Analysis of a herbicide resistant mutant obtained by transformation of the Chlamydomonas chloroplast

A herbicide resistant Chlamydomonas double mutant (I219A264) has been obtained by transforming the psbA deletion mutant FuD7 with a cloned psbA gene fragment containing mutations in codons 219 and 264. Copies from both the recipient (FuD7) genome and the genome carrying the mutated psbA gene persist in the transformant. This stable heteroplasmic state appears to be required for photoautotrophic growth. Comparison of resistance profiles for classical and phenol-type inhibitors of the double mutant and the corresponding single mutants demonstrates independent, additive contributions of both amino acids to herbicide binding. The approach chosen here to modify the psbA gene should be useful in those cases where consequences of psbA gene manipulations are not predictable with respect to inhibitor resistance.Abbreviations DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - DCPIP 2,6-dichlorophenol-indophenol - I₅₀ herbicide concentration giving 50% inhibition - PCR polymerase chain reaction - Q_B secondary electron acceptor of Photosystem II     

Expression of the B subunit of <Emphasis Type="Italic">E. coli</Emphasis> heat-labile enterotoxin in tobacco using a herbicide resistance gene as a selection marker

The B subunit of Escherichia coli heat-labile enterotoxin (LTB) has been transformed to plants for use as an edible vaccine. We have developed a simple and reliable Agrobacterium-mediated transformation method to express synthetic LTB gene in N. tabacum using a phosphinothricin acetyltransferase (bar) gene as a selectable marker. The synthetic LTB gene adapted to the coding sequence of tobacco plants was cloned to a plant expression vector under the control of the ubiquitin promoter and transformed to tobacco by Agrobacterium-mediated transformation. Transgenic plants were selected in the medium supplemented with 5 mg l^-1 phosphinothricin (PPT). The amount of LTB protein detected in the transgenic tobacco was approximately 3.3% of the total soluble protein, approximately 300-fold higher than in the plants generated using the native LTB gene under the control of the CaMV 35S promoter. The transgenic plants that were transferred to a greenhouse had harvested seeds that proved to be resistant to herbicide. Thus, the described protocol could provide a useful tool for the transformation of tobacco plants.     

ISOLATION AND CHARACTERIZATION OF A CELL WALL‐DEFECTIVE MUTANT OF CHLAMYDOMONAS MONOICA (CHLOROPHYTA)1

Cell wall–defective strains of Chlamydomonas have played an important role in the development of transformation protocols for introducing exogenous DNA (foreign genes or cloned Chlamydomonas genes) into C. reinhardtii. To promote the development of similar protocols for transformation of the distantly related homothallic species, C. monoica, we used UV mutagenesis to obtain a mutant strain with a defective cell wall. The mutant, cw‐1, was first identified on the basis of irregular colony shape and was subsequently shown to have reduced plating efficiency and increased sensitivity to lysis by a non‐ionic detergent as compared with wild‐type cells. Tetrad analysis of crosses involving the cw‐1 mutant confirmed 2:2 segregation of the cw:cw⁺ phenotypes, indicating that the wall defect resulted from mutation of a single nuclear gene. The phenotype showed incomplete penetrance and variable expressivity. Although some cells had apparently normal cell walls as viewed by TEM, many cells of the cw‐1 strain had broken cell walls and others were protoplasts completely devoid of a cell wall. Several cw‐1 isolates obtained from crosses involving the original mutant strain showed a marked enhancement of the mutant phenotype and may prove especially useful for future work involving somatic cell fusions or development of transformation protocols.     

Production of herbicide-resistant transgenic sweet potato plants through <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis> method

Transgenic herbicide-resistant sweet potato plants [Ipomoea batatas (L.) Lam.] were produced through Agrobacterium-mediated transformation system. Embryogenic calli derived from shoot apical meristems were infected with Agrobacterium tumefaciens strain EHA105 harboring the pCAMBIA3301 vector containing the bar gene encoding phosphinothricin N-acetyltransferase (PAT) and the gusA gene encoding β-glucuronidase (GUS). The PPT-resistant calli and plants were selected with 5 and 2.5 mg l⁻¹ PPT, respectively. Soil-grown plants were obtained 28–36 weeks after Agrobacterium-mediated transformation. Genetic transformation of the regenerated plants growing under selection was demonstrated by PCR, and Southern blot analysis revealed that one to three copies of the transgene were integrated into the plant genome of each transgenic plant. Expression of the bar gene in transgenic plants was confirmed by RT-PCR and application of herbicide. Transgenic plants sprayed with Basta containing 900 mg l⁻¹ of glufosinate ammonium remained green and healthy. The transformation frequency was 2.8% determined by herbicide application which was high when compared to our previous biolistic method. In addition, possible problems with multiple copies of transgene were also discussed. We therefore report here a successful and reliable Agrobacterium-mediated transformation of the bar gene conferring herbicide-resistance and this method may be useful for routine transformation and has the potential to develop new varieties of sweet potato with several important genes for value-added traits such as enhanced tolerance to the herbicide Basta.     

