卷枝毛霉pyrG基因缺陷突变株的诱变筛选与鉴定

为制备新的遗传筛选标记用于构建高产番茄红素的工程菌株,实验运用化学诱变的手段,以N-甲基-N'-硝基-N-亚硝基胍为诱变剂,以番茄红素生产菌株卷枝毛霉MU616为出发菌株,诱变获得5株尿嘧啶缺陷型突变株,突变株在基本培养基中培养5天后仍不能生长。通过同源转化带有pyrG基因(编码乳清酸核苷-磷酸盐脱羧酶)的质粒pEPM1确定突变株Mt1、Mt4和Mt5为pyrG基因缺陷突变株。随之对pyrG突变株进行生长特性的研究和产番茄红素性能的检测,结果表明,其中突变株Mt4的生物量为(9.0±0.6)g/L,番茄红素产量在黑暗和光照条件下分别为(1 648±185)μg/g和(3 234±281)μg/g,均与出发菌株相似,适宜作为进行卷枝毛霉转化的带有遗传标记的受体菌。pyrG基因缺陷突变菌株的获得对构建高产番茄红素的卷枝毛霉工程菌具有重要的意义。     

烟曲霉菌热稳定性植酸酶在黑曲霉菌中的分泌性表达

在先前克隆获得烟曲霉菌植酸酶phyA基因并构建了重组质粒的基础上,将该质粒转化黑曲霉菌pyrG基因缺陷株M54;同时制备植酸酶多克隆抗体用于植酸酶的免疫学检测。SDS-PAGE和western-blot结果表明,phyA在黑曲霉菌中获得分泌性表达。表达产物活性测定结果显示,重组植酸酶的表达量为597.6 IU/mL。在90℃加热10 min和100℃加热20 min后,重组植酸酶残余酶活分别为74%和70%,具有较好的热稳定性。实现了烟曲霉菌植酸酶在黑曲霉菌中的分泌性表达,表达产物具较高的生物活性和耐热性。     

米曲霉尿嘧啶营养缺陷型突变体筛选及转化体系建立

米曲霉(Aspergillus oryzae)是一种十分重要的工业微生物，但由于其菌丝体和孢子均含有多个细胞核，并且对多种抗性药物具有抗性，导致其遗传转化和分子生物学研究较其他模式丝状真菌困难。目前米曲霉遗传转化主要采用营养缺陷型作为筛选标记。尿嘧啶营养缺陷型是米曲霉转化中最常用的一种营养缺陷型标记，其筛选标记基因pyrG编码乳清酸核苷-5′-磷酸脱羧酶为尿嘧啶的前体尿苷合成所必需。本研究以米曲霉3.042为背景，利用紫外线诱变和5-氟乳清酸(5-FOA)胁迫筛选获得5株尿嘧啶营养缺陷型菌株，经DNA测序5株菌株均为pyrG基因不同位点突变，确定为尿嘧啶营养缺陷型。以筛选到的尿嘧啶营养缺陷型菌株为背景，利用农杆菌介导的米曲霉转化系统，成功构建了米曲霉尿嘧啶营养缺陷型为筛选标记的农杆菌转化体系。     

柠檬明串珠菌及相近种部分持家基因的系统发育分析

[目的]利用16S rRNA、dnaA、murC和pyrG基因分子标记研究Leuconostoc citreum(Leu.citreum)及相近种间的种系发育关系,并比较这些基因序列对Leu.citreum及相近种的区分能力.[方法]以分离自酸面团中的7株Leu.citreum为研究对象,以dnaA、murC和pyrG基因片段为标记,通过PCR扩增、测序,结合已公布的近缘种及亚种相应序列,计算遗传距离,构建系统发育树,并与16S rRNA基因进行比较.[结果]研究发现Leu.citreum及相近种间的dnaA、murC和pyrG基因构建的系统发育树拓扑结构与16S rRNA基因基本一致,区别在于相似性的不同,其分别为75.5％-97.2％、50.2％-99.7％、65.0％-99.8％和98.5％-100％.[结论]在Leu.citreum及相近种间的种系发育关系中,dnaA、murC和pyrG基因与16S rRNA基因系统进化关系都具有很好的一致性,但这三个持家基因的遗传距离显著高于16S rRNA基因.因此,采用dnaA、murC和pyrG基因可以用于Leu.citreum及相近种的分类鉴定.     

