Embryonic Lethals and T-DNA Insertional Mutagenesis in Arabidopsis

T-DNA insertional mutagenesis represents a promising approach to the molecular isolation of genes with essential functions during plant embryo development. We describe in this report the isolation and characterization of 18 mutants of Arabidopsis thaliana defective in embryo development following seed transformation with Agrobacterium tumefaciens. Random T-DNA insertion was expected to result in a high frequency of recessive embryonic lethals because many target genes are required for embryogenesis. The cointegrate Ti plasmid used in these experiments contained the nopaline synthase and neomycin phosphotransferase gene markers. Nopaline assays and resistance to kanamycin were used to estimate the number of functional inserts present in segregating families. Nine families appeared to contain a T-DNA insert either within or adjacent to the mutant gene. Eight families were clearly not tagged with a functional insert and appeared instead to contain mutations induced during the transformation process. DNA gel blot hybridization with internal and right border probes revealed a variety of rearrangements associated with T-DNA insertion. A general strategy is presented to simplify the identification of tagged embryonic mutants and facilitate the molecular isolation of genes required for plant embryogenesis.     

Genetic and molecular characterization of embryonic mutants identified following seed transformation in Arabidopsis

Over 5000 transgenic families of Arabidopsis thaliana produced following seed transformation with Agrobacterium tumefaciens were screened for embryonic lethals, defectives, and pattern mutants. One hundred and seventy-eight mutants with a wide range of developmental abnormalities were identified. Forty-one mutants appear from genetic studies to be tagged (36% of the 115 mutants examined in detail). Mapping with visible markers demonstrated that mutant genes were randomly distributed throughout the genome. Seven mutant families appeared to contain chromosomal translocations because the mutant genes exhibited linkage to visible markers on two different chromosomes. Chromosomal rearrangements may therefore be widespread following seed transformation. DNA gel blot hybridizations with 34 tagged mutants and three T-DNA probes revealed a wide range of insertion patterns. Models of T-DNA structure at each mutant locus were constructed to facilitate gene isolation. The value of such models was demonstrated by using plasmid rescue to clone flanking plant DNA from four tagged mutants. Further analysis of genes isolated from these insertional mutants should help to elucidate the relationship between gene function and plant embryogenesis.     

Genetic and molecular characterization of embryonic mutants identified following seed transformation in Arabidopsis

Over 5000 transgenic families of Arabidopsis thaliana produced following seed transformation with Agrobacterium tumefaciens were screened for embryonic lethals, defectives, and pattern mutants. One hundred and seventy-eight mutants with a wide range of developmental abnormalities were identified. Forty-one mutants appear from genetic studies to be tagged (36% of the 115 mutants examined in detail). Mapping with visible markers demonstrated that mutant genes were randomly distributed throughout the genome. Seven mutant families appeared to contain chromosomal translocations because the mutant genes exhibited linkage to visible markers on two different chromosomes. Chromosomal rearrangements may therefore be widespread following seed transformation. DNA gel blot hybridizations with 34 tagged mutants and three T-DNA probes revealed a wide range of insertion patterns. Models of T-DNA structure at each mutant locus were constructed to facilitate gene isolation. The value of such models was demonstrated by using plasmid rescue to clone flanking plant DNA from four tagged mutants. Further analysis of genes isolated from these insertional mutants should help to elucidate the relationship between gene function and plant embryogenesis.     

Molecular analysis of rice plants harboring a multi-functional T-DNA tagging system

About 25,000 rice T-DNA insertional mutant lines were generated using the vector pCAS04 which has both promoter-trapping and activation-tagging function. Southern blot analysis revealed that about 40% of these mutants were single copy integration and the average T-DNA insertion number was 2.28. By extensive phenotyping in the field, quite a number of agronomically important mutants were obtained. Histochemical GUS assay with 4,310 primary mutants revealed that the GUS-staining frequency was higher than that of the previous reports in various tissues and especially high in flowers. The T-DNA flanking sequences of some mutants were isolated and the T-DNA insertion sites were mapped to the rice genome. The flanking sequence analysis demonstrated the different integration pattern of the right border and left border into rice genome. Compared with Arabidopsis and poplar, it is much varied in the T-DNA border junctions in rice.     

Identification of nuclear genes encoding chloroplast-localized proteins required for embryo development in Arabidopsis

We describe here the diversity of chloroplast proteins required for embryo development in Arabidopsis (Arabidopsis thaliana). Interfering with certain chloroplast functions has long been known to result in embryo lethality. What has not been reported before is a comprehensive screen for embryo-defective (emb) mutants altered in chloroplast proteins. From a collection of transposon and T-DNA insertion lines at the RIKEN chloroplast function database (http://rarge.psc.riken.jp/chloroplast/) that initially appeared to lack homozygotes and segregate for defective seeds, we identified 23 additional examples of EMB genes that likely encode chloroplast-localized proteins. Fourteen gene identities were confirmed with allelism tests involving duplicate mutant alleles. We then queried journal publications and the SeedGenes database (www.seedgenes.org) to establish a comprehensive dataset of 381 nuclear genes encoding chloroplast proteins of Arabidopsis associated with embryo-defective (119 genes), plant pigment (121 genes), gametophyte (three genes), and alternate (138 genes) phenotypes. Loci were ranked based on the level of certainty that the gene responsible for the phenotype had been identified and the protein product localized to chloroplasts. Embryo development is frequently arrested when amino acid, vitamin, or nucleotide biosynthesis is disrupted but proceeds when photosynthesis is compromised and when levels of chlorophyll, carotenoids, or terpenoids are reduced. Chloroplast translation is also required for embryo development, with genes encoding chloroplast ribosomal and pentatricopeptide repeat proteins well represented among EMB datasets. The chloroplast accD locus, which is necessary for fatty acid biosynthesis, is essential in Arabidopsis but not in Brassica napus or maize (Zea mays), where duplicated nuclear genes compensate for its absence or loss of function.     

