piggyBac-based insertional mutagenesis and enhancer detection as a tool for functional insect genomics

Transposon mutagenesis provides a fundamental tool for functional genomics. Here we present a non-species-specific, combined enhancer detection and binary expression system based on the transposable element piggyBac: For the different components of this insertional mutagenesis system, we used widely applicable transposons and distinguishable broad-range transformation markers, which should enable this system to be operational in nonmodel arthropods. In a pilot screen in Drosophila melanogaster, piggyBac mutator elements on the X chromosome were mobilized in males by a Hermes-based jumpstarter element providing piggyBac transposase activity under control of the alpha1-tubulin promoter. As primary reporters in the piggyBac mutator elements, we employed the heterologous transactivators GAL4delta or tTA. To identify larval and adult enhancer detectors, strains carrying UASp-EYFP or TRE-EYFP as secondary reporter elements were used. Tissue-specific enhancer activities were readily observed in the GAL4delta/UASp-based systems, but only rarely in the tTA/TRE system. Novel autosomal insertions were recovered with an average jumping rate of 80%. Of these novel insertions, 3.8% showed homozygous lethality, which was reversible by piggyBac excision. Insertions were found in both coding and noncoding regions of characterized genes and also in noncharacterized and non-P-targeted CG-number genes. This indicates that piggyBac will greatly facilitate the intended saturation mutagenesis in Drosophila.     

A bidirectional gene trap construct suitable for T-DNA and Ds-mediated insertional mutagenesis in rice (Oryza sativa L.)

A construct suitable for genome-wide transfer-DNA (T-DNA) and subsequent transposon-based (Ds) gene trapping has been developed for use in rice (Oryza sativa). This T-DNA/Ds construct contains: Ds terminal sequences immediately inside T-DNA borders for subsequent Ds mobilization; promoterless green fluorescent protein (sgfpS65T) and beta-glucuronidase (uidA) reporter genes, each fused to an intron (from Arabidopsis GPA1 gene) to enable bidirectional gene trapping by T-DNA or Ds; an ampicillin resistance gene (bla) and a bacterial origin of replication (ori) to serve as the plasmid rescue system; an intron-containing hygromycin phosphotransferase gene (hph) as a selectable marker or Ds tracer; and an intron-containing barnase gene in the binary vector backbone (VB) to select against transformants carrying unwanted VB sequences. More than a threefold increase over previously reported reporter gene-based gene trapping efficiencies was observed in primary T-DNA/Ds transformant rice lines, returning an overall reporter gene expression frequency of 23%. Of the plant organs tested, 3.3-7.4% expressed either reporter at varying degrees of organ or tissue specificity. Approximately 70% of the right border (RB) flanking sequence tags (FSTs) retained 1-6 bp of the RB repeat and 30% of the left border (LB) FSTs retained 5-23 bp of the LB repeat. The remaining FSTs carried deletions of 2-84 bp inside the RB or 1-97 bp inside the LB. Transposition of Ds from the original T-DNA was evident in T-DNA/Ds callus lines super-transformed with a transposase gene (Ac) construct, as indicated by gene trap reporter activity and rescue of new FSTs in the resulting double transformant lines.     

Spectrum of T-DNA integrations for insertional mutagenesis of Histoplasma capsulatum

Agrobacterium-mediated transformation is being increasingly used for insertional mutagenesis of fungi. To better evaluate its effectiveness as a mutagen for the fungal pathogen Histoplasma capsulatum, we analyzed a collection of randomly selected T-DNA insertion mutants. Testing of different T-DNA element vectors engineered for transformation of fungi showed that pBHt2 provides the highest transformation efficiency and the lowest rate of vector backbone carryover. Sixty-eight individual T-DNA integrations were characterized by recovery of T-DNA ends and flanking genomic sequences. The right border (RB) end of the T-DNA is largely preserved whereas the left border (LB) end is frequently truncated. Analysis of T-DNA insertion sites confirms the lack of any integration hotspots in the Histoplasma genome. Relative to genes, T-DNA integrations show significant bias towards promoter regions at the expense of coding sequences. With consideration for potential promoter interruption and the demonstrated efficacy of intronic insertions, 61 % of mapped T-DNA insertions should impair gene expression or function. Mapping of T-DNA flanking sequences demonstrates 67 % of T-DNA integrations are integrations at a single chromosomal site and 31 % of T-DNA integrations are associated with large-scale chromosomal rearrangements. This characterization of T-DNA insertions in mutants selected without regard to phenotype supports application of Agrobacterium-mediated transformation as an insertional mutagen for genome-based screens and functional discovery of genes in Histoplasma.     

