High Efficiency Transgene Segregation in Co-Transformed Maize Plants using an Agrobacterium Tumefaciens 2 T-DNA Binary System

For regulatory issues and research purposes it would be desirable to have the ability to segregate transgenes in co-transformed maize. We have developed a highly efficient system to segregate transgenes in maize that was co-transformed using an Agrobacterium tumefaciens 2 T-DNA binary system. Three vector treatments were compared in this study; (1) a 2 T-DNA vector, where the selectable marker gene bar (confers resistance to bialaphos) and the -glucuronidase (GUS) reporter gene are on two separate T-DNA's contained on a single binary vector; (2) a mixed strain treatment, where bar and GUS are contained on single T-DNA vectors in two separate Agrobacterium strains; (3) and a single T-DNA binary vector containing both bar and GUS as control treatment. Bialaphos resistant calli were generated from 52 to 59% of inoculated immature embryos depending on treatment. A total of 93.4% of the bialaphos selected calli from the 2 T-DNA vector treatment exhibited GUS activity compared to 11.7% for the mixed strain treatment and 98.2% for the cis control vector treatment. For the 2 T-DNA vector treatment, 86.7% of the bialaphos resistant/GUS active calli produced R₀ plants exhibiting both transgenic phenotypes compared to 10% for the mixed strain treatment and 99% for the single T-DNA control vector treatment. A total of 87 Liberty herbicide (contains bialaphos as the active ingredient) resistant/GUS active R₀ events from the 2 T-DNA binary vector treatment were evaluated for phenotypic segregation of these traits in the R₁ generation. Of these R₀ events, 71.4% exhibited segregation of Liberty resistance and GUS activity in the R₁ generation. A total of 64.4% of the R₀ 2 T-DNA vector events produced Liberty sensitive/GUS active (indicating selectable-marker-free) R₁ progeny. A high frequency of phenotypic segregation was also observed using the mixed strain approach, but a low frequency of calli producing R₀ plants displaying both transgenic phenotypes makes this method less efficient. Molecular analyses were then used to confirm that the observed segregation of R₁ phenotypes were highly correlated to genetic segregation of the bar and GUS genes. A high efficiency system to segregate transgenes in co-transformed maize plants has now been demonstrated.     

Characterization of the aldehyde dehydrogenase gene families of Zea mays and Arabidopsis

Cytoplasmic male sterility is a maternally transmitted inability to produce viable pollen. Male sterility occurs in Texas (T) cytoplasm maize as a consequence of the premature degeneration of the tapetal cell layer during microspore development. This sterility can be overcome by the combined action of two nuclear restorer genes, rf1 and rf2a. The rf2a gene encodes a mitochondrial aldehyde dehydrogenase (ALDH) that is capable of oxidizing a variety of aldehydes. Six additional ALDH genes were cloned from maize and Arabidopsis. In vivo complementation assays and in vitro enzyme analyses demonstrated that all six genes encode functional ALDHs. Some of these ALDHs are predicted to accumulate in the mitochondria, others in the cytosol. The intron/exon boundaries of these genes are highly conserved across maize and Arabidopsis and between mitochondrial and cytosolic ALDHs. Although animal, fungal, and plant genomes each encode both mitochondrial and cytosolic ALDHs, it appears that either the gene duplications that generated the mitochondrial and the cytosolic ALDHs occurred independently within each lineage or that homogenizing gene conversion-like events have occurred independently within each lineage. All studied plant genomes contain two confirmed or predicted mitochondrial ALDHs. It appears that these mitochondrial ALDH genes arose via independent duplications after the divergence of monocots and dicots or that independent gene conversion-like events have homogenized the mitochondrial ALDH genes in the monocot and dicot lineages. A computation approach was used to identify amino acid residues likely to be responsible for functional differences between mitochondrial and cytosolic ALDHs.     

