促甲状腺激素释放激素受体mRNA在大鼠睾丸中的表达(英文)

本应用原位杂交技术在大鼠睾丸恒冷箱切片上研究了促甲状腺激素释放激素受体（TRH－R）RNA的表达和定位。由DNA合成仪合成两个含48个碱基的寡核苷酸探针,两个探针分别与小鼠垂体TRH－R1005－1052和1332－1379区段的cDNA互补,生物素在5′末端标记寡核苷酸探针。结果显示TRH－R寡核苷酸探针与其互补的mRNA杂交信号集中在大鼠睾丸的间质细胞中,生精细胞地交信号,杂交信号的强度依探针浓度而增加,该结果表明RTH可能通过自分泌调节生殖功能和发育,TRH－R作用途径可能与在垂体的作用类似。     

分子信标及其应用研究进展

分子信标作为一种核酸荧光探针,其最初的设计目的是用来检测核酸,特别是用作实时荧光PCR反应的探针。由于其特殊的发夹结构,决定的其具有多种的用途。不仅能在液相或固相对核酸进行定性、定量分析,还能用于多种DNA－蛋白质、DNA－DNA损伤试剂之间相互作用等的研究。     

HBV耐药相关DNA突变体的构建及在寡核苷酸基因芯片分析中的应用

拉米呋啶（Lamivudine)是近年来开发成功的一种治疗HBV慢性感染病人的核苷类药物。随着拉米呋啶在临床上广泛使用,HBV耐药现象已成为临床实践中的棘手问题。建立HBV耐药测定技术成为HBV基础研究和临床实践中企待解决的一个重要问题。寡核苷酸芯片（Oligochip)是近年来发展并逐步成熟的一种高通量基因检测技术,已成功应用于基因突变快速和高通量检测。本研究在构建HBV拉米呋啶抗性相关HBV多聚酶基因突变体基础上,设计并制备了HBV耐药寡核苷酸芯片（HBV－Lam Oligochip)。根据HBV拉米呋啶抗药性相关突变主要位于HBV多聚酶基因的L526、A546、M550和V553等氨基酸位点,设计了28条寡核苷酸探针。探针对应序列为HBV DNA聚合酶基因反义链,HBVHBVhb长度为15－18nt。探针合成时在其3′端接氨基和间隔臂（spacer)等特定修饰;探针纯化与定量后,用基因芯片点样仪点到醛基修饰载玻片上,制成HBV耐药寡核苷酸芯片（HBV－Lam Oligochip)。为了分析基因芯片的性能,克隆了833号HBV病人血清中HBV DNA聚合酶基因,测序证实为HBV DNA野生型（即未发生突变）。以该DNA为模板,用PCR法构建了HBV耐药相关突变体。用HBV－Lam Oligochip对HBV野生型DNA和人工构建DNA突变体进行了检测。结果发现,HBV－Lam Oligochip能有效检测出野生型DNA序列。检测突变体时,HBV－Lam Oligochip检测结果与相应突变体DNA序列一致,表明HBV－Lam Oligochip不仅可检测出野生型序列,而且可有效地分辨出单碱基突变,可应用于HBV耐药基因突变临床检测。     

DNA指纹技术的应用及局限性

DNA指纹图谱是一种在一单一实验中可检测出大量DNA位点差异性的分子生物学技术。自1985年Jeffeys et al,从人的肌红蛋白位点获得第一个多位点探针并用于检查人类基回的VNTRs以来,由于各种高水平探针如微卫星探针、寡聚核苷酸探针的相继问世,使DNA指纹技术在动植物科学研究、遗传疾病的诊断、基因图谱的绘制,遗传标记的寻找及法医学等方面得到广泛应用,充分表现了此技术的优越性。当然此技术还需进一步完善。     

谷氨酸棒状杆菌启动子在枯草芽孢杆菌中的克隆及其序列分析

利用枯草芽孢杆菌启动子探针质粒pPL 603为载体,从谷氨酸棒状杆菌1014—6染色体DNA上克隆到一个有较强启动功能的DNA片段。生物素标记的DNA—DNA分子杂交实验证明该片段确实来自谷氨酸棒状杆菌1014—6染色体DNA。在绘制该片段限制酶酶切图谱的基础上,经另一个启动子探针质粒pPL 703的亚克隆,将其启动子功能区定位在Bamm酶切片段中。对后者进行了核苷酸序列分析,发现它具有棒状杆菌类启动子的－35区和－10医序列。     

酶标DNA探针与Y染色体DNA的探测

 用辣根过氧化物酶标记DNA的技术,制备了酶标基因探针。研究了酶标过程和产物的电泳行为;用斑点杂交和southern印迹杂交探测了单链、双链DNA,灵敏度可达pg水平,以此酶标的Y染色体特异的DNA片段作探针,进行了DNA杂交的性别分析,证明该探针能清楚地区别两性基因组DNA,这对基因的研究和诊断有一定实用价值。     