根癌农杆菌介导的球毛壳菌遗传转化及T-DNA插入突变体的获得

根癌农杆菌介导的遗传转化系统是植物基因工程常用方法,目前已将这一转化系统应用到酵母、丝状真菌以及人类细胞的转化。利用这一转化系统,成功地实现了丝状真菌球毛壳菌（Chaetomium globosum）的遗传转化,转化率约为60～180个转化子/10.7个孢子 。通过对转化子的PCR检测和Southern 杂交分析表明,TDNA已整合进毛壳菌基因组中,而且在所检测的转化子中都是以单拷贝的形式整合,转化子都能够稳定遗传。根癌农杆菌介导的遗传转化具有转化率高、低拷贝、遗传稳定、操作简便等优点,因此有可能成为丝状真菌遗传转化和功能基因组研究的有力工具。     

Large-scale T-DNA mutagenesis in Arabidopsis for functional genomic analysis

In planta Agrobacterium-mediated transformation combined with a soil-based herbicide selection for transgenic plants was used to recover large numbers of transgenic Arabidopsis plants for functional genomic studies. A tissue-culture-free system for generating transgenic plants was achieved by infiltrating Arabidopsis plants with Agrobacterium tumefaciens harboring a binary T-DNA vector containing the phosphinothricin acetyltransferase gene from Streptomyces hygroscopicus, and by selecting transgenic Arabidopsis growing in soil by foliar application of the herbicide Finale (phosphinothricin). Analysis of herbicide-resistant plants indicated that all were transgenic and that the T-DNA transformation process occurred late during flower development, resulting in a preponderance of independently derived T-DNA insertions. T-DNA insertions were usually integrated in a concatenated, rearranged form, and using linkage analysis, we estimated that T1 plants carried between one and five T-DNA loci. Using pooling strategies, both DNA and seed pools were generated from about 38,000 Arabidopsis plants representing over 115,000 independent T-DNA insertions. We show the utility of these transgenic lines for identifying insertion mutations using gene sequence and PCR-based screening. Electronic Publication     

An insertional mutagenesis system for analyzing the Chinese cabbage genome using Agrobacterium T-DNA

In this study, we applied insertional mutagenesis using Agrobacterium transfer DNA to functionally characterize the gene of Brassica rapa L. ssp. pekinensis. The specific objectives were to: (i) develop and apply a gene tagging system using plasmid rescue and inverse PCR, (ii) select and analyze mutant lines, and (iii) analyze the phenotypic characteristics of mutants. A total of 3,400 insertional mutant lines were obtained from the Chinese cabbage cultivar, ’seoul’, using optimized condition. Plasmid rescue was performed successfully for transgenic plants with multiple T-DNA insertions, and inverse PCR was performed for plants with a single copy. The isolated flanking DNA sequences were blasted against the NCBI database and mapped to a linkage map. We determined the genetic loci in B. rapa with two methods: RFLP using the rescue clones themselves and sequence homology analysis to the B. rapa sequence database by queries of rescued clones sequences. Compared to wild type, the T₁ progenies of mutant lines showed variable phenotypes, including hairless and wrinkled leaves, rosette-type leaves, and chlorosis symptoms. T-DNA inserted mutant lines were the first population that we developed and will be very useful for functional genomics studies of Chinese cabbage.     

Functional genomics of eukaryotic photosynthesis using insertional mutagenesis of Chlamydomonas reinhardtii

The unicellular green alga Chlamydomonas reinhardtii is a widely used model organism for studies of oxygenic photosynthesis in eukaryotes. Here we describe the development of a resource for functional genomics of photosynthesis using insertional mutagenesis of the Chlamydomonas nuclear genome. Chlamydomonas cells were transformed with either of two plasmids conferring zeocin resistance, and insertional mutants were selected in the dark on acetate-containing medium to recover light-sensitive and nonphotosynthetic mutants. The population of insertional mutants was subjected to a battery of primary and secondary phenotypic screens to identify photosynthesis-related mutants that were pigment deficient, light sensitive, nonphotosynthetic, or hypersensitive to reactive oxygen species. Approximately 9% of the insertional mutants exhibited 1 or more of these phenotypes. Molecular analysis showed that each mutant line contains an average of 1.4 insertions, and genetic analysis indicated that approximately 50% of the mutations are tagged by the transforming DNA. Flanking DNA was isolated from the mutants, and sequence data for the insertion sites in 50 mutants are presented and discussed.     