Retrograde regulation of nuclear gene expression in CW-CMS of rice

The CW-cytoplasmic male sterility (CMS) line has the cytoplasm of Oryza rufipogon Griff, and mature pollen is morphologically normal under an optical microscope but lacks the ability to germinate; restorer gene Rf17 has been identified as restoring this ability. The difference between nuclear gene expression in mature anthers was compared for the CW-CMS line, [cms-CW] rf17rf17, and a maintainer line with normal cytoplasm of Oryza sativa L., [normal] rf17rf17. Using a 22-k rice oligoarray we detected 58 genes that were up-regulated more than threefold in the CW-CMS line. Expression in other organs was further investigated for 20 genes using RT-PCR. Five genes, including genes for alternative oxidase, were found to be preferentially expressed in [cms-CW] rf17rf17 but not in [normal] rf17rf17 or [cms-CW] Rf17Rf17. Such [cms-CW] rf17rf17-specific gene expression was only observed in mature anthers but not in leaves, stems, or roots, indicating the presence of anther-specific mitochondrial retrograde regulation of nuclear gene expression, and that Rf17 has a role in restoring the ectopic gene expression. We also used a proteomic approach to discover the retrograde regulated proteins and identified six proteins that were accumulated differently. These results reveal organ-specific induced mitochondrial retrograde pathways affecting nuclear gene expression possibly related to CMS. Electronic Supplementary Material Supplementary material is available to authorised users in the online version of this article at .     

A complete sequence of the rice sucrose synthase-1 (RSs1) gene

Using a fragment of the maize sucrose synthase gene Sh-1 as probe, the rice genome was shown to contain at least three genes encoding sucrose synthase. One of these genes was isolated from a genomic library, and its full sequence, including 1.7 kb of 5 flanking sequence and 0.9 kb of 3 flanking sequence, is reported. The new rice gene, designated RSs1, is highly homologous to maize Sh-1 (approx. 94% identity in derived amino acid sequence), and contains an identical intron-exon structure (16 exons and 15 introns). Both RSs1 and maize Sh-1 show similar sequence homologies to a second rice sucrose synthase gene described recently (designated RSs2, Yu et al. (1992) Plant Mol Biol 18: 139–142), although both the rice genes predict an extra 6 amino acids at the C-terminus of the protein when compared to the maize gene. The RSs1 5 flanking sequence contains a number of promoter-like sequences, including putative protein-binding regions similar to maize zein genes.     

The myrosinase gene family in Arabidopsis thaliana: gene organization,expression and evolution

Myrosinase (thioglucoside glucohydrolase, EC 3.2.3.1.) is in Brassicaceae species such as Brassica napus and Sinapis alba encoded by two differentially expressed gene families, MA and MB, consisting of about 4 and 10 genes, respectively. Southern blot analysis showed that Arabidopsis thaliana contains three myrosinase genes. These genes were isolated from a genomic library and two of them, TGG1 and TGG2, were sequenced. They were found to be located in an inverted mode with their 3 ends 4.4 kb apart. Their organization was highly conserved with 12 exons and 11 short introns. Comparison of nucleotide sequences of TGG1 and TGG2 exons revealed an overall 75% similarity. In contrast, the overall nucleotide sequence similarity in introns was only 42%. In intron 1 the unusual 5 splice border GC was used. Phylogenetic analyses using both distance matrix and parsimony programs suggested that the Arabidopsis genes could not be grouped with either MA or MB genes. Consequently, these two gene families arose only after Arabidopsis had diverged from the other Brassicaceae species. In situ hybridization experiments showed that TGG1 and TGG2 expressing cells are present in leaf, sepal, petal, and gynoecium. In developing seeds, a few cells reacting with the TGG1 probe, but not with the TGG2 probe, were found indicating a partly different expression of these genes.     

Electroporation of immature maize zygotic embryos and regeneration of transgenic plants

Using the pulse-discharging electroporation system HPES-3, we have transferred the neomycin phosphotransferase II (nptII) gene and -glucuronidase (gus) gene into mechanically-woulded immature zygotic embryo cells of an elite local maize cultivar Huanong Supersweet No. 42 and have produced transgenic maize plants. DNA hybridization and NPTII dot assay showed that the foreign genes were integrated into the genomes and expressed stably in the cells of the transgenic calluses and plants.     

