基因克隆及组装技术的研究进展

随着测序技术的发展,已知的DNA序列数量呈指数性增加,为了能更快的探索其未知的生物功能,一些简化组装流程的DNA克隆及组装新技术争相发展起来。其中大部分需要在菌体外构建重组体,但重组酶纯化过程复杂,运送和保存方法要求严格,致使成本较高。最近研究者开发了一些在菌体内进行DNA组装的简易、低成本的新方法。主要对各类基因克隆及组装方法的研究现状、原理和优缺点等进行综述,并结合实际的工作内容展望了未来的发展趋势,希望能为进一步研究开发新技术提供参考。     

DNA组装技术

DNA组装是合成生物学研究的核心技术。随着合成生物学的发展,研究者开发了依赖于DNA聚合酶或DNA连接酶的不同DNA组装技术;为了降低组装成本和便于实现DNA组装的自动化,也发展了一些非酶依赖的DNA组装技术;而几百kb到Mb的大片段DNA的组装则多数依赖于微生物体内重组。文中主要综述了酶依赖、非酶依赖和体内同源重组三类DNA组装技术及其发展情况。     

用于通路(Gateway)克隆技术的植物表达载体研究进展

通路(Gateway)克隆技术是根据λ噬菌体基因组和大肠杆菌基因组之间的位点专一性重组分子机制开发的一套分子克隆新技术.利用该技术LR反应构建目的基因的表达载体时不需要经过酶切和连接等繁琐而又费时的过程,因此,可以节省很多时间.为了扩大Gateway技术在植物基因工程领域的应用,最近有很多研究机构和研究小组开发了能用于组成型或诱导型表达目的基因、基因沉默、启动子分析、蛋白质亚细胞定位、蛋白质/蛋白质相互作用、多个DNA片段的模块化组装和DNA组片段功能验证等研究用的植物表达载体.该文对这些技术的研究进展进行了综述.     

DNA重组技术的研究综述

DNA重组技术,即DNA克隆技术的研究和运用是现代生物学发展的一个重要分支,是分子生物学发展的突出领域。本文介绍了DNA重组的类型及相关的生物学概念;综述了目前已报道的传统的酶切-连接经典克隆方法、位点特异性重组克隆方法、以及同源重组克隆方法,重点阐述了各自的原理、步骤、特点及实际应用等方面;最后归纳总结了各种方法的优缺点和应用范围,并对该技术的科研成果进行了回顾和对未来的研究进行了展望。     

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。     

DNA克隆和组装技术研究进展

DNA克隆和组装技术是重要的分子生物学工具。近年来,随着合成生物学的飞速发展,对大片段DNA元件的快速有效组装就显得尤为关键。同时,各种DNA克隆和组装技术也竞相发展起来。通过对基于非典型酶切连接、PCR、同源重组、单链退火拼接等原理发展起来的各种DNA克隆和组装技术进行综述,为合成生物学的进一步发展提供有效的操作工具。     

一种简便快速检测质粒DNA的方法

简便、快速检测质粒DNA的方法,无论是 在筛选、鉴定新质粒或重组质粒的研究中都具有重要的意义。我们在将质粒pBR 322 DNA与 A DNA进行体外重组、筛选和鉴定重组子过程 中,摸索了一种简便快速检测质粒及重组质粒 DNA的方法。此法对菌体和溶菌产物不需任 何处理,比较简便易行     

体内直接克隆大片段DNA的研究进展

基因组序列的功能分析以及代谢途径的构建改造等都需要克隆目的DNA。获得大片段DNA序列的方法有构建和筛选基因文库,PCR扩增,体外大片段DNA合成和组装等,但体内重组直接克隆的方法在操作、克隆长片段和应用等方面更具优势。介绍了Red/ET重组介导的大片段DNA体内直接克隆的主要方法及其应用。     

DNA重组技术生产人胰岛素

人胰岛素是用DNA重组技术生产的第一个药物。这个产品的研究始于管理大规模DNA重组的联邦条例制订或DNA重组技术产品的商业开发之前。本文叙述了为争取大规模进行生产的许可与保证重组DNA产品的鉴定及安全所采取的措施。DNA重组技术的基础研究将继续在生命科学研究中发生巨大的影响,而在它在商业上的应用将取决于经济状况与投入的资本的效益。     

大肠杆菌重组工程

源于噬菌体的大肠杆菌同源重组系统不需要限制性内切酶和DNA连接酶就可以进行DNA克隆和亚克隆,还能快速地改造质粒、细菌人工染色体及细菌基因组染色体,是基因工程技术的一大突破,被称为重组基因工程或重组工程。该技术操作简单,效率较高,可望为功能基因组学研究提供一个有力的工具。     

Stable integration and expression of mosquito-larvicidal genes from Bacillus thuringiensis subsp. israelensis and Bacillus sphaericus into the chromosome of Enterobacter amnigenus: a potential breakthrough in mosquito biocontrol