分子倒置探针技术的研究进展及应用

分子倒置探针技术是一项新发展起来的用于目标序列捕获的分子生物学技术,该技术通过设计特异的探针对已知的特定目的基因组序列进行捕获,将目标序列DNA富集后再利用芯片杂交或测序进行检测。此技术有助于研究人员对大样本中的基因组重要区域进行研究,避免了全基因组研究费用高、分析困难等问题。并且,分子倒置探针技术弥补了杂交捕获技术、PCR捕获技术等分子捕获手段的不足,为动植物及病原菌重要DNA片段的研究提供强有力的技术支持。目前,分子倒置探针技术广泛应用于单核苷酸多态性(SNP)分型、外显子测序、拷贝数变异、杂合性丢失、体细胞突变、DNA甲基化和可变剪接等方面的研究。由于其特异性强、重复性好、操作简单、费用低廉,并对DNA完整度要求不高,适用于福尔马林石蜡包埋样本分析等特点,分子倒置探针技术的应用越来越广泛。然而,分子倒置探针技术在探针设计及数据分析软件研发等方面仍存在一些不足,还需要进一步的优化完善。为促进相关领域学者全面了解该技术,综述了分子倒置探针技术的基本原理、发展历程、技术特点及在疾病研究领域的应用,讨论了分子倒置探针技术的价值及存在的问题。     

10月开始HCV检测用DNA探针诊断药的临床试验

第一化学药品公司从10月开始与美国 Chiron 公司共同开发的丙型肝炎病毒(HCV)检测用 DNA 探针诊断药的临床试验。HCV 检测 DNA 探针诊断药进入临床试验还是首次。进入临床的 DNA 探针诊断药采用 Chiron 公司和第一化学药品公司开发的分歧 DNA 技术。这种技术     

牙龈卟啉菌特异性克隆探针的筛选

目的 从牙龈卟啉菌47A－1的基因文库中筛选出特异性片段,制备成特异性克隆探针,方法 将牙龈卟啉菌47A－1基因文库中的重组质粒大量扩增和纯化,采用地高辛标记法制备成探针,与口腔中14种常见细菌DNA进行杂交鉴定,检测其特异性,从中筛选出对牙龈叶卟啉菌具有特异性的克隆探针。结果 重组质粒pZJ1与牙龈卟啉菌47A－1杂交,而与其它细菌DNA均不杂交,包括牙龈卟啉菌ATCC33277和W83。结论 重组质粒pZJI可制备成高特异笥克隆探针。     

DNA探针技术在肠道细菌检定中的应用

<正>随着生物化学和分子生物学的发展,DNA探针技术应运而生。它作为一个有效的检测手段,已广泛应用于分子生物学和遗传工程中的基因检测等工作中。近年来,医学微生物工作者已将探针技术应用于致病性细菌鉴定、分类和流行病学调查等工作。本文拟就DNA探针技术在肠道细菌检定中的应用情况作一综述。     

虫生真菌研究与应用的发展与机遇 ——2012年虫生真菌专刊序言

我国从事虫生真菌基础及应用研究的人员众多,各方面均取得了重要的研究成果。2012年“虫生真菌专刊”收集了国内关于虫生真菌资源、基因克隆及活性成分代谢等方面的最新研究进展。进一步的基础研究,应加强致病基因功能和真菌-昆虫分子相互作用等研究,以提高真菌杀虫剂或药用虫生真菌的应用效率。     

广西南宁市柑橘木虱虫生真菌种类调查

自柑橘木虱Diaphorina citri体上分离虫生真菌,通过形态特征和ITS序列分析,鉴定虫生真菌的种类;通过致病性测定,明确昆虫病原真菌种类。结果发现,自死体柑橘木虱上分离到18个虫生真菌菌株,隶属4种真菌;自活体上分离到985个虫生真菌菌株,隶属25种真菌。致病性测定结果发现,仅4种真菌对柑橘木虱成虫有致病性,包括刀孢蜡蚧菌Lecanicillium psalliotae、球孢白僵菌Beauveria bassiana、爪哇虫草Cordyceps javanica和淡紫紫孢菌Purpureocillium lilacinum,该4种真菌的分生孢子悬浮液（浓度5×10^7个孢子/mL）接种柑橘木虱成虫后10 d的累计死亡率分别为100%、100%、98.89%和43.33%。其中爪哇虫草仅自死体柑橘木虱上分离到,刀孢蜡蚧菌仅在活体上分离到,球孢白僵菌和淡紫紫孢菌在死体和活体上均被分离到。可见,柑橘木虱活体上的虫生真菌种类丰富。生产上应加强保护和利用昆虫病原真菌,提高其对柑橘木虱的自然抑制力。     

Pathogenesis-related genes of entomopathogenic fungi

All living things on Earth experience various diseases such as those caused by viruses, bacteria, and fungi. Insects are no exception to this rule, and fungi that cause disease in insects are called entomopathogenic fungi. These fungi have been developed as microbial insecticides and are used to control various pests. Generally, the mode of action of entomopathogenic fungi is divided into the attachment of conidia, germination, penetration, growth, and generation of secondary infectious conidia. In each of these steps, that entomopathogenic fungi use genes in a complex manner (specific or diverse) has been shown by gene knock-out and RNA-sequencing analysis. In this review, the information mechanism of entomopathogenic fungi was divided into six steps: (1) attachment of conidia to host, (2) germination and appressorium, (3) penetration, (4) fungal growth in hemolymph, (5) conidia production on host, and (6) transmission and dispersal. The strategy used by the fungi in each step was described at the genetic level. In addition, an approach for studying the mode of action of the fungi is presented.     