一种安全高效大豆转化筛选体系的建立

以灭草烟作为筛选剂,利用基因枪法建立一种安全高效的大豆遗传转化体系.比较不同筛选剂对大豆胚尖外植体丛生芽诱导数目的影响.与卡那霉素、潮霉素和草胺膦等传统筛选剂相比,以灭草烟作为筛选剂可使丛生芽的数目增加1倍以上.克隆了拟南芥突变体csrl-2中突变的乙酰羟基酸合成酶基因(ahas),以其作为筛选标记基因,构建可利用灭草烟作为筛选剂的植物表达载体.利用基因枪法将该载体转化大豆,获得6棵灭草烟抗性植株,分子检测证明外源ahas基因整合到5棵转基因大豆植株的基因组中.     

Cloning, mutagenesis, and characterization of the microalga Parietochloris incisa acetohydroxyacid synthase, and its possible use as an endogenous selection marker

Parietochloris incisa is an oleaginous fresh water green microalga that accumulates an unusually high content of the valuable long-chain polyunsaturated fatty acid (LC-PUFA) arachidonic acid within triacylglycerols in cytoplasmic lipid bodies. Here, we describe cloning and mutagenesis of the P. incisa acetohydroxyacid synthase (PiAHAS) gene for use as an herbicide resistance selection marker for transformation. Use of an endogenous gene circumvents the risks and regulatory difficulties of cultivating antibiotic-resistant organisms. AHAS is present in plants and microorganisms where it catalyzes the first essential step in the synthesis of branched-chain amino acids. It is the target enzyme of the herbicide sulfometuron methyl (SMM), which effectively inhibits growth of bacteria and plants. Several point mutations of AHAS are known to confer herbicide resistance. We cloned the cDNA that encodes PiAHAS and introduced a W605S point mutation (PimAHAS). Catalytic activity and herbicide resistance of the wild-type and mutant proteins were characterized in the AHAS-deficient E. coli, BUM1 strain. Cloned PiAHAS wild-type and mutant genes complemented AHAS-deficient bacterial growth. Furthermore, bacteria expressing the mutant PiAHAS exhibited high resistance to SMM. Purified PiAHAS wild-type and mutant proteins were assayed for enzymatic activity and herbicide resistance. The W605S mutation was shown to cause a twofold decrease in enzymatic activity and in affinity for the Pyruvate substrate. However, the mutant exhibited 7 orders of magnitude higher resistance to the SMM herbicide than that of the wild type.     

Glyphosate as a selective agent for the production of fertile transgenic maize (Zea mays L.) plants

Efficient and reproducible selection of transgenic cells is an essential component of a good transformation system. In this paper, we describe the development of glyphosate as a selective agent for the recovery of transgenic embryogenic corn callus and the production of plants tolerant to Roundup^® herbicide. Glyphosate, the active ingredient in Roundup^® herbicide inhibits the enzyme 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) and thus prevents the synthesis of chorismate-derived aromatic amino acids and secondary metabolites in plants. A maize EPSPS gene has been cloned, mutated to produce a modified enzyme resistant to inhibition by glyphosate, and engineered into a monocot expression vector. In addition, a bacterial gene which degrades glyphosate (glyphosate oxidoreductase, or GOX) was also cloned into a similar expression vector. Stably transformed callus has been reproducibly recovered following introduction of mutant maize EPSPS and GOX genes into tissue culture cells by particle bombardment and selection on glyphosate-containing medium. Plants have been regenerated both on and off glyphosate selection medium, and are tolerant to normally lethal levels of Roundup^®. Excellent seed set has been obtained from both self and outcross pollinations from both sprayed and unsprayed regenerated plants. Progeny tests have demonstrated normal Mendelian transmission and tolerance to the herbicide for some of the transgenic events.     

Construction of a Homologous Selectable Marker Gene for Lentinula edodes Transformation

We cloned a gene for the iron sulfur protein (Ip) subunit from an edible mushroom, Lentinula edodes, and introduced a point mutation that confers carboxin resistance into it. The mutant gene successfully transformed L. edodes with high efficiency (9 transformants/2.5 μg vector DNA). Restriction enzyme-mediated integration (REMI) increased the transformation efficiency by about two-fold.     

