泡桐丛枝和枣疯病植原体tuf基因上游序列结构、功能和遗传变异比较分析

【目的】探究泡桐丛枝和枣疯病植原体tuf基因上游序列结构、功能差异及其遗传多样性。【方法】利用热不对称交错式PCR(TAIL-PCR)扩增枣疯病植原体tuf基因上游未知序列,利用启动子探针载体pSUPV4构建了泡桐丛枝和枣疯病植原体tuf基因上游序列的大肠杆菌异源表达体系,分析泡桐丛枝、苦楝丛枝、莴苣黄化、桑萎缩、长春花绿变等16SrI组和枣疯病、樱桃致死黄化、重阳木丛枝等16SrV组株系tuf基因上游调控序列的遗传变异特征和启动子活性。【结果】泡桐丛枝等16SrI组植原体株系tuf基因和其上游fus A基因之间的间区序列长129-130 bp,预测有完整的启动子保守结构。泡桐丛枝植原体tuf基因上游130 bp片段具有启动子活性,此间区序列在5种35株16SrI组株系中存在4种变异类型;枣疯病植原体等16SrV组株系fusA和tuf基因间区长53-54 bp,未预测到完整启动子结构。枣疯病植原体tuf基因上游144 bp和346 bp片段均未检测到启动子活性,fus A和tuf基因间区序列在3种20株16SrV组株系中存在2种变异类型。fus A-tuf基因间区序列相对保守,基于此序列构建的进化树可清晰区分不同组别的植原体株系。【结论】研究方法和结果为深入研究植原体基因表达与调控、揭示植原体生长繁殖规律及其致病机理等奠定了良好的基础。     

苦楝丛枝植原体质粒的测定与分子特征

摘要: 【目的】测定苦楝丛枝植原体(CWB 植原体)质粒并分析其分子特征。【方法】扩增苦楝丛枝植原体质粒片段并进行系统进化分析,预测质粒编码蛋白的跨膜区、亚细胞定位;以质粒pCWBFq repA为模板制备探针,利用Southern blot 方法检测苦楝丛枝植原体及其他几种植原体的质粒。【结果】测定了苦楝丛枝植原体福清株系的一个质粒pCWBFq,该质粒全长4446 bp,A + T含量为73.5%,编码6个蛋白,其中pCWBFq P2-P5 五个编码蛋白分别含有3、2、1 和2个跨膜区,其信号肽(Singnal Peptide,SP)信号值分别为0.989、0.505、0.918和0.914。氨基酸序列相似性比较表明: pCWBFq RepA 蛋白与其他植原体质粒RepA 的同源性在9.6%－85.6%之间,而pCWBFq SSB 蛋白与其他植原体质粒SSB 的同源性在74.0%－89.4%之间。用pCWBFq repA 探针检测到苦楝丛枝植原体中质粒的存在,同时也能够检测到16SrI组的泡桐丛枝植原体(PaWBNy),海南长春花绿变植原体(PeVHn),苦楝丛枝植原体福州株系(CWBFz)和桑树萎缩植原体濮阳株系(MDPy)中的质粒,但16SrV 组的枣疯植原体北京株系(JWBBj)、樱桃致死黄化西昌株系(CLYXc)和重阳 木丛枝南昌株系(BiWBNc) 中未能检测到任何杂交信号。【结论】苦楝丛枝植原体质粒pCWBFq编码的6个蛋白中,除与质粒复制有关的RepA和SSB外,另外4个均为含有疏水结构的分泌蛋白或膜蛋白。植原体质粒的同源基因间存在程度不同的变异,其中repA 基因在所有植原体质粒上均存在,但变异性相对较大,而ssb基因仅存在于16SrI 组植原体质粒中,且变异相对较小。16SrI 组的CWBFq、PaWBNy、PeVHn、CWBFz和MDPy 中均存在数量和大小不同的质粒,而16SrV 组的JWBBj、CLYXc和BiWBNc 中或含有的质粒因与pCWBFq repA 探针的同源性较低而不能被检测到。     

泡桐丛枝植原体16S rDNA和延伸因子基因序列分析

对采自陕西、山西、甘肃、河南、河北、山东各省的泡桐丛枝病材料,利用巢式扩增,均得到16S rDNA基因片段约1.2kb;扩增得到植原体延伸因子(EF-Tu)tuf基因,长度约为850bp.。通过将16S rDNA基因片段和延伸因子(EF-Tu)tuf基因序列与已知植原体16Sr各组成员进行同源性比较,确定我国大陆泡桐主栽区陕西、山西、甘肃、河南、河北、山东各省与已经报道的台湾省泡桐丛枝植原体基本一致,均为同一个种,没有株系的分化,全部归属于植原体16SrI-D组,从而确定了其分类地位。     

