嗜酸氧化亚铁硫杆菌启动子探针载体的构建

目的：为了研究嗜酸氧化亚铁硫杆菌（Acidithiobacillus ferrooxidans）启动子结构与功能。方法：以pSV-β-galactosidase质粒为骨架,通过定点突变的方法引入一个新的BstBⅠ单酶切位点,构建能在大肠杆菌（Escherichia coli）中正常复制的启动子探针载体。利用PCR的方法将A.ferrooxidans菌cycA2基因上游5＇段上游DNA片段克隆到探针载体β-半乳糖苷酶基因上游以替代其原有启动子（gpt启动子）,并将重组的质粒转化E.coliDH5α菌株。通过检测宿主细胞的β-半乳糖苷酶活性,来鉴定启动子片段,并分析了启动子探针质粒载体的功能及启动子的强度。结果：pSV-β-galactosidase质粒被正确突变,成功构建了启动子探针载体pSVB。来源于A. ferrooxidans菌的启动子片段可驱动β-半乳糖苷酶基因在E.coli细胞中表达,转化子酶活性约为gpt 启动子驱动下活性的70 %。结论：启动子探针载体（pSVB）可用于A. ferrooxidans菌或者其它原核生物启动子的分离及进一步的分析研究。酶活性分析结果表明,来源于A. ferrooxidans菌cycA2基因上游5＇段上游DNA片段具有显著启动子活性。     

丙酮酸脱羧酶基因和乙醇脱氢酶基因表达载体的构建

以运动发酵单胞菌(Zymomonas mobilis)的总DNA为模板,PCR扩增运动发酵单胞菌中的丙酮酸脱羧酶( Pyruvate decarboxylase,PDC)基因和乙醇脱氢酶Ⅱ(Alcohol dehydrogenaseⅡ,ADHⅡ)基因.将丙酮酸脱羧酶基因和含核糖体结合位点(RBS)的乙醇脱氢酶基因串联起来置于T7启动子控制下,构成多顺反子表达质粒pQR-PRA.经酶切和PCR验证,表达载体构建成功.将pQR-PRA转入大肠埃希菌(Escherichia coli)BL21中,转化子的定性检测表明有酶表达,并且初步测定了2种酶的表达量.     

大肠杆菌tyrR基因对aroP的表达调控

大肠杆菌的tyrR基因编码芳香氨基酸生物合成和运输途径中的一种全局性调控蛋白质,该蛋白质控制着包括自身编码基因tyrR在内的涉及苯丙氨酸、酪氨酸、色氨酸合成与运输的8个转录单位的转录。大肠杆菌aroP基因编码一种跨质膜主动运输芳香氨基酸的透性酶,其转录也受TyrR蛋白抑制。利用PCR反应从E.coli K12基因组中分别克隆了aroP(p)基因(携带自身的启动子)、aroP基因(不携带自身的启动子)和tyrR基因,并将它们导入苯丙氨酸生产菌E.coli WT5中。通过大细胞法检测到这3个基因的表达,并分别测定了相应的酶活力。结果:发现导入aroP(p)和aroP基因的大肠杆菌吸收苯丙氨酸的能力分别提高到原来的1．40和1．46倍,这表明利用pλPR质粒能够表达aroP基因,该质粒中的λ右向启动子(R)和aroP自身启动子(p)的表达效率几乎同样;导入tyrR基因的E．coli WT5 ATP酶活力提高到原来的1．69倍。将两种基因串联克隆在同一质粒中共表达时,携带aroP-tyr串联的大肠杆菌株运输苯丙氨酸的能力明显高于携带aroP(p)-tyrR串联的大肠杆菌株。以E．coli WT5为对照,其AroP的活性定为1,前者的Atop相对酶活力为1．31,后者为0．95,这一结果显示TyrR蛋白可能是通过与aroP基因自身启动子的结合作用来负调控aroP基因的表达。     

利用木糖和葡萄糖合成乙醇的新型重组大肠杆菌的研究

利用PCR方法从运动发酵单孢菌染色体DNA扩增出乙醇合成途径的关键酶基因pdc、adhB,分别用tac启动子控制表达,构建了可以在Escherichia coli JM109中表达的重组质粒pKK-PA、pEtac-PA.初步的乙醇发酵结果表明,在E.coli中只引入adhB基因不能拓宽其中的产乙醇途径,引入pdc基因可以与宿主自身的ADH酶协同作用,使碳流有效导向产乙醇方向.同时引入pdc、adhB基因可以在宿主E.coli中成功建立产乙醇途径.     