Different mechanisms generating sequence variability are revealed in distinct regions of the hydroxyproline-rich glycoprotein gene from maize and related specie*

Summary The sequences of the genes coding for a hydroxyproline-rich glycoprotein from two varieties of maize (Zea mays, Ac1503 and W22), a teosinte (Zea diploperennis) and sorghum (Sorghum vulgare) have been obtained and compared. Distinct patterns of variability have been observed along their sequences. The 500 by region immediately upstream of the TATA box is highly conserved in theZea species and contains stretches of sequences also found in the sorghum gene. Further upstream, significant rearrangements are observed, even between the two maize varieties. These observations allow definition of a 5 region, which is common to the four genes and is probably essential for their expression. The 3 end shows variability, mostly due to small duplications and single nucleotide substitutions. There is an intron present in this region showing a high degree of sequence conservation among the four genes analyzed. The coding region is the most divergent, but variability arises from duplications of fragments coding for similar protein blocks and from single nucleotide substitutions. These results indicate that a number of distinct mechanisms (probably point mutation, transposon insertion and excision, homologous recombination and unequal crossing-over) are active in the production of sequence variability in maize and related species. They are revealed in different parts of the gene, probably as the result of the different types of functional constraints acting on them, and of the specific nature of the sequence in each region.The sequences reported in this paper have been deposited in the EMBL/GenBank Database (Bolt, Beranek, and Newman Laboratories, Cambridge, Mass., and EMBL, Heidelberg), accession nos. M36635 (maize Ac1503), X63134 (maize W22), X64173 (teosinte) and X56010 (sorghum)     

Transfer of E. coli gutD gene into maize and regeneration of salt-tolerant transgenic plants

GutD gene, encoding a key enzyme (glucitol-6-phosphate dehydrogenase) of sugar alcohol metabolic pathway inE. coli, was transferred into maize. Results of Southern and Western blotting analysis certified that this gene had integrated and been expressed in transgenic maize plants and their progeny. The synthesis and accumulation of sorbitol were detected in transgenic maize plants and a preliminary nutrient solution culture experiment showed thatgutD transgenic maize plants had an increased tolerance to salt stress compared with nontransgenic ones. Project supported by the National Natural Science Foundation of China (Grant No. 39670413) and “863” State High Technology Development Program.     

玉米ZmPHO2基因家族克隆及其低磷胁迫下的表达

PHO2(编码一个泛素结合酶E2)作为磷高亲和转运体PHT1的负调控子,在维持植物体内磷的动态平衡中发挥重要作用。该研究以拟南芥和水稻中的PHO2为基础,从玉米自交系B73基因组中鉴定出9个ZmPHO2基因家族成员,在系统进化关系上将其分为3类。在玉米自交系178中克隆了上述9个基因的CDS全长序列,保守结构域分析发现,ZmPHO2蛋白质序列中均有1个由约130个氨基酸组成的泛素结合酶E2催化结构域(UBCc),其中包含1个重要的保守氨基酸(半胱氨酸)。实时荧光定量结果表明,低磷胁迫处理后,所有ZmPHO2基因均有表达,并呈现不同的表达模式,主要表现为叶与根之间的组织差异和玉米自交系178与9782之间的基因型差异,而在同一组织多数基因间的表达差异不明显。其中,ZmPHO2;H2在自交系9782的根中持续下调表达,但在叶中持续上调表达,表明ZmPHO2;H2可能参与调控磷素在叶与根之间的运输,以维持地上部分和地下部分磷的平衡。     

Cytogenetic studies in the genus Zea

Summary Premeiotic colchicine treatment brings about the production of one to five quadrivalents in Zea mays ssp. mays (maize, 2n=20) and an increase in the number of quadrivalents from five to ten in Zea perennis (2n=40). The results confirm the allotetraploid nature of maize and suggest that the species possesses two homoeologous genomes (A2A2 B2B2) that fail to pair, probably due to the presence of Ph-like genes. Moreover, the autoallooctoploid nature of Zea perennis, with a genome formula A1A1 A1A1 C1C1 C2C2, is supported by the present results.     

