重组工程系统研究进展

大肠杆菌的同源重组依靠内源性的RecA蛋白。RecBCD降解双链线性DNA分子,因此必须构建环状质粒打靶载体才能完成体内重组,操作过程繁琐,所需同源臂长。最近建立起的依赖于Rac噬菌体的ET重组系统和基于入噬菌体的Red重组系统,可有效利用线性DNA片段作为打靶分子,对大肠杆菌染色体DNA和BAC、PAC载体中包含的真核细胞基因组DNA进行基因敲除、敲入、替换、单碱基突变及体内基因克隆等修饰。这2种系统重组效率高,用PCR方法便可合成双链线性DNA打靶分子,不需要限制酶和连接酶,操作过程简单、精确、快速、经济,大大缩短了构建打靶载体的时间,成为功能基因组研究的有力工具。     

Red体内同源重组介导的egfp、kan基因融合和重组质粒构建

重组工程是近年来建立的一种基于高效率体内同源重组的新型遗传工程技术,可应用于靶DNA序列的敲入、敲除和基因克隆等。在应用重组工程技术进行基因亚克隆时发现,体外重叠PCR法难以获得高质量的目的DNA打靶片段,严重影响重组效率。为了解决上述问题,根据Red重组酶介导的体内同源重组工作原理进行了技术改进。先用PCR方法合成egfp和kan两条末端互补的线性DNA片段,然后将其电击共转化进入携带Red重组酶和pcDNA3．1载体DNA的大肠杆菌DY331菌株内,经体内同源重组直接产生的pcDNA3.1—egfp-kan环状重组质粒DNA分子可通过抗生素标记筛选获得,阳性率可达到45％。瞬时转染pcDNA3．1-egfp-kan可获得绿色荧光蛋白在293细胞中的表达。     

Red体内同源重组介导的egfp、kan基因融合和重组质粒构建

重组工程是近年来建立的一种基于高效率体内同源重组的新型遗传工程技术,可应用于靶DNA序列的敲入、敲除和基因克隆等。在应用重组工程技术进行基因亚克隆时发现,体外重叠PCR法难以获得高质量的目的DNA打靶片段,严重影响重组效率。为了解决上述问题,根据Red重组酶介导的体内同源重组工作原理进行了技术改进。先用PCR方法合成egfp和kan两条末端互补的线性DNA片段,然后将其电击共转化进入携带Red重组酶和pcDNA3.1载体DNA的大肠杆菌DY331菌株内,经体内同源重组直接产生的pcDNA3.1-egfp-kan环状重组质粒DNA分子可通过抗生素标记筛选获得,阳性率可达到45%,瞬时转染pcDNA3.1-egfp-kan可获得绿色荧光蛋白在293细胞中的表达。     

Red体内同源重组介导的egfp、kan基因融合和重组质粒构建

重组工程是近年来建立的一种基于高效率体内同源重组的新型遗传工程技术,可应用于靶DNA序列的敲入、敲除和基因克隆等。在应用重组工程技术进行基因亚克隆时发现,体外重叠PCR法难以获得高质量的目的DNA打靶片段,严重影响重组效率。为了解决上述问题,根据Red重组酶介导的体内同源重组工作原理进行了技术改进。先用PCR方法合成egfp和kan两条末端互补的线性DNA片段,然后将其电击共转化进入携带Red重组酶和pcDNA3.1载体DNA的大肠杆菌DY331菌株内,经体内同源重组直接产生的pcDNA3.1-egfp-kan环状重组质粒DNA分子可通过抗生素标记筛选获得,阳性率可达到45%,瞬时转染pcDNA3.1-egfp-kan可获得绿色荧光蛋白在293细胞中的表达。     

Gap-Repair方式建立一种基于pBR322-Red的新型重组工程系统

应用Gap—Repair新技术,以pBR322为载体,在λ噬菌体Red重组酶的作用下,通过同源重组直接从大肠杆菌DY330染色体上亚克隆了长度为6．7kb的包含Red重组酶基因的λ噬菌体左向操纵子基因序列。建立了一种能够随意在不同细菌宿主中转移的基于pBR322-Red的重组工程系统。为了验证pBR322-Red的生物功能,以大肠杆菌染色体上的galk基因为靶标,用Red介导的单链DNA重组技术敲入T→G单碱基突变,使galk基因内编码第145位氨基酸的密码子由TAT转变成TAG,产生了一个琥珀突变。确定了pBR322-Red系统的重组功能。     