Expression of a bacterial gene in transgenic tobacco plants confers resistance to the herbicide 2,4-dichlorophenoxyacetic acid

Plants resistant to the herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) were produced through the genetic engineering of a novel detoxification pathway into the cells of a species normally sensitive to 2,4-D. We cloned the gene for 2,4-D monooxygenase, the first enzyme in the plasmid-encoded 2,4-D degradative pathway of the bacterium Alcaligenes eutrophus, into a cauliflower mosaic virus 35S promoter expression vector and introduced it into tobacco plants by Agrobacterium-mediated transformation. Transgenic tobacco plants expressing the highest levels of the monooxygenase enzyme exhibited increased tolerance to 2,4-D in leaf disc and seed germination assays, and young plants survived spraying with levels of herbicide up to eight times the usual field application rate. The introduction of the gene for 2,4-D monooxygenase into broad-leaved crop plants, such as cotton, should eventually allow 2,4-D to be used as an inexpensive post-emergence herbicide on economically important dicot crops.     

Herbicide-resistant Acala and Coker cottons transformed with a native gene encoding mutant forms of acetohydroxyacid synthase

Herbicide-resistant transgenic cotton (Gossypium hirsutum L.) plants carrying mutant forms of a native acetohydroxyacid synthase (AHAS) gene have been obtained by Agrobacterium and biolistic transformation. The native gene, A19, was mutated in vitro to create amino acid substitutions at residue 563 or residue 642 of the precursor polypeptide. Transformation with the mutated forms of the A19 gene produced resistance to imidazolinone and sulfonylurea herbicides (563 substitution), or imidazolinones only (642 substitution). The herbicide-resistant phenotype of transformants was also manifested in their in vitro AHAS activity. Seedling explants of both Coker and Acala cotton varieties were transformed with the mutated forms of the A19 gene using Agrobacterium. In these experiments, hundreds of transformation events were obtained with the Coker varieties, while the Acala varieties were transformed with an efficiency about one-tenth that of Coker. Herbicide-resistant Coker and Acala plants were regenerated from a subset of transformation events. Embryonic cell suspension cultures of both Coker and Acala varieties were biolistically transformed at high frequencies using cloned cotton DNA fragments carrying the mutated forms of the A19 gene. In these transformation experiments the mutated A19 gene served as the selectable marker, and the efficiency of selection was comparable to that obtained with the NPT II gene marker of vector Bin 19. Using this method, transgenic Acala plants resistant to imidazolinone herbicides were obtained. Southern blot analyses indicated the presence of two copies of the mutated A19 transgene in one of the biolistically transformed R₀ plants, and a single copy in one of the R₀ plants transformed with Agrobacterium. As expected. progeny seedlings derived from outcrosses involving the R₀ plant transformed with Agrobacterium segregated in a 1:1 ratio with respect to herbicide resistance. The resistant progeny grew normally after irrigation with 175 g/l of the imidazolinone herbicide imazaquin, which is five times the field application rate. In contrast, untransformed sibling plants were severely stunted.Abbreviations AHAS acetohydroxyacid synthase - CaMV cauliflower mosaic virus - ELISA enzyme linked immunosorbent assay - FW fresh weight - GUS -glucuronidase - IC₅₀ herbicide concentration that produces a 50% reduction in the fresh weight growth of cells - NAA -naphthaleneacetic acid - NPT II neomycin phosphotransferase II - MS Murashige and Skoog (1962)     

Isolation of Hem3 mutants from Candida albicans by sequential gene disruption

Summary Molecular methods for directed mutagenesis in Candida albicans have relied on a combination of gene disruption by transformation to inactivate one allele and UV-induced mitotic recombination or point mutation to produce lesions in the second allele. An alternate method which uses two sequential gene disruptions was developed and used to construct a C. albicans mutant defective in a gene essential for synthesizing tetrapyrrole (uroporphyrinogen I synthase). The Candida gene was cloned from a random library by complementation of the hem3 mutation in Saccharomyces cerevisiae. The complementing region was limited to a 2.0 kb fragment by subcloning and a BglII site was determined to be within an essential region. Linear fragments containing either the Candida URA3 or LEU2 gene inserted into the BglII site were used to disrupt both alleles of a leu2, ura3 mutant by sequential transformation. Ura⁺, Leu⁺ heme-requiring strains were recovered and identified as hem3 mutants by Southern hybridization, transformation to heme independence by the cloned gene, and enzyme assays.     