Previously, we have successfully integrated a spectinomycin/streptomycin resistance gene into Enterobacter amnigenus strain An11, a potential host for mosquito control, using in vivo recombination via homologous recombination (An11S4::Omega). We now report the successful transfer of two mosquito-larvicidal genes, cry4B from Bacillus thuringiensis subsp. israelensis and binary toxin genes from Bacillus sphaericus, into the host genome. To facilitate the screening procedure, the E. amnigenus derivative, An11S4::Omega, was used as a host. The integration of both toxin genes by two successive crossover events interrupted the Omega region yielding two integrants designated An11S4::cry4B and An11S4::Omega::bin, respectively. Differences in the integration efficiency of these toxin genes were observed. The presence of both genes in the target sites of the host genome was verified by PCR. Cry4B was expressed weakly from An11S4::cry4B, but no expression of the binary toxin gene could be detected from An11S4::Omega::bin. Nevertheless, these two integrants exhibited mosquito-larvicidal activity against Aedes and Culex, suggesting that both proteins were expressed, but at very low levels.     

一种快速、精确构建大肠杆菌组氨酸营养缺陷型的方法

将表达Red体内重组蛋白的质粒pKD46转化大肠杆菌：DH5α,用5′端与组氨酸基因同源,3′端与卡那霉素抗性基因同源的引物获得具有卡那霉素抗性基因的PCR产物,然后电击转化DH5α,在λRed重组系统的帮助下,通过卡那霉素抗性基因两侧的组氨酸基因序列在体内与大肠杆菌染色体上的组氨酸基因发生同源重组,置换了DH5α组氨酸操纵元中的hisDCB基因,最后利用卡那霉素抗性基因两端的FRT位点,通过FTP位点专一性重组将卡那霉素抗性基因去除,最终获得了不具抗性的大肠杆菌组氨酸营养缺陷型菌株。为在大肠杆菌及其他菌株中快速、精确的构建营养缺陷型菌株提供了有益的参考。     

Characterization of in vivo recombination activities in the mouse embryo

Homologous recombination makes use of sequence homology to repair DNA and to rearrange genetic material. In mammals, these processes have mainly been characterized using cultured cell systems. We have developed an assay that allows us to quantitatively analyze homologous recombination in vivo in the mouse embryo. Transgenic mouse lines were generated by microinjection into a fertilized mouse ovum of a vector containing two homologous LINE-1 (L1) sequences arranged as a direct repeat: these sequences can recombine with each other and with endogenous L1 sequences before, during or after integration of the vector into the genome. Using a plasmid rescue procedure, we determined the composition of the integrated vector array in several transgenic mice and their descendants. Homologous recombination frequencies were found to be strikingly high, involving 70% of integrated vectors in some arrays, with homologous deletions being five times more frequent than gene conversion without crossing-over. Interestingly, non-homologous recombination was found to be much less frequent. We also found that endogenous L1 sequences could be involved in homologous recombination events in the mouse embryo, and that the integrated arrays could be modified from generation to generation by homologous recombination between the integrated L1 sequences.     

Consequences of unique double-stranded breaks in yeast chromosomes: death or homozygosis

We have developed a system in which a unique double-stranded break (DSB) can be introduced into a yeast chromosome during mitotic growth. The recognition site for the endonuclease I-SceI was inserted at different places in the yeast genome in haploid and diploid cells expressing this endonuclease. Induction of the break in haploids results in cell death if no intact copy of the cleaved region is present in the cell. If such a copy is provided on a plasmid, as an ectopic gene duplication, or on a homologous chromosome, the break can be repaired. Repair results in two identical copies in the genome of the locus which has been cut. We call this phenomenon homozygotization by reference to diploids heterozygous for the cut site in which repair leads to homozygosis at this site. We have compared the efficiencies of repair in the various topological situations examined, and conclude that some mechanism must search for regions of homology to both sides of the DSB and that repair is successful only if the homologies are provided by the same template molecule.     

Construction of a series of ompC-ompF chimeric genes by in vivo homologous recombination in Escherichia coli and characterization of their translational products

Summary OmpC and OmpF are major outer membrane proteins and although they are homologous proteins, they function differently in several respects. As an approach to elucidate the submolecular structures that determine their differences, we have constructed a series of ompC-ompF chimeric genes by in vivo homologous recombination between these two genes, which are adjacent on a plasmid. The recombination sites in the chimeric genes were localized by means of restriction endonuclease analysis and nucleotide sequence determination. Most of the chimeric gene products were accumulated in the outer membrane. One of the chimeric gene products, with a fusion site in a central region between the OmpC and OmpF proteins, was normally expressed but not accumulated in the outer membrane. The trimeric structures of some of the chimeric gene products appeared to be extremely unstable in a SDS solution. From these results, domains contributing to the formation of specific structures in which the OmpC and OmpF proteins differ were identified. Bacterial cells possessing the chimeric gene products were also investigated as to their sensitivity to phages that require either OmpC or OmpF as a receptor component. With the aid of the chimeric gene products, the immunogenic determinants for three anti-OmpC monoclonal antibodies were found to be localized at different portions of the OmpC polypeptide: the N-terminal, central and C-terminal portions, respectively.     

