大肠杆菌基因无痕敲除技术及策略

基因敲除技术是大肠杆菌基因组减小和代谢途径改造的有效手段,其中基于同源重组原理的基因无痕敲除技术显现出其他技术所不具备的应用优势和发展潜力。该技术可以快速敲除大肠杆菌基因组中的目标基因,并且在基因组中不残留任何外源片段,所以不会干扰后续的基因操作。我们分类介绍了无痕敲除技术中所涉及载体的结构、功能及其相应的敲除策略,着重介绍了无痕敲除技术的原理及载体构建方法。     

基因组编辑:植物生物技术的机遇与挑战

基于序列特异性核酸酶的基因组编辑技术可以在不同物种中对目标基因进行定点敲除,并可实现特定基因片段置换,基因的定点插入等基因组靶向修饰。基因组编辑是一种精准和高效的基因工程方法,近年来快速发展并得到了广泛的应用,并将改变生物技术的现状。目前,基因组编辑在不同植物,特别是农作物中的技术体系已建立,初步展示了其在植物生物技术领域的巨大潜力。介绍了不同基因组编辑系统的工作原理,并对基因组编辑技术在植物研究中的应用及成功案例进行了综述,最后对基因组编辑在植物生物技术领域所面临的机遇与挑战进行了讨论。     

基因组编辑:植物生物技术的机遇与挑战北大核心CSCD

基于序列特异性核酸酶的基因组编辑技术可以在不同物种中对目标基因进行定点敲除,并可实现特定基因片段置换,基因的定点插入等基因组靶向修饰。基因组编辑是一种精准和高效的基因工程方法,近年来快速发展并得到了广泛的应用,并将改变生物技术的现状。目前,基因组编辑在不同植物,特别是农作物中的技术体系已建立,初步展示了其在植物生物技术领域的巨大潜力。介绍了不同基因组编辑系统的工作原理,并对基因组编辑技术在植物研究中的应用及成功案例进行了综述,最后对基因组编辑在植物生物技术领域所面临的机遇与挑战进行了讨论。     

CRISPR/Cas9编辑棉花精氨酸酶基因促进侧根形成和发育

基因敲除是植物功能基因组研究和农艺性状定向遗传改良的关键步骤,近年来,CRISPR/Cas9介导的目标基因敲除在拟南芥(Arabidopsis thaliana)、水稻(Oryza sativa)等植物中取得飞速的发展,然而在棉花(Gossypium spp.)中目前尚没有成功的报道.陆地棉(G.hirsutum L.)是典型的异源四倍体模式作物,包括A亚基因组和D亚基因组.利用CRISPR/Cas9技术同时成功地敲除A和D亚基因组精氨酸酶编码基因(GhARG),Gh_A05G2143和Gh_D05G2397.无论高氮还是低氮条件下,T1代双基因纯和株系侧根生长发育显著地提高.因此,本研究的CRISPR/Cas9系统可以同时高效地编辑棉花中多个同源基因,从而实现关键农艺性状简单快速得改良.     

CRISPR⁃Cas9技术在植物次生代谢物生产中的研究进展

基因编辑（gene editing）技术可以对目的基因进行定点插入、敲除和置换。基于CRISPR-Cas9的基因编辑技术是继锌指核酸酶和转录激活样效应物核酸酶之后的第3代基因编辑技术。近年来,CRISPR-Cas9系统作为研究的热点被广泛应用于医学、药学、植物学、动物学和微生物学等领域,但其在植物次生代谢物领域的应用还处于探索时期。阐述了基于CRISPR-Cas9基因编辑技术的发展历程、工作原理和几种常用的基因编辑方法及其应用实例,总结了CRISPR-Cas9技术在对植物次生代谢产物研究方面的应用。利用CRISPR-Cas9系统可对植物基因组进行定点敲除、突变和插入,以达到提高植物次生代谢物含量、改良作物品质和提高植物抗性等目的。该技术已在植物次生代谢物生物合成关键酶基因的编辑等方面显示出越来越重要的作用。     

基因组信息的整合与植物功能基因组学研究的策略

近年来,随着许多植物基因组测序和可利用序列的增加,相继建立了一些基于靶基因诱变的“反向”遗传学研究策略,如T—DNA诱变、基因敲除、基因沉默和超表达分析等。同时,DNA微阵列和基因芯片技术的发展使得快速、定量检测植物发育不同时期和不同组织器官的基因转录时空变化成为现实。作图技术的改进和来自不同物种基因组信息的整合也正在加速图谱克隆程序的简化和发展。因此,随着生物基因组测序工作日益增多,整合不同类群植物基因组的信息和资源,在植物功能基因组学研究中的重要性日趋显著。     