Non-Mendelian Inheritance of 3-(3,4-Dichlorophenyl)-1,1-dimethylurea-Resistant Thylakoid Membrane Properties in Chlamydomonas

A uniparentally inherited 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU)-resistant mutant of Chlamydomonas reinhardii, Dr2, which has a resistance mechanism of the type defined as `primary,' has been isolated. In vitro Hill reactions catalyzed by isolated thylakoid membranes reveal a reduced apparent affinity of the thylakoids for DCMU. These changes in membrane properties quantitatively account for the resistance of mutant Dr2 to herbicide inhibition of growth. The properties of this mutant show that all of the Hill reaction-inhibiting DCMU binding sites are under identical genetic control. Mutant Dr2 is a useful new uniparental genetic marker, since it has a novel phenotype and it may be possible to identify its altered gene product. The low cross-resistance of Dr2 to atrazine suggests that there may be considerable flexibility in exploiting induced herbicide resistance of crop plants for improving herbicide specificity.     

Cloning and characterization of the trpC gene from an aflatoxigenic strain of Aspergillus parasiticus

The trpC gene in the tryptophan biosynthetic pathway was isolated from an aflatoxigenic Aspergillus parasiticus by complementation of an Escherichia coli trpC mutant lacking phosphoribosylanthranilate isomerase (PRAI) activity. The cloned gene complemented an E. coli trpC mutant deficient in indoleglycerolphosphate synthase (IGPS) activity as well as an Aspergillus nidulans mutant strain that was defective in all three enzymatic activities of the trpC gene (glutamine amidotransferase, IGPS, and PRAI), thus indicating the presence of a complete and functional trpC gene. The location and organization of the A. parasiticus trpC gene on the cloned DNA fragment were determined by deletion mapping and by hybridization to heterologous DNA probes that were prepared from cloned trpC genes of A. nidulans and Aspergillus niger. These experiments suggested that the A. parasiticus trpC gene encoded a trifunctional polypeptide with a functional domain structure organized identically to those of analogous genes from other filamentous fungi. The A. parasiticus trpC gene was expressed constitutively regardless of the nutritional status of the culture medium. This gene should be useful as a selectable marker in developing a DNA-mediated transformation system to analyze the aflatoxin biosynthetic pathway of A. parasiticus.     

Cloning and characterization of the trpC gene from an aflatoxigenic strain of Aspergillus parasiticus.

The trpC gene in the tryptophan biosynthetic pathway was isolated from an aflatoxigenic Aspergillus parasiticus by complementation of an Escherichia coli trpC mutant lacking phosphoribosylanthranilate isomerase (PRAI) activity. The cloned gene complemented an E. coli trpC mutant deficient in indoleglycerolphosphate synthase (IGPS) activity as well as an Aspergillus nidulans mutant strain that was defective in all three enzymatic activities of the trpC gene (glutamine amidotransferase, IGPS, and PRAI), thus indicating the presence of a complete and functional trpC gene. The location and organization of the A. parasiticus trpC gene on the cloned DNA fragment were determined by deletion mapping and by hybridization to heterologous DNA probes that were prepared from cloned trpC genes of A. nidulans and Aspergillus niger. These experiments suggested that the A. parasiticus trpC gene encoded a trifunctional polypeptide with a functional domain structure organized identically to those of analogous genes from other filamentous fungi. The A. parasiticus trpC gene was expressed constitutively regardless of the nutritional status of the culture medium. This gene should be useful as a selectable marker in developing a DNA-mediated transformation system to analyze the aflatoxin biosynthetic pathway of A. parasiticus.     

A New GA-Insensitive Semidwarf Mutant of Rice (Oryza sativa L.) with a Missense Mutation in the SDG Gene

A semidwarf line of Indica rice, Xinguiai, was derived from the progeny of a cross between the double dwarf mutant Xinguiaishuangai and the wild-type variety Nanjing 6. The semidwarf phenotype was controlled by the semidwarf gene, sdg. The second sheath and shoot elongation responses of the dwarf mutant to exogenous gibberellin (GA₃) showed that sdg was insensitive to gibberellin (GA), and its endogenous GAs content was higher than that in wild-type cultivars. The SDG gene was cloned by a map-based cloning method and sequencing analysis revealed that the coding region of sdg had a single nucleotide substitution resulting in a single amino acid change from alanine to threonine. A cleaved amplified polymorphic sequence marker was designed according to sequences from mutant and wild-type materials. This sequence marker could be used to distinguish wild types and mutants, and thus, could be used for molecular marker-assisted selection. The dwarf phenotype of the sdg mutant was restored to a normal phenotype by introducing the wild-type SDG gene. Rice transformation experiments and GUS staining demonstrated that the SDG gene was predominantly expressed in vegetative organs.     

