插入突变在水稻功能基因组学中的研究进展

构建饱和的基因突变体库是最直接、有效的分析鉴定基因功能的方法.根据插入突变源不同可分为T-DNA插入突变、转座子插入突变等.主要介绍这两种方法的原理及其在水稻功能基因组学研究中的应用和进展,并分析和讨论了插入突变在水稻功能基因组学研究中存在的困难和发展趋势.     

插入突变在功能基因组学研究中的应用

插入突变库的构建是功能基因组学研究的一个重要内容,可为确定基因的功能提供最直接的证据。构建插入突变库的方法有T-DNA插入突变、转座子插入突变和质粒介导的插入突变。本文分别介绍三种方法的原理及其在功能基因组学研究中的应用和研究进展。     

水稻T-DNA插入突变体库的构建及突变类型的分析

利用农杆菌介导的转化系统转化中花11成熟胚愈伤组织,获得1489个独立转化的T-DNA插入再生株系。PCR和Southern杂交的结果表明,69．8％转化株系被整合了T-DNA,通过Tail-PCR也从转化植株中扩增出T-DNA侧翼序列。同时对1066个T1转化株系的抽穗期、株高、单株穗数的调查结果表明,不同株系中分离出了突变植株。     

水稻T-DNA插入突变体库的筛选及遗传分析

T-DNA标签技术是分离和研究植物功能基因的有效方法,寻找T-DNA插入表型突变体是进一步开展研究的关键所在。文章对以ZH11、ZH15为受体亲本构建的4416份T,代标签系进行了表型鉴定,发现存在拟纯合突变和系内分离突变两种类型,突变表型涉及株高、生育期、叶形、叶色、分蘖力、植株松紧度、穗颈节、穗形、颖花、粒形、类病变、雄性不育、生长极性等14类性状。其中,株高、生育期、叶色、雄性不育有着相对较高的突变频率（超过1％）,株高和叶色的突变频率在品种及年度间表现稳定,而生育期、雄性不育波动较大,表明这类性状的表型易受到环境的影响。通过T1、T2连续世代的共分离分析,筛选出3个与穗部或颖花发育相关的T-DNA插入突变体,为分离相关功能基因奠定基础。随机选择42份有表型突变的标签系,通过质粒拯救和TAIL-PCR的方法分离其侧翼序列,从39个标签系中获得40条序列,其中25条为载体序列,14条与水稻基因组有很好的同源性,BlastN分析结果表明T-DNA有优先整合进植物功能基因内部的特性。     

水稻Ds插入纯合体的筛选和鉴定

采用Basta抗性鉴定、潮霉素抗性鉴定和PCR检测相结合的方法筛选和鉴定了水稻Ds插入纯合体。在T1代236个转化株系中,有16个株系的全部植株表现出对Basta的敏感,其余220个株系的植株表现出对Basta的抗性。经过3代的纯合筛选,共鉴定出Ds插入纯合体203个．这些Ds插入纯合体可用于构建Ac／Ds系统和对Ds插入突变体进行筛选和鉴定,为水稻功能基因组学研究提供了材料。     

水稻Ds插入淡绿叶突变体的鉴定和遗传分析

Ac/Ds插入突变是水稻基因功能鉴定的有力工具之一。文章从水稻中花11 Ds-T-DNA转化纯合体与Ac-T-DNA 转化纯合体的杂交群体中筛选到一个淡绿叶突变体。该突变体在三叶期由绿苗转为淡绿叶苗, 自然光照下突变体迅速焦枯, 但是在弱光照条件下, 突变体能缓慢生长至开花结实; 突变体光合作用特性研究表明该突变是典型的光抑制突变体。遗传分析表明该突变为Ds插入导致的隐性突变。     

T-DNA插入产生的水稻迟抽穗突变体的遗传分析

在筛选和鉴定水稻T-DNA插入纯合体的过程中,观察到一个迟抽穗的突变体.对该突变体进行遗传分析的结果表明,分离群体后代中出现正常抽穗、迟抽穗和特迟抽穗3种类型,分离比率为1:2:1,符合一对基因的不完全显性遗传;对突变体及其后代分离群体做Basta抗性检测及PCR分子检测,证实该突变体是由T-DNA插入所引起的,突变性状与T-DNA共分离.该材料可用于插入座位的基因克隆.     