多元自动化基因组工程

基因组编辑技术在基因组工程研究中应用广泛,其中位点特异性核酸酶编辑技术和CRISPR/Cas系统在单基因编辑方面贡献卓越,但由于基因组的庞大,这些技术又有一定的局限性。多元自动化基因组工程(MAGE)是一种新型基因组编辑技术,可同时作用于多个基因,具有快速、高效的特点,已被用于大肠杆菌的基因敲除和基因替换。主要介绍了MAGE的原理、具体操作流程及技术进展,并结合MAGE技术的应用,讨论其发展趋势。     

Latour system精简酿酒酵母染色体方法的建立及应用

酿酒酵母是第一个完成全基因组测序的真核生物,具有广泛的科研应用价值。利用酿酒酵母的全基因组序列可以进行精确的基因定位及敲除,从而达到对其基因组进行精简的目的,为合成生物学最小基因组的研究工作打下基础。根据Latour system 设计敲除所需引物,构建敲除盒,筛选重组体和缺失体,成功敲除酿酒酵母a型单倍体染色体XIII中339301-352281 nt包含的8个基因,为酿酒酵母染色体精简奠定基础,同时证明了Latour system 可以应用于酿酒酵母大片段敲除。     

基因敲除技术在微生物中的应用

随着分子生物学技术的发展,基因敲除技术越来越广泛地应用于动植物、微生物领域,成为研究生物基因功能最有力的工具之一。基因敲除技术在改造动植物、微生物基因组、研究发育生物学、鉴定新基因新功能、育种以及医疗领域都有应用价值。针对微生物方面,对实现基因敲除的各种原理方法,RecA系统同源重组法, Red系统同源重组法,基于自杀载体的同源重组法,基于温敏型质粒的同源重组法, CRISPR/Cas系统介导的基因敲除方法进行了总结,比较各自的优缺点,并提供一些成功案例以及各种方法相关的发明专利,以期对了解基因敲除技术的方法与发展提供参考。     

基因网络中基因关系图谱的构建及其应用

尽快破解基因组所包含基因的功能是一项费力但又很重要的工作。一个基因功能的实现依赖于该基因与其它基因间的相互作用。基因网络是一组基因的集合体,这些基因通过相互协作来控制生物体重要的生命过程。通过基因敲除、RNA干扰或其它方法改变基因网络中某个基因的表达水平,将会引起该网络中其它基因表达水平的变化。而这种变化可以方便地通过基因表达差异显示技术检测相应mRNA含量变化来反映。因此,将这两类方法组合在一起,可以在基因组水平上有效地检测出基因网络中的基因关系。这种策略对基因功能研究方法是一个重要补充。     

CRISPR/Cas9系统在培育抗病毒植物新种质中的应用

随着CRISPR/Cas9系统在基因组编辑技术上的开发和完善,CRISPR/Cas9系统在应用于动物病毒感染性疾病防治并取得相当成效的同时,也逐步被应用到对植物病毒基因组进行高效靶向修饰的研究中。CRISPR/Cas9系统对基因组靶向修饰作用不仅实现了对植物DNA病毒基因组序列的编辑,还展示了其有效作用于植物RNA病毒基因组的潜力,同时CRISPR/Cas9系统还能在基因转录和转录后调控水平发挥作用,说明该系统具有通过多种途径调控植物病毒复制的潜能。相对其他植物病毒病防治策略,该系统对病毒基因组的编辑更精准、对基因表达的调控更稳定,对病毒病的抗性也更为广谱。本文将CRISPR/Cas9系统与其他植物病毒病防治策略进行了比较,概述了该系统在培育植物抗病毒病新种质中的优势,分析了其具体应用在该领域中面临的主要问题,讨论了该系统在培育抗病毒植物新种质应用中的发展趋势。     

Gene expression during the male gametophytic phase

Abstract

In recent years a number of experimental findings have indicated that in higher plants the gametophytic phase is able to express its own genetic information, a large part of which it shares with the sporophytic generation. Quantitative estimates of haploid and haplodiploid gene expression have been obtained by mRNA and isozyme analysis in several plant species: 60-70% of the genes are expressed in both pollen and plant, about 10% are pollen-specific, and 20% represent the sporophytic domain. Moreover, it has been demonstrated that stage-specific genes are expressed in the gametophytic generation: at least two sets of genes are activated during pollen development, others are expressed only in the postshedding period, during germination and tube growth. Studies have been made to ascertain the role played by gametophyte-expressed genes in pollen development; the in vivo and in vitro pollen tube growth rate has been revealed to be controlled by the gametophyte genome itself. Differential effects of specific chromosomal deficiencies on the development of maize pollen grains have indicated that components of normal microspore development are controlled by genes located in specific parts of the genome. For single gene analysis, gene transfer can be used; on the contrary, for traits with a multifactorial genetic control, direct proof of gene expression both in the gametophytic and the sporophytic generation can be obtained when selection is applied to the pollen population of a hybrid plant, and response to selection is observed in the resulting sporophytic progeny. Response to selection, applied at different stages of the gametophytic phase, has been described in the sporophytic progeny and this with regard to many adaptive traits; thus the phenomenon can have an important bearing on the genetic structure of natural populations and on higher plant evolution, it can also be used as a breeding tool to increase the efficiency of conventional selection methods.     

