆菌中异源表达,用紫色杆菌CV026和根癌农杆菌NTL4做显色实验。构建基因敲除菌株和回补菌株,发酵测量突变株的生长曲线与总吩嗪产量。构建转录融合质粒,测定吩嗪合成基因启动子的转录水平。【结果】显色实验显示,aurI能产生多种信号分子,使CV026显紫色、NTL4显蓝色。分别单独敲除aurI和aurR基因,同时敲除aurI/aurR基因,吩嗪产量均会升高,而回补菌株吩嗪产量降为野生型水平。β-半乳糖苷酶活性测定结果显示,突变株的酶活比野生型高。【结论】aurI/aurR负调控GP72的吩嗪合成,通过抑制吩嗪合成启动子的转录而影响吩嗪类物质的产量。     

生防菌密旋链霉菌Act12中SPA7074缺失突变株的构建及其次级代谢产物鉴定

【目的】通过基因工程手段构建生防菌Act12转录调控因子SPA7074缺失突变株,并挖掘其中活性次级代谢产物资源和探讨其活性机理。【方法】利用同源重组方法敲除Act12基因组中可能的Tet R家族转录调控因子编码基因spa7074(accession number:KU955325),平板实验检测缺失突变株发酵液抑菌活性的变化,并通过HPLC比较代谢图谱,然后通过质谱及核磁共振对差异峰对应化合物进行结构鉴定。【结果】SPA7074缺失突变株对几种病原真菌的拮抗活性显著增强,比较代谢图谱表明出现数个差异峰,将最显著差异峰所对应化合物进行分离纯化鉴定,结果为寡霉素D。【结论】本研究通过基因工程手段敲除生防菌株Act12中的负转录调控转录因子,使得突变菌株抑菌活性显著增强,并获得了产量达野生型菌株7倍的寡霉素D高产菌株Δspa7074。     

铜绿假单胞菌M18 rsmY突变株的构建及其对PCA和Plt的区别性调控

【目的】在假单胞菌中,小RNA(sRNA)参与初级和次级代谢产物、多种毒素因子以及菌群传感系统的调控,通过在植物根际促生铜绿假单胞菌M18中研究RsmY对吩嗪-1-羧酸(PCA)和藤黄绿菌素(Plt)两种抗生素的调控作用,深入了解假单胞菌中次级代谢的途径并为构建高产抗生素工程菌株提供了一定的理论基础。【方法】运用同源重组技术,构建了铜绿假单胞菌M18株的rsmY突变菌株M18RY,通过基因过表达、lacZ报告基因融合分析实验,进一步验证了RsmY对抗生素合成基因的调控作用。【结果】比较野生型M18和突变株M18RY中PCA和Plt在同一培养条件下的生物合成量,突变菌株M18RY中PCA的产量显著增加,为野生型菌株的5倍左右,而Plt的产量降为野生型的1/8。LacZ报告基因融合分析进一步证明了RsmY对PCA的负调控作用主要是通过phz2基因簇来实现的。【结论】结果表明,rsmY基因区别性调控PCA和Plt的生物合成。     

基于pDS132的基因敲除中创伤弧菌氯霉素的耐药性变化及影响

【背景】基于自杀载体的基因敲除在单基因敲除上的应用较为常见,但在多基因敲除过程中细菌耐药性的变化及对后续敲除的影响尚未明确。【目的】探究基于自杀载体pDS132的创伤弧菌vvhA与rtxA1双基因敲除株构建过程中,创伤弧菌对氯霉素耐药性的变化及对后续基因敲除的影响。【方法】基于自杀载体pDS132的同源重组法构建创伤弧菌YJ016的单基因敲除株YJ016-ΔvvhA、YJ016-ΔrtxA1和双基因敲除株YJ016-ΔvvhAΔrtxA1,记录单交换筛选时的氯霉素浓度与筛选平板上成功重组的菌株所占比例。琼脂稀释法测定野生型与敲除株的氯霉素最低抑菌浓度(Minimum InhibitoryConcentration,MIC)和抗性突变频率,纸片扩散法测定菌株对其他药物的敏感性,分析其对单交换筛选的影响。【结果】单基因敲除株的氯霉素MIC及氯霉素抗性突变频率高于野生型;双基因敲除株的庆大霉素抑菌圈直径小于野生型。单基因敲除时,单交换重组菌株的占比为100%(20/20);在YJ016-ΔvvhA上敲除rtxA1基因,氯霉素筛选浓度为2、4μg/mL时,单交换重组菌株的占比分别为40%(8/20)、5%(1/20);在YJ016-ΔrtxA1上敲除vvhA基因,氯霉素筛选浓度为2、4μg/mL时,单交换重组菌株的占比均为0%(0/20)。【结论】基于自杀载体pDS132的基因敲除中创伤弧菌氯霉素的耐药性升高,可能影响后续单交换重组的筛选,该结果为基于自杀载体的同源重组技术应用于多基因敲除提供了参考。     

鸡白痢沙门菌S06004株致病岛-2缺失株的构建及其生物学特性

摘要:【目的】了解致病岛-2(Salmonella Pathogenicity Island 2,SPI-2)对鸡白痢沙门菌致病性的影响,初步探讨研制安全有效的鸡白痢沙门菌减毒株的可行性。【方法】采用λ-red 同源重组系统构建鸡白痢沙门菌S06004株的SPI-2(约40 kb)缺失株S06004ΔSPI2。并与野生型相比较,对该缺失株的生长特性、生化特性、遗传稳定性和致病性等基本生物学特性进行鉴定。【结果】成功构建SPI-2缺失株S06004ΔSPI2,SPI-2的缺失不影响鸡白痢沙门菌的生长特性和生化特性,且该缺失株具有良好的遗传稳定性,其对2日龄雏鸡的LD50是野生株的252 倍。【结论】SPI-2的缺失引起鸡白痢沙门菌毒力的明显下降,这为进一步研究鸡白痢沙门菌SPI-2的功能及制备减毒疫苗奠定了基础。     

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.