Engineering herbicide-resistant maize using chimeric RNA/DNA oligonucleotides.

Maize plants resistant to imidazolinone herbicides were engineered through targeted modification of endogenous genes using chimeric RNA/DNA oligonucleotides. A precise single-point mutation was introduced into genes encoding acetohydroxyacid synthase (AHAS), at a position known to confer imidazolinone resistance. Phenotypically normal plants from the converted events (C0) were regenerated from resistant calli and grown to maturity. Herbicide leaf painting confirmed the resistance phenotype in C0 plants and demonstrated the anticipated segregation pattern in C1 progeny. DNA cloning and sequencing of the targeted region in resistant calli and derived C0 and C1 plants confirmed the expected mutation. These results demonstrate that oligonucleotide-mediated gene manipulation can be applied to crop improvement. This approach does not involve genomic integration of transgenes. Since the new trait is obtained through modifying a gene within its normal chromosomal context, position effects, transgene silencing, or other concerns that arise as part of developing transgenic events are avoided.

Targeted manipulation of maize genes in vivo using chimeric RNA/DNA oligonucleotides.

Site-specific heritable mutations in maize genes were engineered by introducing chimeric RNA/DNA oligonucleotides. Two independent targets within the endogenous maize acetohydroxyacid synthase gene sequence were modified in a site-specific fashion, thereby conferring resistance to either imidazolinone or sulfonylurea herbicides. Similarly, an engineered green fluorescence protein transgene was site-specifically modified in vivo. Expression of the introduced inactive green fluorescence protein was restored, and plants containing the modified transgene were regenerated. Progeny analysis indicated Mendelian transmission of the converted transgene. The efficiency of gene conversion mediated by chimeric oligonucleotides in maize was estimated as 10(-4), which is 1-3 orders of magnitude higher than frequencies reported for gene targeting by homologous recombination in plants. The heritable changes in maize genes engineered by this approach create opportunities for basic studies of plant gene function and agricultural trait manipulation and also provide a system for studying mismatch repair mechanisms in maize.

Bnm1, a Brassica pollen-specific gene.

cDNA and genomic clones of a new pollen-specific gene, Bnm1, have been isolated from Brassica napus cv. Topas. The gene contains an open reading frame of 546 bp and a single intron of 362 bp. A comparison of the deduced amino acid sequence with sequences in data banks did not show similarity with known proteins. Northern blot analysis of developing pollen showed that Bnm1 mRNA was first detected in bicellular pollen and accumulated to higher levels in tricellular pollen. Bnm1 mRNA was not detected in leaves, stems, roots, pistils, seeds or pollen-derived embryos. RNA in situ hybridization of whole flower buds confirmed that Bnm1 was pollen-specific and expressed late in development. A promoter fragment of the Bnm1 gene fused to the gusA reporter gene yielded similar patterns of tissue specificity and developmental regulation in transgenic B. napus cv. Westar plants; however, the promoter was also active during the early stages of pollen development. The Bnm1 gene, cloned in this study, was derived from the A genome of the allotetraploid species B. napus (AACC). Southern blot analysis indicated that sequences similar to the Bnm1 gene were found in both A and C Brassica genomes. Related sequences were found in all 10 members of the Brassiceae tribe examined, but were not present in all tribes of the Brassicaceae family.

The 5[prime] Flanking Regions of Vicilin and Napin Storage Protein Genes Are Down-Regulated by Desiccation in Transgenic Tobacco.

Drying of seeds, when imposed prematurely, elicits a switch in metabolism; events unique to development, such as synthesis of storage protein, are terminated, whereas syntheses associated with germination and growth are initiated. To determine the role of desiccation in down-regulating the expression of genes for storage proteins, the desiccation responsiveness of the 5[prime] and 3[prime] regulatory regions of the genes encoding the pea storage protein vicilin and the Brassica napus storage protein napin was tested in transgenic tobacco seed. Chimeric genes were introduced into tobacco; these genes consisted of the coding region of the reporter gene for [beta]-glucuronidase (GUS) and 5[prime] and/or 3[prime] regions from the vicilin or napin genes or, as controls, the same regions derived from constitutively expressed genes, presumed to be desiccation insensitive. In transgenic seed expressing the gene constructs containing the vicilin or napin promoters, GUS activities declined during late seed development, and more dramatically, after imbibition of mature dry seed or prematurely dried seed. In contrast, GUS activities increased after seed rehydration when the constitutive viral promoter replaced the storage-protein gene 5[prime] region. Transient expression assays support the hypothesis that premature drying down-regulates the expression of the storage-protein gene promoter. Following desiccation, this region may become insensitive to positive controlling factors; alternatively, changes to trans-acting factors may occur.