医学昆虫的病原真菌

昆虫病原真菌在自然环境中对害虫数量起着抑制作用,在害虫的微生物防治中也占有独特的地位。长期以来,在应用昆虫病原真菌防治人畜害虫方面研究进展较为缓慢,六十年代以来才有较大的发展。本文介绍了近十年的发展概况,并讨论了影响这个研究领域迅速发展的原因及病原真菌在生物防治中应用的潜力。认为尽管至今对这类真菌了解还较少,但通过今后加强有关方面的研究工作,其应用前景是广阔的。     

医学昆虫的病原真菌*

昆虫病原真菌在自然环境中对害虫数量起着抑制作用,在害虫的微生物防治中也占有独特的地位。长期以来,在应用昆虫病原真菌防治人畜害虫方面研究进展较为缓慢,六十年代以来才有较大的发展。本文介绍了近十年的发展概况,并讨论了影响这个研究领域迅速发展的原因及病原真菌在生物防治中应用的潜力。认为尽管至今对这类真菌了解还较少,但通过今后加强有关方面的研究工作,其应用前景是广阔的。     

Fungal proteinase expression in the interaction of the plant pathogen Magnaporthe poae with its host.

Infection by pathogenic fungi involves breaching the outer layer of the host by either mechanical or enzymatic means. Subtilisin-like proteinases are considered to be important in the infection process of entomopathogenic, nematophagous, and mycoparasitic fungi. Little is known regarding the expression of such proteinases by plant pathogenic fungi. Magnaporthe poae, a fungal pathogen of Kentucky bluegrass, expressed a subtilisin-like proteinase, proteinase Mp1, in the infected roots. Antibody was produced against the purified enzyme. From immunoblot analysis, expression of the proteinase in infected roots correlated with increasing severity of disease symptoms. Sequence analysis of a genomic clone indicated proteinase Mp1 was homologous to other fungal subtilisin-like proteinases. DNA gel blot analysis indicated proteinase Mp1 was encoded by a small gene family.     

Entomopathogenic fungi isolated from suspended-soil-inhabiting arthropods in East Kalimantan,Indonesia

As a preliminary survey to develop suspendedsoil arthropods as a new isolation source of entomopathogenic fungi, we investigated the entomopathogenic fungi of these arthropods. Fifty-five suspended-soil arthropods were collected from lowland tropical rainforests in East Kalimantan, and ten fungal isolates belonging to seven entomopathogenic species, including two undescribed species, were isolated from nine of the arthropods. Only two of the seven entomopathogenic species were commonly found from the arthropods inhabiting the ground soil in the same forests. The percentage of entomopathogenic fungi-positive arthropods from suspended soil was similar to that from ground soil of the same and another forest of the region, and lower than that from ground soil of Japan. However, the number of entomopathogenic species isolated from the suspended-soil arthropods was larger than that from ground-soil arthropods. This result suggests that suspended-soil arthropods can be a new isolation source of entomopathogenic fungi.     

Correlation of fruit tree rhizosphere soils with entomopathogenic fungi

Entomopathogenic fungi are microorganisms that control the density of host insects in nature; they are being studied as environmentally friendly alternatives to chemical insecticides for controlling insect pests. The main habitat of entomopathogenic fungi is soil, and the correlation between the distribution of entomopathogenic fungi and the physicochemical characteristics of soils planted with different trees, including vine (outdoor, greenhouse, and greenhouse shine musket), apple, peach, and pear, were analyzed. The entomopathogenic fungi of the genera Beauveria, Metarhizium, and Purpureocillium investigated in this study were all found in soil samples from vine-greenhouse, apple, and peach trees. Purpureocillium and Beauveria abundances were positively correlated with soil properties; however, Metarhizium abundances were not correlated with soil properties. The Metarhizium isolates discovered in this study showed pathogenicity to cotton aphids (an agricultural pest) and can be employed as sources for biological studies in the future. This study provides data on the diversity and abundance of entomopathogenic fungi related with soil properties, as well as the molecular, biological, and insecticidal characteristics of Metarhizium isolates.     

Genotypic diversity among Brevibacillus laterosporus strains.

In comparison with other entomopathogenic Bacillus species, the genome of Brevibacillus laterosporus is poorly characterized. The aim of this study was to examine genetic variability in B. laterosporus by using a range of typing methodologies. Strains of B. laterosporus were examined for variation in 13 chromosomal genes encoding enzymes by multilocus enzyme electrophoresis. Optimal conditions of pulsed-field gel electrophoresis and randomly amplified polymorphic DNA were established that allowed analysis of the genome of B. laterosporus. None of these techniques allowed the identification of a convenient molecular marker for entomopathogenic strains, although one specific primer amplified only DNA from almost all mosquitocidal strains.