提高水稻插入突变体库利用效率的尝试

T-DNA标签法是一种以农杆菌介导的遗传转化为基础来创造插入突变体库, 从而高通量地分离和克隆植物功能基因的方法。但由于种种原因, 水稻插入突变体库的利用效率较低。为了提高水稻插入突变体库的利用效率, 结合水稻一个双拷贝T-DNA插入突变体的发现和鉴定研究, 通过特异PCR检测、侧翼序列与目标性状的共分离分析, 在1个双插入位点均为杂合的植株的后代株系中分拆了2个插入事件, 分离出目标性状存在遗传分离且只带有1个插入事件的后代株系, 为后续的共分离检测和基因克隆研究打下了重要的基础。由此产生了对插入突变体库中的非串联多拷贝插入标签系进行研究的一些思路和方法, 提出来与同行商榷。     

Insertional mutagenesis to isolate acetate-requiring mutants in Chlamydomonas reinhardtii

Abstract An arg 7 mutant of the green alga Chlamydomonas reinhardtii was transformed with pARG7.8, a plasmid bearing the wild-type ARG 7 gene. Out of 4100 arg⁺ transformants selected on an arginine-free medium supplemented with acetate, nine failed to grow on acetate-free medium (ac⁻ mutants). The results of the genetic and molecular analysis of several ac⁻ mutants are in agreement with the hypothesis that they originated from insertion of the incoming plasmid into the nuclear genome. These mutants should constitute valuable tools for isolating the corresponding wild-type genes after plasmid rescue into Escherichia coli .     

Insertional mutagenesis of Aspergillus fumigatus

We have investigated transformation with heterologous DNA as a method for insertional mutagenesis of Aspergillus fumigatus. Two methods, polyethylene glycol-mediated transformation of protoplasts and electroporation of germinating spores, were used to establish conditions leading to single-copy integration of transforming DNA at different genomic sites. We have assessed the effect of restriction enzyme-mediated integration (REMI) for both methods. Non-REMI protoplast transformation led to integration of multiple copies of transforming DNA in the majority of transformants. Results of REMI with protoplast transformation varied depending on the enzyme used. Low concentrations of several restriction enzymes stimulated transformation, but of ten enzymes investigated only REMI with XhoI and KpnI resulted in single-copy integration of transforming DNA for the majority of transformants. For protoplast transformation with XhoI- or KpnI-based REMI, 50% and 76% of insertions, respectively, were due to integrations at a genomic enzyme site corresponding to the enzyme used for REMI. Electroporation of spores without addition of restriction enzyme resulted in a high transformation efficiency, with up to 67% of transformants containing a single copy of transforming DNA. In contrast to protoplast transformation, electroporation of spores in the presence of a restriction enzyme did not improve transformation efficiency or lead to insertion at genomic restriction sites. Southern analysis indicated that for both protoplast transformation with REMI using KpnI or XhoI and for electroporation of spores without addition of restriction enzymes, transforming DNA inserted at different genomic sites in a high proportion of transformants. Received: 6 March 1998 / Accepted: 25 May 1998     

斑马鱼反转录病毒插入突变技术的发展及其在基因饱和突变与筛选中的应用

用于大规模基因突变与筛选的主要策略有化学诱变、插入突变、基因诱捕。插入突变是一种通过外源DNA整合的方式来获得突变体,并克隆得到对应突变基因的方法。运用反转录病毒介导的插入突变技术,在脊椎动物斑马鱼中已经获得了许多影响胚胎发育和细胞生长过程的突变体,并找到了对应的基因。基因诱捕技术也被运用于反转录病毒载体的构建。这套系统的建立使斑马鱼成为第一个有可能达到基因饱和突变和筛选的脊椎动物。     

An Efficient Rice Mutagenesis System Based on Suspension-Cultured Cells

Plant mutants are important bio-resources for crop breeding and gene functional studies. Conventional methods for generating mutant libraries by mutagenesis of seeds with physical or chemical agents are of low efficiency. Here, we developed a highly-efficient ethyl methanesulfonate (EMS) mutagenesis system based on suspension-cultured cells, with rice (Oryza sativa L.) as an example. We show that treatment of suspension-cultured tiny cell clusters with 0.4% EMS for 18-22h followed by differentiation and regeneration produced as high as 29.4% independent mutant lines with visible phenotypic variations, including a number of important agronomic traits such as grain size, panicle size, grain or panicle shape, tiller number and angle, heading date, male sterility, and disease sensitivity. No mosaic mutant was observed in the mutant lines tested. In this mutant library, we obtained a mutant with an abnormally elongated uppermost internode. Sequencing and functional analysis revealed that this is a new allelic mutant of eui (elongated uppermost internode) caused by two point mutations in the first exon of the EUI gene, representing a successful example of this mutagenesis system.     