A comparison of apomictic reproduction in eastern gamagrass (Tripsacum dactyloides (L.) L.) and maize-Tripsacum hybrids

The expression of gene(s) governing apomictic reproduction inTripsacum provides the best foundation for comparing the effectiveness of apomictic reproduction in a series of maize-Tripsacum hybrids. Several 38-chromosome, apomictic maize-Tripsacum hybrids are available which possess the gene(s) conferring apomictic reproduction fromTripsacum. Without a base line for comparison, studies directed towards discerning the successful transfer or effectiveness of gene expression in a maize background are hampered. The objectives of this study are to compare the reproductive features found in apomicticTripsacum with those in apomictic maize-Tripsacum hybrids. In addition, this study determined the feasibility of utilizing these maize-Tripsacum hybrid materials to continue an attempt to transfer the genes into a pure maize background. The frequency and occurrence of five unique reproductive features found in apomictic accessions ofTripsacum dactyloides were compared to the reproductive behaviours exhibited in the maize-Tripsacum hybrids. Results indicate the genes controlling apomixis in tetraploidTripsacum are fully functional in maize-Tripsacum hybrids with diploid and triploid maize constitutions. The ability of theTripsacum apomictic genes to retain full expression provides evidence to continue their transfer to a diploid or tetraploid maize background.The use of company names in this publication does not imply endorsement by the USDA-ARS, or the product names or criticism of similar ones not mentioned. All programs and services of the U.S. Department of Agriculture are offered on a nondiscriminatory basis without regard to race, color, national origin, religion, sex, age, marital status, or handicap.     

Characterization,expression and phylogenetic study of R2R3-MYB genes in orchid

cDNA fragments representing 21 R2R3-MYB genes were isolated by RT-PCR from the Dendrobiumorchid hybrid Woo Leng. Six full-length cDNA clones were obtained from a flower cDNA library, four of which, DwMYB1, DwMYB2, DwMYB8 and DwMYB10, represent typical plant R2R3-MYB genes. The conceptual DwMYB4 protein is truncated at the C-terminal region and contains the R2 repeat and the N-terminal half of the R3 repeat (R2R3). DwMYB4 expression is restricted to flowers. DwMYB9 contains an 8 amino acid N-terminal deletion in the R2 repeat (R2R3) and is expressed at high levels in mature flower and inflorescence, but at very low levels in young flower buds. DwMYB8 and DwMYB10 show similar expression patterns and share very high sequence similarity in the N-terminal part of the MYB domain. Analysis of amino acid substitution indicated that the pattern and type of substitution between Arabidopsis and maize are quite different. Maize may have more conserved substitution in the MYB^BRH domain than Arabidopsis.     

Analyses of mutants of three genes that influence root hair development in Zea mays (Gramineae) suggest that root hairs are dispensable

Root hairs are specialized epidermal cells that are thought to play an important role in plant nutrition by facilitating the absorption of water and nutrients. Three maize mutants with abnormal root hair morphologies (rthl, rth2, and rth3) have been isolated from Mutator transposon stocks. All three root hair mutant phenotypes are controlled by single recessive alleles. The rthl mutant initiates normal-looking root hair primordia that fail to elongate. The normal-looking root hair primordia of the rth2 mutant elongate to only approximately one-fifth to one-fourth the length of wild type root hairs. Like rth1 primordia, rth3 primordia undergo little elongation. However, unlike the relatively normal-looking rth1 primordia, rth3 primordia are distinctly abnormal when viewed through a scanning electron microscope. The rth1 mutant exhibits pleiotropic nutrient deficiencies, while the rth2 and rth3 mutants grow vigorously. This finding suggests that under some environmental conditions, root hairs are less important to plant growth than has been previously thought. The rthl, rth2, and rth3 genes have been mapped to chromosomes 1L, 5L, and 1S, respectively, via crosses with BA translocation stocks. The rth2 allele exhibits reduced transmission through the male gametophyte, but a normal rate of transmission through female gametophytes; rth1 and rth3 are transmitted at normal rates.     

