多功能降解菌Pseudomonas putida KT2440-DOP的构建与降解特性研究

三唑磷水解酶基因为研究发现的一个新的广谱有机磷水解酶基因,通过PCR从有机磷降解菌株Ochrobactrum sp. Mp4总DNA扩增了tpd,将tpd定向克隆到pBBRMCS5载体上,构建重组质粒pTPD,在辅助质粒pRK2013 的帮助下,通过三亲接合将pTPD转移到模式菌株Pseudomonas putida KT2440中,获得的工程菌Pseudomonas putida KT2440DOP可以降解多种有机磷农药及芳香烃化合物;KT2440DOP的有机磷水解酶活较出发菌株MP4提高了一倍左右,且遗传性状稳定。     

圈卷产色链霉菌尼可霉素生物合成基因——sanH和sanI的研究

通过反向遗传学方法克隆到圈卷产色链霉菌尼可霉素生物合成基因簇中约7.0kb的DNA片段。该片段除含有尼可霉素生物合成基因sanF外,对sanF上游约22kb的BglⅡDNA片段进行序列测定及分析表明,还含有两个完整的开放阅读框(ORF)。ORF1由1233个核苷酸组成,ORF2由195个核苷酸组成,它们分别编码由410个氨基酸残基和64个氨基酸残基组成的蛋白质,依次命名为sanH和sanI。蛋白序列数据库比较结果表明,SanH和SanI与浅灰链霉菌(\%Streptomyces griseolus)\%中共转录的细胞色素P450(cytochrome P450)和铁氧还蛋白(ferredoxin)有较高的同源性,一致性分别为46%和56%,相似性分别为62%和70%。基因功能研究表明,sanH基因的破坏虽不影响圈卷产色链霉菌产生的尼可霉素的生物活性,但该基因可能参与了尼可霉素羟基化反应的生物合成。     

钝齿棒杆菌天冬氨酸激酶基因的克隆和序列分析

运用PCR方法,从野生型钝齿棒杆菌株(Corynebacterium crenatum)AS1542及具有AEC抗性的突变株CD945染色体上分别扩增出天冬氨酸激酶(AK)基因(ask),构建了重组质粒。核苷酸序列分析表明,C.crenatum AS1542AK基因与C.crenatum CD945相比,第1199位的碱基由T变为C,引起酶蛋白β亚基第80位氨基酸从亮氨酸变成脯氨酸。该氨基酸的突变在蛋白结构上位于ACT结构域内,该区受赖氨酸调控。C.crenatum AS1542的AK基因的编码区核苷酸序列与C.glutamicum\,C.flavum及B.lactofermentum相比,同源性分别为97.23%、97.55%和97.62%,酶蛋白氨基酸序列的同源性分别为99.76%、99.52%和99.76%。但在AK基因的启动子上游序列部分与其它棒杆菌相比有较大差异。     

Sinorhizobium morelensS-5中N_氨甲酰基水解酶基因的克隆、表达和纯化

N_氨甲酰基水解酶是一种非常具有工业应用价值的水解酶,可用于制备光学纯氨基酸。通过LA PCR 从 Sinorhizobium morelens S_5 菌中克隆到13kb的DNA片段,测序表明该片段上含有一个完整的N_氨甲酰基水解酶的基因（hyuC）序列。将hyuC基因克隆到表达载体pET30a上,重组质粒pET30a_HyuC在大肠杆菌中获得了高水平表达。重组的N_氨甲酰基水解酶经过热处理和三步柱色谱分离而纯化。纯化倍数为16.1倍,收率21.2%。该酶为同源四聚体,亚基分子量是38kDa。最适温度是60℃,最适pH为7.0。该酶有较高的热稳定性和氧化稳定性。Fe²⁺和Ca²⁺对酶的活性有一定的促进作用,而金属螯合剂和巯基试剂对酶活无明显影响。     

苜蓿中华根瘤菌与耐盐有关DNA片段的亚克隆和测序分析

将苜蓿中华根瘤菌(Sinorhizobium meliloti)042B与耐盐有关的4kb ClaⅠ DNA片段克隆在pML122上,用HindⅢ酶切下其2.4kb DNA片段,回收后与pBBR1\|MCS2连接,然后转化大肠杆菌(Escherichia coli)DH5α,筛选到转化子GS2〖DK〗。将残留在pML122上16kb ClaⅠ\|HindⅢ DNA片段连同质粒一起回收,让其自连,转化大肠杆菌S17-1,得到转化子GS0。以GS0为供体,042B的盐敏感突变株GZ17为受体,进行二亲本杂交,没有得到接合子。以GS2为供体,GZ17为受体,在辅助质粒pRK2013的协助下,进行三亲本杂交,筛选到接合子GG2,获得2.4kb HindⅢ与耐盐有关的DNA片段。将此片段连接到测序载体pGEM\|7Zf(+)上进行测序。测序结果表明,该24kb HindⅢ DNA片段含有3个开放阅读框(ORF)。在此基础上再一次亚克隆,获得19kb与耐盐有关的DNA片段。     