Transposome Insertional Mutagenesis and Direct Sequencing of Microbial Genomes

Preformed transposase-transposon complexes called Transposomes have been electroporated into bacterial cells. The magnesium dependent process of insertion of the transposable element into bacterial chromosomal DNA occurs in vivo. The transposition efficiency of a Transposome containing a kanamycin marker was between 1.0×10⁴and 1.0×10⁷kanamycin resistant clones per microgram of transposon DNA in three gram-negative enteric bacterial species. Transposon integration sites were examined by direct genome sequencing of chromosomal DNA. Genomic DNA was isolated from transposition clones and directly cycle sequenced with primers specific for the ends of the transposon. The precise location of genome interruption for a transposition clone was identified by homology to known genes or sequences. Mutant phenotypes were rapidly correlated with genomic insertions sites.     

Analysis of gene-trap Ds rice populations in Korea

Insertional mutagen-mediated gene tagging populations have been essential resources for analyzing the function of plant genes. In rice, maize transposable elements have been successfully utilized to produce transposant populations. However, many generations and substantial field space are required to obtain a sufficiently sized transposant population. In rice, the japonica and indica subspecies are phenotypically and genetically divergent. Here, callus cultures with seeds carrying Ac and Ds were used to produce 89,700 lines of Dongjin, a japonica cultivar, and 6,200 lines of MGRI079, whose genome is composed of a mixture of the genetic backgrounds of japonica and indica. Of the more than 3,000 lines examined, 67% had Ds elements. Among the Ds-carrying lines, 81% of Dongjin and 63% of MGRI079 contained transposed Ds, with an average of around 2.0 copies. By examining more than 15,000 lines, it was found that 12% expressed the reporter gene GUS during the early-seedling stage. GUS was expressed in root hairs and crown root initials at estimated frequencies of 0.78% and 0.34%, respectively. The 5,271 analyzed Ds loci were found to be randomly distributed over all of the rice chromosomes. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. Sung Han Park, Nam Soo Jun, Chul Min Kim are contributed equally to this paper     

A Medium-Copy-Number Plasmid for Insertional Mutagenesis ofStreptococcus mutans

We have constructed a plasmid useful for insertional mutagenesis inStreptococcus mutans.The molecule, pSU20Erm, is based on a derivative of pACYC184 known as pSU20. The plasmid described here is approximately 3.7 kb in size and has the following properties: it replicates inEscherichia coli,does not replicate inS. mutans,contains an erythromycin-resistance marker which can be selected inE. colior the streptococci, contains a multiple cloning site with few restriction sites in the remainder of the molecule, and can be screened on X-Gal-containing medium for the presence of insertions into the multiple cloning site. We have used the plasmid to construct a library ofS. mutansDNA inE. coliand show that the clones can be reintegrated into theS. mutanschromosome via homologous recombination, thereby interrupting native genes. The plasmid has been used to clone part of a homologue of theE. coli drpAgene, encoding a global regulatory element for RNA synthesis. Further, we have identified an element closely linked todrpAinS. mutanswith high homology to IS861.     

植物诱变技术的研究进展

植物诱变技术是指利用外界因素加快物种遗传变异,在短期内获得有利用价值的突变体,为培育新种质、新品种及基因功能的研究等创造条件。相对于自然的选择,诱变技术具有高频率、广突变、周期短等特性。主要论述了常用的化学诱变、物理诱变、空间诱变和生物诱变等诱变手段,并详细介绍了上述诱变技术的诱变机理、生物学效应、常用的诱变方式及其在植物应用中的研究现状。针对现行各种诱变技术的不足提出了今后努力的方向。     

水稻耐储藏特性研究进展

水稻的耐储藏特性存在品种间差异,从基因型着手培育耐储藏水稻新品种具有重要的意义。从评价体系、遗传因素和基因定位几个方面对近年来水稻稻耐储性的相关研究进行了较系统的综述。