Mapped Ds/T-DNA launch pads for functional genomics in barley

A system for targeted gene tagging and local saturation mutagenesis based on maize transposable elements (Ac/Ds) was developed in barley (Hordeum vulgare L.). We generated large numbers of transgenic barley lines carrying a single copy of the non-autonomous maize Ds element at defined positions in the genome. Independent Ds lines were either generated by activating Ds elements in existing single-copy lines after crossing with AcTPase-expressing plants or by Agrobacterium-mediated transformation. Genomic DNA flanking Ds and T-DNA insertion sites from over 200 independent lines was isolated and sequenced, and was used for a sequence based mapping strategy in a barley reference population. More than 100 independent Ds insertion sites were mapped and can be used as launch pads for future targeted tagging of genes in the vicinity of the insertion sites. Sequence analysis of Ds and T-DNA flanking regions revealed a sevenfold preference of both mutagens for insertion into non-redundant, gene-containing regions of the barley genome. However, whilst transposed Ds elements preferentially inserted adjacent to regions with a high number of predicted and experimentally validated matrix attachment regions (nuclear MARs), this was not the case for T-DNA integration sites. These findings and an observed high transposition frequency from mapped launch pads demonstrate the future potential of gene tagging for functional genomics and gene discovery in barley.     

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。     

Highly efficient production and characterization of T-DNA plants for rice ( Oryza sativa L.) functional genomics

We investigated the potential of an improved Agrobacterium tumefaciens-mediated transformation procedure of japonica rice ( Oryza sativa L.) for generating large numbers of T-DNA plants that are required for functional analysis of this model genome. Using a T-DNA construct bearing the hygromycin resistance ( hpt), green fluorescent protein ( gfp) and beta-glucuronidase ( gusA) genes, each individually driven by a CaMV 35S promoter, we established a highly efficient seed-embryo callus transformation procedure that results both in a high frequency (75-95%) of co-cultured calli yielding resistant cell lines and the generation of multiple (10 to more than 20) resistant cell lines per co-cultured callus. Efficiencies ranged from four to ten independent transformants per co-cultivated callus in various japonica cultivars. We further analysed the T-DNA integration patterns within a population of more than 200 transgenic plants. In the three cultivars studied, 30-40% of the T(0) plants were found to have integrated a single T-DNA copy. Analyses of segregation for hygromycin resistance in T(1) progenies showed that 30-50% of the lines harbouring multiple T-DNA insertions exhibited hpt gene silencing, whereas only 10% of lines harbouring a single T-DNA insertion was prone to silencing. Most of the lines silenced for hpt also exhibited apparent silencing of the gus and gfp genes borne by the T-DNA. The genomic regions flanking the left border of T-DNA insertion points were recovered in 477 plants and sequenced. Adapter-ligation Polymerase chain reaction analysis proved to be an efficient and reliable method to identify these sequences. By homology search, 77 T-DNA insertion sites were localized on BAC/PAC rice Nipponbare sequences. The influence of the organization of T-DNA integration on subsequent identification of T-DNA insertion sites and gene expression detection systems is discussed.     

Early and multiple Ac transpositions in rice suitable for efficient insertional mutagenesis

A GFP excision assay was developed to monitor the excision of Ac introduced into rice by Agrobacterium-mediated transformation. The presence of a strong double enhancer element of the CaMV 35S promoter adjacent to the Ac promoter induced very early excision, directly after transformation into the plant cell, exemplified by the absence of Ac in the T-DNA loci. Excision fingerprint analysis and characterization of transposition events from related regenerants revealed an inverse correlation between the number of excision events and transposed Ac copies, with single early excisions after transformation generating Ac amplification. New transpositions were generated at a frequency of 15–50% in different lines, yielding genotypes bearing multiple insertions, many of which were inherited in the progeny. The sequence of DNA flanking Ac in three representative lines provided a database of insertion tagged sites suitable for the identification of mutants of sequenced genes that can be examined for phenotypes in a reverse genetics strategy to elucidate gene function. Remarkably, two-thirds of Ac tagged sites showing homology to sequences in public databases were in predicted genes. A clear preference of transposon insertions in genes that are either predicted by protein coding capacity or by similarity to ESTs suggests that the efficiency of recovering knockout mutants of genes could be about three times higher than random. Linked Ac transposition, suitable for targeted tagging, was documented by segregation analysis of a crippled Ac element and by recovery of a set of six insertions in a contiguous sequence of 70 kb from chromosome 6 of rice.     