新疆小叶白蜡丛枝病植原体的鉴定及16S rRNA基因序列分析

【目的】对新疆小叶白蜡丛枝病植原体进行检测,通过其16S rRNA基因分析确定其分类地位。【方法】利用苯胺蓝和4′,6-二脒基-2-苯基吲哚(DAPI)染色,在荧光显微镜下观察新疆小叶白蜡嫩茎横切片;采用植原体16S rRNA基因的通用引物对P1/P7和R16F2n/R16R2进行直接和巢式PCR扩增,对得到的16S rRNA基因的序列进行RFLP和构建系统进化树分析。【结果】表现丛枝病症状的新疆小叶白蜡中存在植原体,暂命名为Fraxinus sogdianaBunge witches’broom phytoplasma(Fraxinus sogdianaBunge WB);其16S rRNA基因的序列GenBank登录号为KF061042,RFLP图谱与16Sr V-B亚组的枣疯病植原体相同,系统进化地位与枣疯病菌株AB052876相同。【结论】新疆小叶白蜡丛枝病植原体为16Sr V-B亚组成员。     

花生丛枝植原体免疫膜蛋白基因克隆及细菌双杂交诱饵质粒构建

初步分析植原体免疫膜蛋白的结构,以Imp构建诱饵质粒,为研究植原体与寄主互作的分子机理,探讨植原体传播、侵染及在寄主内的运输方式奠定基础。根据本实验室获得的花生丛枝植原体免疫膜蛋白基因序列设计特异性引物Imp-F/Imp-R,通过PCR扩增获得花生丛枝植原体免疫膜蛋白基因,大小519 bp,编码蛋白含有172个氨基酸残基,与SPWB膜蛋白的核苷酸差异一个碱基,氨基酸序列相同。另外与WBDL膜蛋白的核苷酸和氨基酸序列同源性分别为79.8%和70.2%。对其进行系统发育树分析;初步分析imp基因编码蛋白的跨膜区和疏水区。分析结果表明：花生丛枝植原体免疫膜蛋白C端有一跨膜锚定区,N端主要为膜内亲水区,没有前导信号序列。预测花生丛枝植原体免疫膜蛋白是一类C端跨膜的植原体免疫膜蛋白。将imp基因克隆到带有λcI基因的pBT质粒上,构建诱饵载体pBT-Imp,并通过IPTG诱导表达,western blot检测和自激活检验对其进行检测。结果显示：所构建的诱饵载体pBT-Imp可用于细菌双杂交的进一步实验。     

杏褪绿卷叶植原体新疆分离物核糖体蛋白基因的克隆与序列分析

【目的】了解杏褪绿卷叶植原体新疆分离物的系统发育关系及遗传分化,确定其分类地位。【方法】利用植原体核糖体蛋白(rp)基因的特异性引物rpF1/rpR1对新疆轮台县托克逊县杏褪绿卷叶病植株总DNA进行PCR扩增,并对部分扩增片段克隆、测序及序列分析。【结果】获得杏褪绿卷叶植原体新疆分离物rp基因片段大小为1196 bp,该片段包含部分rpS19以及rpL22和rpS3基因的全部序列。序列相似性和系统进化分析表明,杏褪绿卷叶植原体新疆分离物与16SrⅤ-rp亚组中的各代表性植原体的rp基因核苷酸序列相似性达到95.7%~99.3%,其中与rpⅤ-C亚组的甜樱桃绿化植原体和枣疯病植原体的相似性最高,核苷酸及氨基酸相似性分别达到99.2%~99.3%和98.3%~98.4%。进一步虚拟RFLP分析,发现杏褪绿卷叶植原新疆分离物rp基因的酶切图谱与rpⅤ-C亚组成员相似性最高,但在MseⅠ、SspⅠ和TaqⅠ的酶切位点上存在差异。综上初步判断其可能属于16SrⅤ组(榆树黄化组)中的一个新rp亚组。【结论】本研究首次报道了杏褪绿卷叶植原体新疆分离物的rp基因序列,确定了其分类地位,为杏褪绿卷叶病的早期诊断和检测提供了基础。     