Both conserved and non-conserved regions of Spo11 are essential for meiotic recombination initiation in yeast

DNA double-strand breaks (DSBs) are the initiators of most meiotic recombination events. In Saccharomyces cerevisiae, at least ten genes are necessary for meiotic DSB formation. However, the molecular roles of these proteins are not clearly understood. The meiosis-specific Spo11 protein, which shows sequence similarity with a subunit of an archaeal topoisomerase, is believed to catalyze the meiotic DSB formation. Spo11 is also required for induction of meiotic DSBs at long inverted repeats and at large trinucleotide repeat tracts. Here we report the isolation and characterization of temperature-sensitive spo11-mutant alleles to better understand how Spo11 functions, and how meiotic DSBs are generated at various recombination hotspots. Analysis of mutation sites of isolated spo11-mutant alleles indicated that both N-terminal and C-terminal non-conserved residues of Spo11 are essential for the protein’s function, possibly for interaction with other meiotic DSB enzymes. Several of the mutation sites within the conserved region are predicted to lie on the surface of the protein, suggesting that this region is required for activation of the meiotic initiation complex via protein-protein interaction. In addition to the conditional mutants, we isolated partially recombination-defective mutants; analysis of one of these mutants indicated that Ski8, as observed previously, interacts with Spo11 via the latter’s C-terminal residues.     

A new approach to directed gene evolution by recombined extension on truncated templates (RETT)

We describe a new approach to in vitro DNA recombination technique termed recombined extension on truncated templates (RETT). RETT generates random recombinant gene library by template-switching of unidirectionally growing polynucleotides from primers in the presence of unidirectional single-stranded DNA fragments used as templates. RETT was applied to the recombination of two homologous chitinase genes from S. marcescens ATCC 21074 and S. liquefaciens GM1403. When the shuffled genes were examined by restriction mapping and sequence analysis, it was found that chimeric genes were produced at a high frequency (more than 70%) between two chitinase genes with 83% of sequence identity. The number of crossovers within each chimeric gene ranged from one to four, and the recombination points were randomly distributed along entire DNA sequence. We also applied RETT to directed evolution of a chitinase variant for enhancing thermostability. Chimeric chitinases that were more thermostable than the parental enzyme were successfully obtained by RETT-based recombination.     

Physical mapping of translocation breakpoints in a set of wheat-Aegilops umbellulata recombinant lines using in situ hybridization

Aegilops umbellulata Zhuk. carries genes at Glu-U1 loci that code for a pair of high-molecular-weight glutenin subunits not found in common wheat, Triticum aestivum. Wheat-Ae. umbellulata recombinant lines were produced with the aim of transferring genes coding for glutenin subunits from Ae. umbellulata into wheat with minimal flanking material. We used fluorescent genomic in situ hybridization to evaluate the extent of recombination and to map physically the translocation breakpoints on 11 wheat-Ae. umbellulata recombinant lines. In situ hybridization was able to identify alien material in wheat and showed breakpoints not only near the centromeres but also along chromosome arms. To characterize and identify chromosomes further, including deletions along the 1U chromosome, we used simultaneous multiple target in situ hybridization to localize a tandemly repeated DNA sequence (pSc119.2) and the 18S–25S and 5S rRNA genes. One line contained an Ae. umbellulata telocentric chromosome and another two had different terminal deletions, mostly with some wheat chromosome rearrangements. Although from six independent original crosses, the other eight lines included only two types of intercalary wheat-Ae. umbellulata recombination events. Five occurred at the 5S rRNA genes on the short arm of the Ae. umbellulata chromosome with a distal wheat-origin segment, and three breakpoints were proximal to the centromere in the long arm, so most of the long arm was of Ae. umbellulata origin. The results allow characterization of recombination events in the context of the karyotype. They also facilitate the design of crossing programmes to generate lines where smaller Ae. umbellulata chromosome segments are transferred to wheat with the potential to improve bread-making quality by incorporating novel glutenin subunits without undesirable linked genes.     

噬菌体重组酶系统在合成生物学中的应用*

合成生物学技术采用工程化设计理念,对生物体进行有目标的设计、改造乃至重新合成,对重塑非自然功能的“人造生命”具有重要意义。噬菌体重组系统具有高效、精确和广谱适用性等特点,在基因工程、代谢工程以及生物治疗等合成生物学领域得到了广泛的应用。从基因电路、体内遗传改造和体外重组等方面全面阐述了噬菌体重组系统在合成生物学研究的现状及热点,对当前该系统的局限性进行了探讨,并就未来的研究和发展趋势进行了展望。