建立以TK基因为标记将外源基因导人臭曲霉(Aspergillus foetidus)的选择系统及外源基因的表达

本文报道建立以胸腺嘧啶核苷激酶(TK)为标记,利用痘苗病毒TK基因为旁侧序列的外源基因载体,通过体内同源重组的原理将外源基因导入臭曲霉菌,并用5-溴脱氧尿嘧啶核膏(BudR)进行转化株选择的新系统,对黑曲霉菌TK+株的挑选、转化及选择条件等进行了研究。用这一系统,成功地将臭曲霉菌自身分离到的启动基因H8驱动下的乙肝表面抗原(HB8sAg)基因导入臭曲霉菌。Southern blot和多次孢子传代证实,HBsAg基因已与宿主的DNA稳定整合：在发酵上清液中,ELIAS检测HBsAg为阳性(P/N值在20左右),wwstern blot有特异的带存在,免疫电镜观察发现,在发酵液中存在有与人血清中HBssAg颗粒大小、形态相似的22nm的颗粒。这些结果表明,HBAg基因在臭曲霉菌中已获表达。本文建立的基因导入和选择系统可广泛应用于各种外源基因在臭曲霉菌中表达的研究。     

谷氨酸棒杆菌1dh基因的敲除

谷氨酸棒杆菌中ldh基因编码乳酸脱氢酶,可催化丙酮酸转化生成乳酸.利用重叠延伸PCR的方法,获得中间缺失部分序列的dldh基因片段,将其与载体pk 18mobsacB连接,转化大肠杆菌感受态,筛选出阳性转化子后,转化谷氨酸棒杆菌ATCC 13032感受态细胞.分别在卡那霉素抗性平板及10％蔗糖平板上进行两次筛选,利用PCR方法鉴定,成功获得ldh基因缺失的谷氨酸棒杆菌突变株ATCC 13032-(4)ldh.应用荧光定量PCR检测,ATCC 13032-(z)ldh中的ldh基因在转录水平与野生型菌株ATCC 13032相比,相对表达量为O.ldh基因的敲除对菌株的生长造成了一定的影响.     

农杆菌介导的蛹虫草营养缺陷型菌株和遗传转化体系的构建

【背景】天然蛹虫草是蛹虫草菌侵染昆虫蛹或虫形成的子实体,具有非常重要的生物药理活性。目前蛹虫草基因组测序已经完成,但是其分子生物学研究较少。【目的】在蛹虫草中构建一种以尿苷/尿嘧啶营养缺陷型为筛选标记的农杆菌介导的转基因体系。【方法】乳清酸核苷-5′-磷酸（orotidine-5′-monophosphate,OMP）脱羧酶为尿嘧啶合成必需酶,利用根癌农杆菌介导的转化（Agrobacterium tumefaciens-mediated transformation,ATMT）方法,通过同源重组对蛹虫草野生型菌株中该酶的编码基因pyrG进行敲除,构建尿苷/尿嘧啶营养缺陷型突变体。然后,利用本实验室已有的米曲霉pyrG为筛选标记的二元转化载体,通过农杆菌转化法对该营养缺陷型菌株进行遗传转化。【结果】通过同源重组法,成功敲除pyrG构建了尿嘧啶营养缺陷型蛹虫草,以此为背景,在22℃共培养66h,成功对蛹虫草实现转基因,转化效率为（75±35）/10⁶孢子。另外,本研究还发现丝状真菌常用的构巢曲霉3-磷酸甘油醛脱氢酶启动子PgpdA及α淀粉酶启动子PamyB不能在蛹虫草...     