噬菌体重组酶介导的DNA同源重组工程

来源于噬菌体的遗传操作工具在基因工程中具有非常重要的地位,例如位点特异性重组酶、柯斯质粒DNA文库及同源重组酶等。其中,来源于lambda噬菌体的同源重组酶Redα/Redβ和来源于Rac原噬菌体的同源重组酶RecE/RecT能够高效地介导35–50 bp短同源臂之间的重组。基于噬菌体同源重组酶Redα/Redβ和RecE/RecT开发的DNA同源重组工程（Recombineering）能够对靶标DNA分子进行快速、精准、高效的修饰,不受限制性内切酶识别位点和DNA分子大小限制,已发展成为一种新型的基因工程技术。本文主要综述了噬菌体同源重组酶及其作用机制、在大肠杆菌及其他细菌中的应用和开发,以及在微生物次级代谢产物的挖掘、动植物转基因、病毒基因组克隆和修饰等方面的应用。原位激活沉默基因簇需要宿主特异性的DNA同源重组工程进行启动子和调控元件的修饰;异源表达次级代谢产物的首要步骤一般是通过RecET直接克隆大的DNA片段;动植物转基因复杂载体的构建效率在有了Red同源重组系统以后有了革命性的发展;RecET直接克隆和Red同源重组介导的感染性克隆构建和修饰方法,不仅有利于病毒基因组功能研究,同时也为载体疫苗开发提供了最优方案。     

Red/ET同源重组技术及其在微生物基因组挖掘中的应用进展

Red/ET同源重组技术(Red/ET recombineering)是由来源于大肠杆菌λ噬菌体的蛋白对Redα/Redβ或来源于Rac原噬菌体的蛋白对Rec E/Rec T所介导的基于短同源臂(40–50 bp)的同源重组技术,能对宿主DNA序列进行快速、高效、精确的修饰和操作。本文主要综述了2010年以来Red/ET同源重组技术在大肠杆菌及其他细菌中的研究进展,同时简要介绍了该技术在微生物基因组挖掘,尤其是在微生物基因簇的异源表达领域的应用进展。     

分枝杆菌噬菌体重组系统及其应用

噬菌体是微生物遗传学研究的有力工具及源泉.分枝杆菌噬菌体也是构建分枝杆菌,尤其是结核分枝杆菌遗传研究工具的基础.目前,基于分枝杆菌噬菌体重组酶的重组系统是国际热点.总结了近年来基于分枝杆菌噬菌体Che9c重组酶gp60、gp61所构建的分枝杆菌重组工程体系及其在分枝杆菌基因组研究方面的应用,并结合实验室工作展望了其研究前景.该体系不依赖细菌自身的RecA系统,不需要限制性内切核酸酶和DNA连接酶,不需要复杂的体外操作,只需表达分枝杆菌噬菌体重组酶,从而使结核分枝杆菌基因敲除、基因敲入及点突变和构建分枝杆菌噬菌体突变株更方便.这为分枝杆菌及其噬菌体基因诱变及基因功能研究提供了迅捷的新途径.     

Red同源重组技术研究进展

伴随着分子生物学的发展,一种基于λ噬菌体Red重组酶的同源重组系统已应用于大肠杆菌基因工程研究。Red重组系统由三种蛋白组成:Exo蛋白是一种核酸外切酶,结合在双链DNA的末端,从5′端向3′端降解DNA,产生3′突出端;Beta蛋白结合在单链DNA上,介导互补单链DNA退火;Gam蛋白可与RecBCD酶结合,抑制其降解外源DNA的活性。Red同源重组技术具有同源序列短(40～60bp)、重组效率高的特点。这种技术可在DNA靶标分子的任意位点进行基因敲除、敲入、点突变等操作,无需使用限制性内切酶和连接酶。此外,这种新型重组技术可直接将目的基因克隆于载体上,目的基因既可来源于细菌人工染色体也可是基因组DNA。Red同源重组技术使难度较大的基因工程实验顺利进行,大大推动功能基因组研究的发展。     

微生物基因打靶系统的研究进展

基因打靶技术是微生物功能基因组学研究的有力工具之一,通过定向改变微生物的遗传信息可以对目的基因进行有效的功能分析。在大肠杆菌中研究较多的是转座子突变系统、RecBCD^-sbcB重组系统、RecA依赖的重组打靶系统、Chi位点刺激的重组、利用单链DNA进行的重组工程。在酵母中进行基因打靶的策略主要是转座子标记的突变、基于PCR方法的基因删除和转化相关重组。在其他微生物中主要应用转座子突变和自杀载体进行基因打靶。近年来,噬菌体重组系统的发展更使对微生物基因打靶系统的研究进入了新的阶段,主要包括Rac编码的RecET系统、Red重组系统和噬菌体退火蛋白介导的单链寡核苷酸重组系统。     

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.