黄地老虎颗粒体病毒cDNA文库的构建

以感染黄地老虎颗粒体病毒（Agrotis segetum ganulosis virus,AsGV)黄地老虎幼虫为材料提取总RNA,分离mRNA,并反转录合成cDNA ,构建了包括黄地老虎（Agrotis segetum,As)幼虫和黄地老虎颗粒体病毒的cDNA 文库。用Eco RI和HindⅢ限制性内切酶酶切AsGV基因组DNA,制备地高辛探针,与上述总RNA,mRNA,cDNA 杂交,从文库中筛选出AsGV的阳性克隆1081个,经cDNA测序,cDNA 编码序列与基因组编码序列相符。并根据基因组阅读框序列合成引物,PCR扩增出59个阅读框的编码基因,也完全与基因组序列相符。     

黄地老虎颗粒体病毒的研究——Ⅲ.黄地老虎颗粒体病毒的酶联免疫分析

黄地老虎颗粒体病毒(Agrotis segetum granulosis virus简称AsGV)已在防治黄地老虎的危害上获得成功,其杀虫率达95％以上,具有无残毒、用药少、经济简便和长效等优点。我们已对AsGV在黄地老虎细胞中的超微结构,AsGV的包涵体,病毒粒子的分离提纯及其形态结构作了报道。酶联免疫法是近年来发展起来的一种新的血清学技术,具有特异     

黄地老虎颗粒体病毒DNA的基因文库和物理图谱

利用EMBL 4——携带多切点连接序列的λ取代载体,建成了黄地老虎颗粒体病毒(AsGV)DNA基因文库,并绘制出AsGV DNA基因组的物理图谱。当体外包装效率达3 x 104pFU／μg DNA时,Sal和BamHI烈酶完全酶切的EMBL 4和BamHI部分酶切的AsGV DNA,按2：1的接头克分子比,用T4连接酶连接后,在BHB2688与2690体系中进行体外包装,再经在L95和ED 8654中的转染和转导,获得1．5×103噬菌斑。根据Clarke和Carbon公式,筛选概率达到复盖99％的AsGV基因组只需35个重组噬菌斑。我们随机挑取了35个重组噬菌斑,经快速琼脂糖凝胶电泳分析得到13个不同类型的重组噬菌斑,以32P标记的AsGV DNA为探针进行分子杂交,确定了BamHI在AsGV DNA基因组上位点的相邻位置。     

小地老虎β-N-乙酰葡萄糖胺糖苷酶基因cDNA序列的克隆及mRNA组织特异性表达

β-N-乙酰葡萄糖胺糖苷酶是昆虫几丁质代谢中一种关键酶,在昆虫的多种生理活动中发挥重要作用。本文以小地老虎Agrotis ipsilon Hufngel预蛹期幼虫为材料提取总RNA,利用RT-PCR和RACE技术,扩增得到其β-N-乙酰葡萄糖胺糖苷酶基因的cDNA序列。该序列含有2701个碱基,包括一个1788个碱基的开放阅读框,编码一个含595个氨基酸的蛋白,分子量约为68.3ku,等电点为5.48。推导得到的氨基酸序列与其他昆虫,尤其是鳞翅目昆虫的β-N-乙酰葡萄糖胺糖苷酶高度同源。基因cDNA序列已经登录GenBank并获得登录号GU985280。该基因在小地老虎取食期和预蛹期的马氏管、中肠、脂肪体、体壁和去中肠虫体中均含有mRNA水平的表达。     

黄地老虎颖粒体病毒的研究I.颗粒体病毒的超微结构

通过病虫组织和提纯病毒包含体的超薄切片的电镜观察,研究了新疆黄地老虎颗粒体病毒 Agrotis segetum granulosis virus (简称AsGV—XJ),在组织细胞中的超徽结构以及它的装配过程。从被AsGV矗J侵染的脂肪体和皮层中,观察到大量的颗粒体病毒。在前肠、中肠、后肠、马氏管、气管的组织切片中未观察到颗粒体病毒o AsG'v一xJ的装配是在脂肪体细胞的胞质中进行的,从病毒粒子的核髓开始,形成病毒粒子,然后由包含体蛋白以类晶体方式包被,构成一个完整的颗粒体o AsGV—xJ的装配过程证明了核糖体、线粒体、内质网在其增殖中的重要作用,AsGV—xJ的基本形态与国外报道的AsGV大致相同。观察到包含体外围有一层透明膜,以及各种不同排列方式的双粒子所构成的包含体,这是AsGv—xJ的特点。     

甘蓝夜蛾和八字地老虎肌动蛋白基因的克隆与序列分析

肌动蛋白是细胞骨架微丝的主要组成成分,在肌肉收缩、细胞骨架形成、细胞移动等方面起重要作用。以鳞翅目夜蛾科昆虫甘蓝夜蛾Mamestra brassicae L.和八字地老虎Agrotis c-nigrum 3龄幼虫整个虫体为材料提取总RNA,利用RT-PCR和cDNA末端快速扩增技术(RACE),分别扩增得到2种昆虫的肌动蛋白的cDNA序列,甘蓝夜蛾肌动蛋白的cDNA序列含有1441个碱基,而八字地老虎肌动蛋白的cDNA序列含有1411个碱基。2种昆虫的该基因的cDNA序列均包括1个1131个碱基的开放阅读框,编码1个含376个氨基酸的蛋白。甘蓝夜蛾肌动蛋白分子量约为41.8kDa;八字地老虎肌动蛋白分子量约为41.9kDa。Prosite软件分析结果表明,甘蓝夜蛾和八字地老虎肌动蛋白氨基酸序列中存在3个肌动蛋白特征片段。GenBank数据库搜索及序列比对结果表明,甘蓝夜蛾肌动蛋白属于肌肉特异型肌动蛋白,八字地老虎肌动蛋白属于细胞质特异型肌动蛋白。2个基因的cDNA序列已经登录GenBank并获得登录号,甘蓝夜蛾肌动蛋白cDNA序列登录号为EU035314,八字地老虎肌动蛋白cDNA序列登录号为EU035315。利用RT-PCR技术在八字地老虎4龄、5龄、6龄幼虫、蛹期4个不同发育阶段和6龄期的肠道、体壁、脂肪体3种不同组织中都检测到了肌动蛋白基因在mRNA水平的表达。     