The role of plastids and assimilate transport system in the control of plant development

Phylogenetic and ontogenetic relationships between the plastids, cell endoplasmic reticulum, and plant transport communication have been reviewed. The initiating role of plastids (endosymbionts) in the origin of endoplasmic reticulum (buffer zone of endosymbiogenesis) has been shown, as well as a similar role of endoplasmic reticulum in the development of transport communication of xylem and phloem. Plastids, sugars and transport system for their distribution can be interpreted as leading sections in the mechanism of developmental control: gene expression of nuclear genome and genome of organelles, cell and tissue differentiation, and plant morphogenesis. The conflict between the bulk of plant genome and low percentage of its realization is explained as a result of limitation of the nuclear genome realization by plastid genome. The concept of development as applied to plant ontogenesis has been critically analyzed. The possibilities of the concept correction by with the help of symbiogenetic hypothesis are discussed.     

Evolution of plant mitochondrial genomes via substoichiometric intermediates

Comparison of the modern fertile maize mitochondrial genome (N) with an ancestral maize mitochondrial genome (RU) reveals a 12 kb duplication (containing the atpA gene) in the modern genome that is absent from the ancestor. Cloning, mapping, and sequencing of the relevant portions of the ancestral genome shows that this duplication probably arose via a three-stage recombination process involving substoichiometric intermediates. Comparison with analogous observations on yeast mitochondrial genomes suggests that this three-stage model of genome reorganization can be generally applied to plant mitochondrial genomes to explain both deletions and the creation of novel repeats, common features of plant mitochondrial genome evolution.     

基因编辑技术研究进展及其在苜蓿等豆科饲草作物中的应用

基因编辑是对生物基因组进行靶向修饰的一项新型生物技术,可以在不同物种中实现对目标基因的定点敲除、基因片段置换以及基因定点插入等基因定向编辑,目前基因编辑技术已在植物基因功能解析和作物遗传改良研究中得到广泛应用。本文简要回顾基因编辑技术的发展历程,重点介绍新近发展的CRISPR/Cas9技术在植物中的研究进展,并对CRISPR/Cas基因编辑技术在苜蓿等饲草作物中的应用进行探讨和展望。     

The Mutant Form of Acetolactate Synthase Genomic DNA from Rice is an Efficient Selectable Marker for Genetic Transformation

The proper use of a marker gene in a transformation process is critical for the production of transgenic plants. However, consumer concerns and regulatory requirements raise an objection to the presence of exogenous DNA in transgenic plants, especially antibiotic-resistant genes and promoters derived from viruses. One approach to overcome this problem is the elimination of marker genes from the plant genome by using several site-specific recombination systems. We propose an alternative method to solve this problem using a marker gene exclusively derived from the host plant DNA. We cloned a genomic DNA fragment containing regulatory and coding sequences of acetolactate synthase (ALS) gene from rice, and mutagenized the ALS gene into a herbicide-resistant form. After transfer of this construct to the rice genome, transgenic plants were efficiently selected with a herbicide, bispyribac-sodium salt, which inhibits the activity of wild type ALS. We also analyzed the regulatory feature of the rice ALS gene promoter with the gusA reporter gene and revealed that GUS expression was observed constitutively in aerial parts of rice seedlings and root tips. The marker system consisted exclusively of host plant DNA and enabled efficient selection in a monocot crop plant, rice. The selection system can potentially be applied to generate transgenic plants of other crop species and can be expected to be publicly acceptable.     

Gene identification using rice genome sequences

Identifying useful gene(s) is one of the most important objectives of plant geneticists. Various strategies can be used, which are based on the characteristics of plant reproduction and available technology. Rice is the first model crop whose whole genome sequence has been reported. In addition, information on the whole genome sequences of two important rice subspecies (japonica and indica rice) is also available. Rice is a self-pollinating crop and methods of artificial crossing are relatively easy to perform; such methods enable the production of numerous seeds for genetic analyses. Based on these features, a map-based cloning (i.e., positional cloning) strategy has been successfully applied over the last decade to identify rice genes. Recently, advanced next-generation sequencing (NGS) technology was used to ascertain the genome sequences of individual plants, opening up a new strategy for gene identification. This strategy has been used successfully to identify the genes responsible for certain qualitative traits in rice. However, to identify the gene(s) involved in a quantitative trait, a map-based cloning strategy is still required after quantitative trait loci analysis using NGS technology. In this review, we discuss both map-based cloning (which is still the primary strategy used to identify rice genes) and NGS-based strategies.