Characterization of T-DNA insertion sites in Arabidopsis thaliana and the implications for saturation mutagenesis

A key component of a sound functional genomics infrastructure is the availability of a knockout mutant for every gene in the genome. A fruitful approach to systematically knockingout genes in the plant Arabidopsis thaliana has been the use of transferred-DNA (T-DNA) from Agrobacterium tumefaciens as an insertional mutagen. One of the assumptions underlying the use of T-DNA as a mutagen is that the insertion of these DNA elements into the Arabidopsis genome occurs at randomly selected locations. We have directly investigated the distribution of T-DNA insertions sites in populations of transformed Arabidopsis using two different approaches. To begin with, we utilized a polymerase chain reaction (PCR) procedure to systematically catalog the precise locations of all the T-DNA elements inserted within a 65 kb segment of chromosome IV. Of the 47 T-DNA insertions identified, 30% were found within the coding regions of genes. We also documented the insertion of T-DNA elements within the centromeric region of chromosome IV. In addition to these targeted T-DNA screens, we also mapped the genomic locations of 583 randomly chosen T-DNA elements by sequencing the genomic DNA flanking the insertion sites from individual T-DNA-transformed lines. 35% of these randomly chosen T-DNA insertions were located within the coding regions of genes. For comparison, coding sequences account for 44% of the Arabidopsis genome. Our results demonstrate that there is a small bias towards recovering T-DNA insertions within intergenic regions. However, this bias does not limit the utility of T-DNA as an effective insertional mutagen for use in reverse-genetic strategies.     

A high-throughput Arabidopsis reverse genetics system

A collection of Arabidopsis lines with T-DNA insertions in known sites was generated to increase the efficiency of functional genomics. A high-throughput modified thermal asymmetric interlaced (TAIL)-PCR protocol was developed and used to amplify DNA fragments flanking the T-DNA left borders from approximately 100000 transformed lines. A total of 85108 TAIL-PCR products from 52964 T-DNA lines were sequenced and compared with the Arabidopsis genome to determine the positions of T-DNAs in each line. Predicted T-DNA insertion sites, when mapped, showed a bias against predicted coding sequences. Predicted insertion mutations in genes of interest can be identified using Arabidopsis Gene Index name searches or by BLAST (Basic Local Alignment Search Tool) search. Insertions can be confirmed by simple PCR assays on individual lines. Predicted insertions were confirmed in 257 of 340 lines tested (76%). This resource has been named SAIL (Syngenta Arabidopsis Insertion Library) and is available to the scientific community at www.tmri.org.     

激活标签法构建拟南芥突变体库及其表型分析

以拟南芥(ArabMopsis thaliana)野生生态型(Columbia)植株为实验材料,以含有激活标记双元质粒pCB260的农杆菌进行转化,并以抗除草剂Basta为筛选标记,构建了拟南芥激活标签突变体库,所用pCB260双元质粒含有两个Ds位点、一个GFP标记基因与一个抗basta标记基因,可以方便高效地筛选转基因植物.目前经初步筛选获得了约10 000个独立转化株系(T1代),其中约50个株系具有明显的表型变化,包括花期改变、株型变异、叶形特异、育性降低、花发育异常、种子颜色变浅等.运用TAIL-PCR技术,成功获得了其中10个表型特异株系的T-DNA侧翼序列,分别分布于拟南芥基因组的5条染色体上.     

Generation and flanking sequence analysis of a rice T-DNA tagged population

Insertional mutagenesis provides a rapid way to clone a mutated gene. Transfer DNA (T-DNA) of Agrobacterium tumefaciens has been proven to be a successful tool for gene discovery in Arabidopsis and rice (Oryza sativa L. ssp. japonica). Here, we report the generation of 5,200 independent T-DNA tagged rice lines. The T-DNA insertion pattern in the rice genome was investigated, and an initial database was constructed based on T-DNA flanking sequences amplified from randomly selected T-DNA tagged rice lines using Thermal Asymmetric Interlaced PCR (TAIL-PCR). Of 361 T-DNA flanking sequences, 92 showed long T-DNA integration (T-DNA together with non-T-DNA). Another 55 sequences showed complex integration of T-DNA into the rice genome. Besides direct integration, filler sequences and microhomology (one to several nucleotides of homology) were observed between the T-DNA right border and other portions of the vector pCAMBIA1301 in transgenic rice. Preferential insertion of T-DNA into protein-coding regions of the rice genome was detected. Insertion sites mapped onto rice chromosomes were scattered in the genome. Some phenotypic mutants were observed in the T₁ generation of the T-DNA tagged plants. Our mutant population will be useful for studying T-DNA integration patterns and for analyzing gene function in rice.Electronic Supplementary Material Supplementary material is available in the online version of this article at .Communicated by D. Mackill