A New Retroposed Gene in <Emphasis Type="Italic">Drosophila</Emphasis> Heterochromatin Detected by Microarray-Based Comparative Genomic Hybridization

A genomic pattern of new gene origination is often dependent on a genomic method that can efficiently identify a statistically adequate number of recently originated genes. The heterochromatic regions have often been viewed as genomic deserts with low coding potential and thus a low flux of new genes. However, increasing reports revealed unexpected roles of heterochromatic regions in the evolution of genes and genomes. We identified recently retroposed genes that originated in heterochromatic regions in Drosophila, by developing microarray-based comparative genomic hybridization (CGH) with multiple species. This new gene family, named Ifc-2h, originated in the common ancestor of the clade of D. simulans, D. mauritiana, and D. sechellia. The sequence features and phylogenetic distribution indicated that Ifc-2h resulted from the retroposition from its parental gene, Infertile crescent (Ifc), and integrated into heterochromatic region of common ancester of the three sibling species 2 million years ago. Expression analysis revealed that Ifc-2h had developed a new expression pattern by recruiting a putative regulatory element from its target sequence. The distribution of indel variation in Ifc-2h of D. simulans and D. mauritiana revealed a significant sequence constraint, suggesting that the Ifc-2h gene may be functional. These analyses cast fresh insight into the evolution of heterochromatin and the origin of its coding regions. Electronic Supplementary Material Electronic Supplementary material is available for this article at and accessible for authorised users. [Reviewing Editor: Dr. Martin Kreitman]     

Diversity and seasonal phenology of aboveground arthropods in conventional and transgenic maize crops in Central Spain

One of the major concerns regarding the release of Bt maize is its potential negative impact on non-target organisms present in this crop. In this paper, we compare the temporal phenology and community structure of the aboveground arthropods in commercial Bt maize fields in Central Spain with those of conventional maize crops, with or without an insecticide (imidacloprid) seed treatment, over a period of three years. Spiders, harvestmen, centipedes, ground beetles, rove beetles, carrion beetles, click beetles, earwigs and damsel bugs were captured in pitfall traps every year in sufficient number to provide meaningful phenological data. One predator spider and three omnivorous species of ground beetles have been consistently present in the maize fields: Pardosa occidentalis, Poecilus cupreus, Pseudophonus rufipes and Pseudophonus griseus, respectively. Rove beetles were caught to a lesser extent, with three dominant species: Acrotona aterrima, Philonthus varians and Platystethus nitens. The variability in activity–density patterns of the aboveground fauna was mainly influenced by the year, but no detrimental effects could be attributed to Bt maize. The only exception being the changes detected in rove beetles, although these differences were transitory and varied from year to year. No changes in species richness and diversity indices for spiders and ground beetles resulted from treatments. However, imidacloprid-treated maize caused a reduction in species richness of rove beetles, even though the abundance of the main species was not reduced. Our results suggest that Bt maize could be compatible with natural enemies that are common in maize fields in Spain.     

Comparative genomic mapping between a 754 kb region flanking DREB1A in Arabidopsis thaliana and maize

Comparative mapping between model plant species for which the complete genome sequence is known and crop species has been suggested as a new strategy for the isolation of agronomically valuable genes. In this study, we tested whether comparative mapping between Arabidopsisand maize of a small region (754 kb) surrounding the DREB1A gene in Arabidopsis could lead to the identification of an orthologous region in maize containing the DREB1A homologue. The genomic sequence information available for Arabidopsis allowed for the selection of conserved, low-copy genes that were used for the identification of maize homologues in a large EST database. In total, 17 maize homologues were mapped. A second BLAST comparison of these genes to the recently completed Arabidopsis sequence revealed that 15 homologues are likely to be orthologous as the highest similarity score was obtained either with the original Arabidopsis gene or with a highly similar Arabidopsis gene localized on a duplication of the investigated region on chromosome 5. The map position of these genes showed a significant degree of orthology with the Arabidopsis region. Nevertheless, extensive duplications and rearrangements in the Arabidopsisand maize genomes as well as the evolutionary distance between Arabidopsis and maize make it unlikely that orthology and collinearity between these two species are sufficient to aid gene prediction and cloning in maize.