心肌α肌球蛋白重链启动子驱动的CREG蛋白表达载体的构建及鉴定

目的：构建心肌特异性α-肌球蛋白重链（α—myosin heavy chain,α—MHC）启动子启动E1A基因阻遏子（cellular repressor of E1A—stimulated genes,CREG）和增强型绿色荧光蛋白（enhanced green fluorescent protein,EGFP）融合的真核表达载体。绿色荧光蛋白作为报告基因,方便在心肌细胞中直接观察CREG蛋白的表达,为心肌特异性转CREG基因动物模型制备提供载体。方法：用BamHI和EcoRI双酶切pcDNA3．1myc—His／hCREG质粒得到CREG基因,亚克隆入增强绿色荧光蛋白表达质粒pEGFP-N1中,构建pCREG-EGFP-N1;根据Genebank中公布的α-MHC基因的启动子序列,人工合成pUC57-α-MHC启动子基因序列,经AseI和NheI双酶切得到启动子α—MHC,亚克隆入pCREG—EGFP-N1中替代原CMV启动子,构建pα-MHC-CREG—EGFP-N1,测序鉴定。用脂质体法将该质粒转染体外培养的小鼠原代心肌细胞,荧光显微镜下观测绿色荧光蛋白的表达;Western blot检测CREG蛋白的表达。结果：成功构建pα—MHC—CREG-EGFP-N1质粒,酶切及测序结果正确;成功转染入原代培养小鼠心肌细胞,在荧光显微镜下可见绿色荧光蛋白的表达,Westem blot检测到CREG蛋白的表达。结论：重组质粒pα-MHC—CREG—EGFP-N1体外转染入原代培养小鼠心肌细胞后,目的基因能够在心肌细胞中有效表达,检测方法简便可靠,为下一步建立心肌细胞特异性表达CREG的过表达转基因小鼠、深入探讨CREG在心肌疾病发生中的生物学功能研究奠定了基础。     

枯草杆菌表达载体pUB23的构建及地衣杆菌α-淀粉酶基因的表达

将克隆的解淀粉芽胞杆菌强启动子经DNA序列分析后连接到能在枯草杆菌中复制的质粒pUB18上,构建枯草杆菌表达载体pUB23。为了测试构建的表达载体能否表达外源基因,将地衣杆菌抉失了启动子的α-淀粉酶基因接到pUB23上启动子的下游,组建重组质粒,转化枯草杆菌QB1130(amy~-),获得能分泌α-淀粉酶的转化株,证明缺失了启动子的结构基因在pUB23上克隆启动子的启动下获得表达。酶活力测定结果表明,表达水平是用原启动子时的2.5倍.     

心肌α肌球蛋白重链启动子驱动的CREG 蛋白表达载体 的构建及鉴定

目的:构建心肌特异性α-肌球蛋白重链(α-myosin heavy chain,α-MHC)启动子启动E1A基因阻遏子(cellular repressorof E1A-stimulated genes,CREG)和增强型绿色荧光蛋白(enhanced green fluorescent protein,EGFP)融合的真核表达载体。绿色荧光蛋白作为报告基因,方便在心肌细胞中直接观察CREG蛋白的表达,为心肌特异性转CREG基因动物模型制备提供载体。方法:用BamH I和EcoR I双酶切pcDNA3.1 myc-His/hCREG质粒得到CREG基因,亚克隆入增强绿色荧光蛋白表达质粒pEGFP-N1中,构建pCREG-EGFP-N1;根据Genebank中公布的α-MHC基因的启动子序列,人工合成pUC57-α-MHC启动子基因序列,经Ase I和Nhe I双酶切得到启动子α-MHC,亚克隆入pCREG-EGFP-N1中替代原CMV启动子,构建pα-MHC-CREG-EGFP-N1,测序鉴定。用脂质体法将该质粒转染体外培养的小鼠原代心肌细胞,荧光显微镜下观测绿色荧光蛋白的表达;Western blot检测CREG蛋白的表达。结果:成功构建pα-MHC-CREG-EGFP-N1质粒,酶切及测序结果正确;成功转染入原代培养小鼠心肌细胞,在荧光显微镜下可见绿色荧光蛋白的表达,Western blot检测到CREG蛋白的表达。结论:重组质粒pα-MHC-CREG-EGFP-N1体外转染入原代培养小鼠心肌细胞后,目的基因能够在心肌细胞中有效表达,检测方法简便可靠,为下一步建立心肌细胞特异性表达CREG的过表达转基因小鼠、深入探讨CREG在心肌疾病发生中的生物学功能研究奠定了基础。     