Large-scale mutagenesis and functional genomics in yeast

One of the major goals for the post-genome era is determining of the function of proteins predicted in the genome sequence. In many organisms functional assignments have been the results of comparative sequencing, proteomics or expression profiling. In the yeast, Saccharomyces cerevisiae, however, the functional role of a gene can be tested directly by disrupting the gene and examining the phenotype of the mutant. Because precise targeted deletions can be easily constructed, it is also possible to systematically delete every gene in the genome. Here we describe recent progress in yeast genome-wide mutagenesis programs and the results produced from analyzing the mutants created by them. Electronic Publication     

根癌农杆菌介导的轮枝镰孢菌遗传转化及T-DNA插入突变体的筛选

建立并优化了农杆菌介导转化轮枝镰孢菌Fusarium verticillioides获得T-DNA插入突变体的体系,在镰孢菌孢子浓度106个/mL、农杆菌OD600=0.15-0.20、乙酰丁香酮浓度为200μmol/mL的条件下共培养36h转化率最高,可达60-120个/106个孢子。共获得转化子1000多个,连续转接5代能够稳定遗传。PCR验证潮霉素B抗性基因已整合进转化子基因组DNA中,部分转化子表现为生长和形态异常。该转化体系的建立为研究该菌的致病机制和功能基因分析奠定了基础。     

A collection of enhancer trap insertional mutants for functional genomics in tomato

With the completion of genome sequencing projects, the next challenge is to close the gap between gene annotation and gene functional assignment. Genomic tools to identify gene functions are based on the analysis of phenotypic variations between a wild type and its mutant; hence, mutant collections are a valuable resource. In this sense, T‐DNA collections allow for an easy and straightforward identification of the tagged gene, serving as the basis of both forward and reverse genetic strategies. This study reports on the phenotypic and molecular characterization of an enhancer trap T‐DNA collection in tomato (Solanum lycopersicum L.), which has been produced by Agrobacterium‐mediated transformation using a binary vector bearing a minimal promoter fused to the uidA reporter gene. Two genes have been isolated from different T‐DNA mutants, one of these genes codes for a UTP‐glucose‐1‐phosphate uridylyltransferase involved in programmed cell death and leaf development, which means a novel gene function reported in tomato. Together, our results support that enhancer trapping is a powerful tool to identify novel genes and regulatory elements in tomato and that this T‐DNA mutant collection represents a highly valuable resource for functional analyses in this fleshy‐fruited model species.     

根癌农杆菌介导的轮枝镰孢菌遗传转化及T-DNA插入突变体的筛选

建立并优化了农杆菌介导转化轮枝镰孢菌Fusarium verticillioides获得T-DNA插入突变体的体系,在镰孢菌孢子浓度106个/mL、农杆菌OD600=0.15-0.20、乙酰丁香酮浓度为200μmol/mL的条件下共培养36h转化率最高,可达60-120个/106个孢子。共获得转化子1000多个,连续转接5代能够稳定遗传。PCR验证潮霉素B抗性基因已整合进转化子基因组DNA中,部分转化子表现为生长和形态异常。该转化体系的建立为研究该菌的致病机制和功能基因分析奠定了基础。     

Functional genomics in Arabidopsis: large-scale insertional mutagenesis complements the genome sequencing project

The ultimate goal of genome research on the model flowering plant Arabidopsis thaliana is the identification of all of the genes and understanding their functions. A major step towards this goal, the genome sequencing project, is nearing completion; however, functional studies of newly discovered genes have not yet kept up to this pace. Recent progress in large-scale insertional mutagenesis opens new possibilities for functional genomics in Arabidopsis. The number of T-DNA and transposon insertion lines from different laboratories will soon represent insertions into most Arabidopsis genes. Vast resources of gene knockouts are becoming available that can be subjected to different types of reverse genetics screens to deduce the functions of the sequenced genes.