Selection of an atrazine-resistant tobacco cell line having a mutant psbA gene

Summary A mutant cell line that shows high resistance to the photosynthesis-inhibiting herbicide atrazine was selected from cultured photomixotrophic Nicotiana tabacum cv. Samsun NN cells by repeated exposure to toxic levels of the herbicide. This resistance was confirmed by measurements of Hill reaction activity in isolated thylakoid membranes. Nucleotide sequencing revealed that the resistant cell line had a point mutation in its chloroplast psbA gene. The 264th codon, AGT (serine) was changed to ACT (threonine) in this mutant. This new type of mutation also conferred moderate cross-resistance to diuron and subsequently was stable in the absence of continued selection pressure.     

<Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of a low glutelin mutant of ‘Koshihikari’ rice variety using the mutated-acetolactate synthase gene derived from rice genome as a selectable marker

We have developed an efficient rice transformation system that uses only rice genome-derived components. The transgenic ‘Koshihikari’ rice, low-glutelin mutant a123, is capable of accumulating large amounts of bioactive peptides in the endosperm. Agrobacterium-mediated transformation using the mutated-acetolactate synthase (mALS) gene expressed under the control of the callus-specific promoter (CSP) as a selectable marker was used to introduce GFP and an anti-hypertensive hexapeptide into ‘Koshihikari’ a123. The CSP:mALS gene cassette confers pyrimidinyl carboxy herbicide resistance to transgenic rice callus, but is not expressed in regenerated plants. Transformation efficiency of transgenic rice line a123 was improved from about 10% to about 30% by modifying callus induction, callus selection and regeneration media conventionally used for rice tissue culture. An erratum to this article can be found at     

Accessing genes in the tertiary gene pool of rice by direct introduction of total DNA fromZizania palustris (wild rice)

Transfer of useful genes from wild relatives of crop plants has relied upon successful conventional crossing or the availability of the cloned gene. Co-bombardment of rice callus with total genomic DNA from wild rice (Zizania palustris) and a plasmid containing a gene confirming hygromycin resistance allowed recovery under selection of transgenic plants with grain characteristics from wild rice. Amplified Fragment Length Polymorphism (AFLP) analysis suggested that a significant amount of DNA fromZizania was introduced by this procedure. One plant had 16 of a possible 122Zizania specific AFLP markers detected with the primers used. This approach may have potential for introgression of genes from wild relatives in other cases where highly efficient transformation methods are available.     

Mutation in the glmS gene responsible for the cell wall synthesis increases resistance to the herbicide amitrole in the cyanobacterium Synechocystis sp. PCC6803

A DNA fragment transforming the cells of the cyanobacterium Synechocystis sp. PCC 6803 to amitrole (3-amino-1,2,4-triazole) resistance was cloned from the Atr2 mutant resistant to this herbicide. The transforming activity of the cloned fragment was shown to be related to the missence-mutation "Val250-->Leu250" in the glmS gene encoding glucosamine-6-phosphate synthase, a key enzyme of cell wall synthesis. The amino acid substitution is localized in the central nonconservative part of the GlmS protein, far from two reaction centers positioned at the ends of a polypeptide. It is suggested that the mutant protein has lost sensitivity to amitrole. In the wild type strain, this herbicide causes conditional glucosamine auxotrophy (exogenous glucosamine restores ability of the cells to row in the presence of the lethal amitrole concentrations). Val250 is proposed to be allosteric binding site of AM in the GlmS protein of cyanobacterium.     

Stable chloroplast transformation of the unicellular red alga Porphyridium species

Red algae are extremely attractive for biotechnology because they synthesize accessory photosynthetic pigments (phycobilins and carotenoids), unsaturated fatty acids, and unique cell wall sulfated polysaccharides. We report a high-efficiency chloroplast transformation system for the unicellular red microalga Porphyridium sp. This is the first genetic transformation system for Rhodophytes and is based on use of a mutant form of the gene encoding acetohydroxyacid synthase [AHAS(W492S)] as a dominant selectable marker. AHAS is the target enzyme of the herbicide sulfometuron methyl, which effectively inhibits growth of bacteria, fungi, plants, and algae. Biolistic transformation of synchronized Porphyridium sp. cells with the mutant AHAS(W492S) gene that confers herbicide resistance gave a high frequency of sulfomethuron methyl-resistant colonies. The mutant AHAS gene integrated into the chloroplast genome by homologous recombination. This system paves the way for expression of foreign genes in red algae and has important biotechnological implications.