链霉菌和大肠杆菌穿梭质粒载体的构建

本文报道了链霉菌和大肠杆菌穿梭质粒载体pSE-3的构建;把具有双启动子的大肠杆菌的质粒pGEM-3与新霉素抗性基因启动子缺失的链霉菌的探针质粒pIJ486分别用BamHI和BglⅡ酶切,T4 DNA连接酶连接后转化到E.coli HB101(Amp(?),Neo(?)),所得重组质粒能强启动pIJ486质粒上的氨基糖苷磷酸转移酶基因(aph),并使新霉素抗性基因在大肠杆菌中得到强表达。此重组质粒被命名为pSE-3,当其转化到变青链霉菌TK54(Tsr(?),Neo(?))的原生质体前,新霉素抗性基因亦能得到强表达。酶切结果表明,构建的具有两个启动子的穿梭质粒载体pSE-3上有HindⅢ和EcoRI的单酶位点,拷贝数约为39。经再转化和传代50代等研究表明,穿梭质粒载体pSE-3在链霉菌和大肠杆菌中均是稳定的。为某些有应用价值的目的基因在大肠杆菌和链霉菌中的克隆与表达提供了一个有价值的穿梭质粒载体。     

地衣芽孢杆菌高温α-淀粉酶异源表达研究

目的：以地衣芽孢杆菌高温α-淀粉酶基因（amyL）为报告基因,构建含不同启动子的枯草杆菌表达载体,转化枯草杆菌,并对重组菌的酶活进行分析,比较不同启动子对amyL基因在枯草杆菌中表达的影响。方法：以高温α-淀粉酶高产菌株B.licheniformis0204染色体DNA为模板,PCR扩增得到amyL并分别与PQ启动子和P43启动子进行连接构建表达载体pUB-PQ-amyL和pUB-P43-amyL,化学法转化枯草杆菌1A717,筛选得到重组转化子后对重组菌的表达产物进行SDS-PAGE和酶活检测。结果：重组菌摇瓶发酵105h后测定高温α-淀粉酶酶活,B.subtilis1A717（pUB-PQ-amyL）的最高酶活为280.1U/mL,B.subtilis1A717（pUB-P43-amyL）的最高酶活为190.5U/mL。结论：PQ启动子调控的高温α-淀粉酶最高表达水平是P43启动子调控的最高表达水平的1.47倍,说明PQ启动子能使amyL基因在枯草杆菌中更高效地表达。     

基于大肠杆菌受体菌DH5α △asd缺失株的构建及作为基因转化、表达操作系统的评估

基于大肠杆菌(E.coli)染色体上asd基因的已知序列,利用λ噬菌体的Red同源重组系统一步法构建E.coliDH5α的asd基因缺失突变株DH5α△asd::cat,在二次重组中利用携带能够表达FLP位点特异性重组酶的质粒pCP20介导二次同源重组,以去除上述缺失突变株中氯霉素抗性筛选基因。结合PCR扩增和测序结果,证明DH5α△asd缺失突变株的正确构建。该缺失突变株失去了在普通LB培养基上生长的能力,只有添加DAP或导入表达asd基因的质粒(asd基因互补试验)才能在LB培养基上生长,与原型DH5α比较,其生长速度和生长对数期、接受不同拷贝数质粒的转化效率几乎相一致。基于该缺失突变株构建出以asd营养基因为标志的大肠杆菌染色体-质粒平衡致死系统。体外培养连续传代50代次,pnirBMisL-fedF-asd质粒不丢失,并功能性表达F18大肠杆菌黏附素FedF。     

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.