Expression of the BnmNAP subfamily of napin genes coincides with the induction of Brassica microspore embryogenesis.

Brassica napus cv. Topas microspores can be diverted from pollen development toward haploid embryo formation in culture by subjecting them to a heat stress treatment. We show that this switch in developmental pathways is accompanied by the induction of high levels of napin seed storage protein gene expression. Changes in the plant growth or microspore culture conditions were not by themselves sufficient to induce napin gene expression. Specific members of the napin multigene family were cloned from a cDNA library prepared from microspores that had been induced to undergo embryogenesis. The majority of napin clones represented three members (BnmNAP2, BnmNAP3 and BnmNAP4) that, along with a previously isolated napin genomic clone (BngNAP1), constitute the highly conserved BnmNAP subfamily of napin genes. Both RNA gel blot analysis, using a subfamily-specific probe, and histochemical analysis of transgenic plants expressing a BngNAP1 promoter-beta-glucuronidase gene fusion demonstrated that the BnmNAP subfamily is expressed in embryogenic microspores as well as during subsequent stages of microsporic embryo development.

Isolation of Lhcb3 sequences from Brassica napus: evidence for conserved genes encoding LHCII type III chlorophyll a/b binding proteins.

Three closely related sequences were isolated from Brassica napus genomic DNA and were identified as Lhcb3 (genes encoding type III chlorophyll a/b binding proteins of LHCII, the major light-harvesting complex of photosystem II). These genes, as was observed for a tomato Lhcb3, contain two introns and yield both divergent and conserved predicted amino acid segments as compared with type I and type II polypeptides. One of the B. napus genes, designated Lhcb3*1, is transcribed in vivo, since it is identical to corresponding sequences in a cDNA clone. The protein deduced from another sequence, Lhcb3*2, appears as the most divergent type III so far characterized. The partial sequence of a third gene, Lhcb3*3, was also recovered. The 5' noncoding sequences of Lhcb3*1 and Lhcb3*2, in the far upstream region, are characterized by an extremely high AT content and extensive direct repeats. In the near upstream region, two long Lhcb3*2 segments are very similar to a segment proposed as containing regulatory signals in Lhcb3*1. Specific binding of nuclear proteins to Lhcb3*1 promoter fragments was detected by electrophoretic mobility-shift assays. The evolutionary relationship between genes for type III polypeptides and the other types present in LHCII is discussed.

Identification of an S-locus glycoprotein allele introgressed from B. napus ssp. rapifera to B. napus ssp. oleifera.

Self-incompatible Brassica napus ssp. oleifera lines were generated by introgressing the S-locus from the self-incompatible B. napus ssp. rapifera Z line into the self-compatible cultivars, Topas and Regent, resulting in T2 and R2, respectively. Screening of a cDNA library made from R2 stigma RNA produced several candidate SLG (S-locus glycoprotein) cDNAs. One of the cDNAs, A14, was found to be represented in only the R2, T2 and Z lines. In addition, the corresponding A14 gene was demonstrated to segregate with the T2 self-incompatibility phenotype in an F2 population derived from a cross between T2 and Topas, and to exhibit high mRNA levels in the stigmas prior to anthesis. Sequence analysis of the A14 cDNA revealed close homology to B. oleracea SLG alleles associated with a Class I high activity self-incompatibility phenotype.

Evaluation of transgenic canola plants under field conditions.

Eleven independent transgenic canola (Brassica napus ssp. oleifera L. cv. Westar and Regent) lines were evaluated in the field. The plants carried a neomycin phosphotransferase (NPTII) gene for kanamycin resistance that was introduced via Agrobacterium-mediated transformation. NPTII enzyme assays, Southern blot by hybridizations and progeny analysis, confirmed the stable, heritable integration and expression of the introduced NPTII gene. A number of agronomic characteristics evaluated under field conditions, including maturity yield, and oil and protein content, were all statistically comparable between the transformed and nontransforemd platns. These results indicate that canola can be genetically engineered successfully, and that the Agrobacterium-based transformation system employed does not induced any adverse effects on the intrinsic agronomic and qualitative traits critical to the agricultural industry.

Promoter for a Brassica napus ribulose bisphosphate carboxylase/oxygenase small subunit gene binds multiple nuclear factors and contains a negative-strand open reading frame encoding a putative transmembrane protein.

Using a fractionated genomic bank, we have cloned and characterized a Brassica napus gene (rbcSF1) encoding the small sub-unit of ribulose 1,5-bisphosphate carboxylase. The promoter of this gene contains a 29 bp direct repeat capable of forming a single or a double hairpin loop, and three elements that are recognized by leaf nuclear proteins in vitro. The most upstream are the S-box, a small A/T-rich sequence between -516 and -512, and the F-box between -492 and -475. Finally, we have also observed binding to the G-box, a regulatory element common to numerous plant promoters. The promoter of rbcSF1 also has a 113 amino acids open reading frame (ORF113) in the non-coding strand. When used to probe a northern blot of leaf RNA, this ORF hybridizes to a 1.5 kb transcript. The protein encoded by ORF113 contains a transmembrane domain.