滇重楼寄生菌的研究

从滇重楼(Paris polyphylla var.yunnanensis)地下茎中分离和鉴定出两种细菌——蜡状芽孢杆菌(Bacillus cereus)和产碱假单胞菌(Pseudomonas alcaligenes),以及三种真菌——黑团孢霉(Periconia sp.)、白色厚顶孢霉(Pachnocybe albida)和重楼索霉(Hormomyces paridiphilus)。对蜡状芽孢杆菌、产碱假单胞菌和重楼索霉进行了液体培养并测定了胞外多糖含量,结果表明重楼索霉可分泌大量胞外多糖,这可能是导致滇重楼地下茎胶质化和多糖含量增加的原因。     

杜氏盐藻psaB基因cDNA的克隆与序列分析

根据真核生物莱茵衣藻(Chlamydomonas reinhardtii)、Chlamydomonas moewusii、Chlorella vulgaris以及Mesostigma viride的psaB基因的氨基酸高度保守序列,设计一对简并引物,利用TRIzol试剂提取杜氏盐藻（Dunaliella salina）细胞的总RNA,通过RT-PCR,得到的一段长为1.8kb左右的cDNA片段。PCR产物经T-A克隆并测序分析以及测序结果推导成氨基酸序列进行同源性比较,表明所克隆的1815bp序列为杜氏盐藻psaB cDNA片段,GenBank收录号为AY820754。根据已经得到的psaB序列推导成氨基酸序列与一些已知物种的psaB基因相比较,同源性分别为Chlamydomonas reinhardtii 92%,Chlamydomonas moewusii 91%,Chlorella vulgaris 86% , Mesostigma viride 85%,Physcomitrella patens subsp.Patens 85%, Nephroselmis olivacea 84%。据此可推断本实验中所克隆的序列为杜氏盐藻psaBcDNA序列。     

头状链轮丝菌染色体复制起始区的克隆和对变铅青链霉菌的转化

头状链轮丝菌(Streptoverticillum caespitosus) ATCC27422是抗肿瘤药物——丝裂霉素A的产生菌,根据高GC含量革兰氏阳性菌染色体复制起始区两端基因序列的保守性,采用PCR方法从该菌中克隆了一段包含染色体复制起始区(oriC)的1.3 kb片段。序列分析发现,克隆片段与天蓝色链霉菌的oriC及邻近区域的同源性达80％以上;头状链轮丝菌oriC中含有22个DnaA-box,保守序列为TTGTCCACA。以克隆片段构建的质粒可以跨属转化变铅青链霉菌(S. lividans)ZX7,原生质体转化效率为3.2×10²个/μgDNA;质粒在S. lividans ZX7中能以低拷贝形式稳定存在;转化子的菌落和菌丝形态均正常。头状链轮丝菌oriC序列与几种链霉菌的oriC有较高的同源性,以及在变铅青链霉菌中仍具有复制起始活性等说明链轮丝菌属与链霉菌属在系统发生上关系较近;但采用最大似然法分析oriC序列构建的头状链轮丝菌与几种链霉菌的系统进化树表明,头状链轮丝菌与几种链霉菌之间的分化距离远大于链霉菌之间的分化距离。该证据支持链轮丝菌属的独立分属。     

环状芽孢杆菌C-2几丁酶基因的克隆

环状芽孢杆菌(Bacillus circulans)C-2总DNA经PstI部分酶切后分离2～10kb的片段,插入质粒pUC19的PstI位点,转化大肠杆菌(Escherichia coli),利用几丁质平板从约8000个重组子中筛选到一个几丁酶基因阳性克隆(命名为pCHT1)。用12种限制酶对重组质粒进行的酶切分析表明,重组质粒中的插入片段长3.0kb,其中各有一个KpnI,SacI和SspI位点。把该克隆片段反向插入pUC19的PstI位点所得到的重组子同样具有几丁酶基因表达活性,说明此片段含有一个完整的几丁酶基因,其自身的启动子能被大肠杆菌转录系统所识别。Southern杂交证实了该片段来自于B.circulans C-2基因组,且以单拷贝形式存在,它不能与来自于其它7株几丁酶产生菌的总DNA杂交。     

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.