蜜蜂螺原体细胞骨架相关基因mreB的克隆表达及在螺原体二种形态时的表达差异

【目的】在大肠杆菌中克隆表达蜜蜂螺原体细胞骨架相关基因mreB1?5,并预测所编码蛋白的理化性质,分析这些基因在螺原体螺旋状和非螺旋状时的表达水平,为进一步分析该基因的功能奠定基础。【方法】通过PCR扩增,从Spiroplasma melliferum CH-1基因组中获得mreB1?5基因,构建的重组表达载体pETmreB1?5分别在大肠杆菌BL21中诱导表达,利用镍亲和树脂纯化重组蛋白,通过在线工具预测MreB蛋白质的理化性质和功能域。利用Real-Time PCR比较螺原体CH-1在两种不同形态时mreB1?5基因的表达量。【结果】成功克隆到5个mreB基因,并在大肠杆菌BL21中高效表达。MreB蛋白分子量分别为36、23、23、37和25 kD,可能均为疏水性的蛋白,属于MreB/Mbl蛋白质家族。荧光定量PCR结果显示,螺原体在非螺旋状时mreB1?5基因的表达水平均远低于在螺旋状时基因的表达水平。【结论】本文第一次克隆表达了螺原体细胞骨架相关基因mreB1?5,初步表明这些基因在螺原体形态方面可能具有重要作用,为后续研究螺原体mreB基因在其运动和形态方面的功能提供了重要信息。     

长春花黄化植原体核糖体蛋白基因片段序列分析*

对自然表现典型黄化症的长春花植株总RNA进行植原体核糖体蛋白基因（ribosomal protein gene,rp gene）PCR扩增,得到约1.3kb的特异片段。将此特异片段与pGEM-T Easy载体连接并转化到大肠杆菌JM109感受态细胞中,通过PCR鉴定、限制性内切酶(EcoRI)酶切分析、核苷酸序列测定及分析,结果表明该株系核糖体蛋白基因片段长1,44bp,包含rp122、rps3基因,分别编码129和252个氨基酸,且这两个基因为重叠基因。该植原体核糖体蛋白基因特性与其它植原体相似。     

红球菌R04苯甲酸转运相关膜蛋白RHOGL009301的生理功能

【目的】探究红球菌(Rhodococcus sp.)R04膜蛋白RHOGL009301的生理功能和突变菌株的代谢特性,确定该膜蛋白的生理功能与苯甲酸转运的关系。【方法】将RHOGL009301基因与绿色荧光蛋白基因在Rhodococcus erythropolis进行融合表达,Delta Vision观察该基因蛋白产物的定位。通过基因同源重组敲除RHOGL009301基因,并对比野生型菌株和缺陷型菌株在不同碳源培养下的生长情况。HPLC测定红球菌R04野生型菌株和缺陷型菌株代谢联苯和苯甲酸时细胞内外代谢物,分析不同生长条件下代谢物的浓度变化。【结果】RHOGL009301基因与绿色荧光蛋白基因在Rhodococcus erythropolis中实现共表达,并定位在细胞膜上。获得了RHOGL009301基因的缺陷型菌株R04ΔMP,与野生型菌株相比,缺陷型菌株在联苯和苯甲酸培养条件下的生物量明显降低,生长速度减慢。HPLC分析表明RHOGL009301基因的缺失抑制了苯甲酸的转运。【结论】膜蛋白RHOGL009301是苯甲酸代谢和转运相关的蛋白,基于序列同源性分析,该膜蛋白是一种新型的苯甲酸转运蛋白。     

中国各地不同枣树品种上枣疯病植原体的PCR检测及分子变异分析

摘要：【目的】检测不同地区枣树品种上的枣疯植原体侵染及保守基因序列的变异。【方法】利用植原体16S rDNA的通用引物R16mF2／R16mR1、16S-23S间区序列(SR)的通用引物SR1/SR及secY基因引物FD9f/r,通过PCR检测采自国内7个地区14个枣树品种上的32个枣疯病和4个酸枣丛枝病样品。将PCR产物进行直接或克隆测序,结合已报导的测序数据,进行序列同源性和系统进化分析。【结果】所有枣疯病样品中均检测到植原体;皆属于榆树黄化16S rV-B亚组,与我国重阳木丛枝和樱桃致死黄化遗传关系     

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.