食品上的应用

942407革兰氏阴性专性甲醇营养株糖原甲基杆菌的高丝氨酸脱氢酶基因和苏氨酸合成酶基因的克隆和核苷酸顺序[英]/Motoyama,H.…//Appl.Environ.Microbi01.一1994,60(1).一;111～119E译自DBA,1994,13(7),94—04035] 通过大肠杆菌缺HD突变株的互补作用,由产氨基酸的革兰氏阴性专性甲醇营养株糖原甲基杆菌(Methylobacillus glycogenes)ATCC 21276~IATCC 21371克隆高丝氨酸脱氢酶(HD)基因(horn)和苏氨酸合成酶基因(thr c)。构建了ATCC 21276和ATCC 21371的基因库,大肠杆菌缺HD突变株Gifl02被转化。将转化细胞涂布在含有L一苏氨酸和…     

原生质体介导马尔尼菲青霉基因转化体系的优化

目的优化原生质体介导的马尔尼菲青霉转化体系,为其基因功能研究提供良好的平台。方法通过原生质体介导将构巢曲霉(Aspergillus nidulans)pyrG基因插入马尔尼菲青霉尿嘧啶缺陷株ligD(pyrG-,ligD-)中,在不含尿嘧啶的培养基中筛选阳性转化子,运用PCR验证重组子,通过改变影响转化效率的酶解时间、培养基浓度、质粒浓度、不同配方的原生质体洗涤液STC和不同配方的原生质体助融剂PTC 5个条件对体系进行优化。结果 PCR验证A.nidulans pyrG基因成功的插入ligD中,转化子可稳定传代。最适合ligD原生质体转化效率的条件为:酶解时间6h,PTC(60%PEG-4000,100mmol/L Tris-HCl pH8.0,100mmol/L CaCl2),每100μL原生质体(106~107/mL)加入2.5μg质粒,0.6 mol/L蔗糖SD/SDU筛选/再生培养基,每1μg质粒转化子可达68个左右。结论成功优化了原生质体介导马尔尼菲青霉转化体系的条件,优化后该方法转化效率高,为基因功能研究提供良好平台。     

利用可重复使用的URA3标记基因建立热带假丝酵母基因敲除系统

热带假丝酵母(Candida tropicalis)在发酵工业中具有重要的应用潜力,但二倍体遗传结构和较低的遗传转化效率限制了其代谢工程育种技术的应用。建立可靠的遗传转化技术并高效的删除目的基因是代谢工程改造热带假丝酵母的重要前提。文章以C. tropicalis ATCC 20336为出发菌株,通过化学诱变筛选获得了尿嘧啶缺陷型突变株C. tropicalis XZX(ura3/ura3)。以丙酮酸脱羧酶(Pyruvate decarboxylase,PDC)基因作为靶基因构建了两端包含同源臂并在选择性标记C. tropicalis URA3(Orotidine-5′-phosphate decarboxylase,乳清酸核苷-5-磷酸脱羧酶)基因两侧同向插入源于沙门氏菌(Salmonella typhimurium)的hisG序列的基因敲除盒PDC1-hisG-URA3-hisG- PDC1(PHUHP),并转化宿主菌株C. tropicalis XZX,筛选获得PHUHP片段正确整合到染色体的PDC基因位点的转化子XZX02。在此基础上,将转化子XZX02涂布于5-FOA(5-氟乳清酸)选择培养基上,筛选得到URA3基因从PHUHP片段中丢失的营养缺陷型菌株XZX03。进一步构建了第2个PDC等位基因的删除表达盒PDCm- URA3-PDCm,并转化C. tropicalis XZX03菌株,获得转化子C. tropicalis XZX04。经PCR和DNA测序确认转化子C. tropicalis XZX04细胞染色体上的两个PDC等位基因被成功敲除。文章建立了一种营养缺陷型标记可重复使用的热带假丝酵母遗传转化技术,利用该技术成功敲除了细胞的PDC基因,为进一步利用代谢工程改造热带假丝酵母奠定了基础。     

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.