一株中华绒螯蟹呼肠孤病毒RNA1 cDNA文库构建及其RNA聚合酶基因部分序列分析

在中华绒螯蟹体内分离到一株呼肠孤病毒(命名为EsRV905株).采用Trizol试剂提取病毒核酸,经聚丙烯酰胺凝胶电泳,碎胶法回收基因组各节段.随机引物法合成第一节段的cDNA文库,胶回收试剂盒去除小片段,平端连接于载体,化学转化,利用蓝白斑筛选阳性克隆子,酶切鉴定重组质粒.从基因组第一节段的重组质粒中选择2个插入片段约为1.5kb的质粒测序,结果得到包括RNA聚合酶主要特征性结构的一段序列.结果说明,这株蟹呼肠孤病毒的RNA聚合酶定位于基因组第一节段.     

棉铃虫核型多角体病毒多角体蛋白聚集体特性及与其它包涵体蛋白的血清学关系

纯化的多角体碱解释放多角体蛋白,经等电点沉淀和柱层析对多角体蛋白进行分离纯化,结合SDS-PAGE、免疫双向扩散、免疫电镜等方法,证明棉铃虫核型多角体病毒(HaNPV)的多角体蛋白以聚集体形式存在。用ELISA法检测包涵体蛋白之间的血清学关系,结果表明,与黄地老虎颗粒体病毒(AsGV)和粘虫颗粒体病毒(PsGV)颗粒体蛋白相比较,HaNPV多角体蛋白与葡萄天蛾核型多角体病毒(ArNPV)和黄地老虎核型多角体病毒(AsNPV)多角体蛋白之间的血清学关系更为密切。     

八字地老虎和粘虫蜕皮激素接受子3(HR3)基因cDNA序列的克隆与序列分析

蜕皮激素接受子3(hormone receptor 3,HR3),是一种蜕皮调节转录因子,调控蜕皮过程中相关基因的表达,是蜕皮级联反应中的关键因子。本文以八字地老虎Agrotisc-nigrumL.和粘虫Mythimna separata Walker预蛹期幼虫为材料,分别提取总RNA,利用RT-PCR和cDNA末端快速扩增技术(RACE),分别扩增得到2种昆虫蜕皮激素接受子3(HR3)的5′非编码区和完整开放读码框在内的cDNA序列,其中八字地老虎的HR3 cDNA序列含有1729个碱基,包括一个1533个碱基的开放阅读框,编码一个含510个氨基酸的蛋白,分子量约为57.5ku。粘虫的HR3cDNA序列含有1743个碱基,包括一个1536个碱基的开放阅读框,编码一个含511个氨基酸的蛋白,分子量约为57.9ku。这2种昆虫HR3 cDNA序列推导的氨基酸序列均具有昆虫核受体超家族特征性结构域,与其他昆虫,尤其是鳞翅目昆虫的蜕皮激素接受子3的氨基酸序列高度同源。获得的基因cDNA序列已经登录GenBank并获得登录号,八字地老虎HR3登录号为GU188853,粘虫HR3登录号为GU188854。     

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.     

A Unique Protease Cleavage Site Predicted in the Spike Protein of the Novel Pneumonia Coronavirus (2019-nCoV) Potentially Related to Viral Transmissibility

正Dear Editor,In December 2019, a novel human coronavirus caused an epidemic of severe pneumonia(Coronavirus Disease 2019,COVID-19) in Wuhan, Hubei, China(Wu et al. 2020; Zhu et al. 2020). So far, this virus has spread to all areas of China and even to other countries. The epidemic has caused 67,102 confirmed infections with 1526 fatal cases     

Curcuminoids as inhibitors of thioredoxin reductase: A receptor based pharmacophore study with distance mapping of the active site

Curcumin is the yellow pigment of turmeric that interacts irreversibly forming an adduct with thioredoxin reductase (TrxR), an enzyme responsible for redox control of cell and defence against oxidative stress. Docking at both the active sites of TrxR was performed to compare the potency of three naturally occurring curcuminoids, namely curcumin, demethoxy curcumin and bis-demethoxy curcumin. Results show that active sites of TrxR occur at the junction of E and F chains. Volume and area of both cavities is predicted. It has been concluded by distance mapping of the most active conformations that Se atom of catalytic residue SeCYS498, is at a distance of 3.56 from C13 of demethoxy curcumin at the E chain active site, whereas C13 carbon atom forms adduct with Se atom of SeCys 498. We report that at least one methoxy group in curcuminoids is necessary for interation with catalytic residues of thioredoxin. Pharmacophore of both active sites of the TrxR receptor for curcumin and demethoxy curcumin molecules has been drawn and proposed for design and synthesis of most probable potent antiproliferative synthetic drugs.     

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.