A rapid immunoprecipitation assay for neomycin phosphotransferase II expression in transformed bacteria and plant tissues.

Anti-kanamycin antibodies produced in rabbits, following coupling of the antibiotic to bovine serum albumin, were used to immunoprecipitate radioactively labelled phosphorylated kanamycin from transformed bacterial or plant extracts in a novel assay system, for the detection of neomycin phosphotransferase II (NPTII) activity. Radioactive counts in the immunoprecipitated pellet give a semiquantitative measure of the kanamycin phosphorylation and hence the amount of NPTII activity. This assay is sensitive, uses very small amounts of radioactivity, and is very rapid, allowing many samples to be processed within a few hours. Immunoprecipitated counts from reactions with bacteria carrying a kanamycin resistance gene or from tobacco and Brassica napus plants transformed with NPTII gene-containing vectors were consistently higher than counts from nontransformed controls. Results obtained with this assay correlate well with those from the previously described gel overlay and dot-blot assays, but can be obtained in an appreciably shorter time frame.

Terminal protein association and sequence homology in linear mitochondrial plasmid-like DNAs of sorghum and maize.

The linear extrachromosomal mitochondrial plasmid-like DNAs from the Ru cytoplasm of maize, and M35-1 and IS1112C cytoplasms of sorghum, possess 5' terminally-attached proteins. These molecules required proteinase K treatment for mobility in agarose gels and were susceptible to exonuclease III but not lambda exonuclease cleavage. Hybridizations, under stringent conditions, indicated that the sorghum plasmid-like DNAs, N1 and N2, did not possess DNA sequence homology to cloned central regions of S1 and S2, the linear mitochondrial plasmid-like DNAs present in S cytoplasm of maize. In addition, a novel 4.2kb, DNAase sensitive, RNAase insensitive band, exhibiting homology to internal sequences from maize S2, was observed in the sorghum IS1112C cytoplasm only.

Regulation of gene expression in corn (Zea mays L.) by heat shock. II. In vitro analysis of RNAs from heat-shocked seedlings.

Five-day-old maize seedlings subjected to heat shock exhibit a dramatic enhancement in the synthesis of a small group of polypeptides. Isolation of total RNA from control and heat-shocked maize plumules, fractionation of poly(A)+ mRNA by oligo(dT)-cellulose chromatography, and in vitro translations of the RNAs in both the rabbit reticulocyte and the wheat germ systems indicates that there is remarkable fidelity of the mRNA pool obtained from heat-shocked plumules to reproduce in vitro those same polypeptides whose synthesis is greatly elevated in the intact, heat-shocked plumule. Moreover, these heat-shock polypeptides with molecular masses of 108 000, 89 000, 84 000, 73 000, and 18 000 are translated from polyadenylated mRNAs. The absence of a 76 000 dalton heat-shock polypeptide (HSP) and the presence of fewer isoelectric point variants of the 89 000 and 84 000 dalton HSPs among the in vitro translation products suggests that translational and (or) posttranslational regulatory mechanisms might be operative in determining the final spectrum of the maize heat-shock proteins.

Regulation of gene expression in corn (Zea Mays L.) by heat shock.

Subjecting 5-day-old plumules of corn (Zea mays L.) to elevated temperatures for brief periods of time causes the pattern of protein synthesis to shift from the production of a broad spectrum of proteins to the new and (or) enhanced synthesis of a small number of heat-shock polypeptides (HSPs). Most notable is the depressed synthesis of a major polypeptide (relative mass (Mr) = 93 000 and isoelectric point = 8.0) normally made at 27 degrees C and the enhanced and (or) new synthesis of polypeptides with MrS of 108 000, 89 000, 84 000, 76 000, 73 000, and 18 000, following 1 h of heat shock. These six HSPs is observed within 120 min following heat shock. Recovery from heat shock is rapid; after 6 to 8 h at 27 degrees C following heat shock, the polypeptide pattern is indistinguishable from the control. Extracts from individual heat-shocked shoots produced polypeptide synthetic patterns identical to those from extracts from 20 shoots, regardless of whether single shoots were intact or excised during labelling. Single 5-day-old primary roots exhibited polypeptide synthetic patterns and responded to heat shock in a manner similar to shoots. This is the first demonstration of the induction of heat-shock polypeptides in a whole, intact higher plant.