Establishment of a soybean (Glycine max Merr. L) transposon-based mutagenesis repository

Soybean is a major crop species providing valuable feedstock for food, feed and biofuel. In recent years, considerable progress has been made in developing genomic resources for soybean, including on-going efforts to sequence the genome. These efforts have identified a large number of soybean genes, most with unknown function. Therefore, a major research priority is determining the function of these genes, especially those involved in agronomic performance and seed traits. One means to study gene function is through mutagenesis and the study of the resulting phenotypes. Transposon-tagging has been used successfully in both model and crop plants to support studies of gene function. In this report, we describe efforts to generate a transposon-based mutant collection of soybean. The Ds transposon system was used to create activation-tagging, gene and enhancer trap elements. Currently, the repository houses approximately 900 soybean events, with flanking sequence data derived from 200 of these events. Analysis of the insertions revealed approximately 70% disrupted known genes, with the majority matching sequences derived from either Glycine max or Medicago truncatula sequences. Among the mutants generated, one resulted in male-sterility and was shown to disrupt the strictosidine synthase gene. This example clearly demonstrates that it is possible to disrupt soybean gene function by insertional mutagenesis and to derive useful mutants by this approach in spite of the tetraploid nature of the soybean genome.     

Molecular evolution of glycinin and β-conglycinin gene families in soybean (Glycine max L. Merr.)

There are two main classes of multi-subunit seed storage proteins, glycinin (11S) and β-conglycinin (7S), which account for approximately 70% of the total protein in a typical soybean seed. The subunits of these two protein classes are encoded by a number of genes. The genomic organization of these genes follows a complex evolutionary history. This research was designed to describe the origin and maintenance of genes in each of these gene families by analyzing the synteny, phylogenies, selection pressure and duplications of the genes in each gene family. The ancestral glycinin gene initially experienced a tandem duplication event; then, the genome underwent two subsequent rounds of whole-genome duplication, thereby resulting in duplication of the glycinin genes, and finally a tandem duplication likely gave rise to the Gy1 and Gy2 genes. The β-conglycinin genes primarily originated through the more recent whole-genome duplication and several tandem duplications. Purifying selection has had a key role in the maintenance of genes in both gene families. In addition, positive selection in the glycinin genes and a large deletion in a β-conglycinin exon contribute to the diversity of the duplicate genes. In summary, our results suggest that the duplicated genes in both gene families prefer to retain similar function throughout evolution and therefore may contribute to phenotypic robustness.     

Expression of recombinant human IFN-γ protein in soybean (Glycine max L.)

Plant Cell, Tissue and Organ Culture (PCTOC) - Globular androgenic haploid embryos of TV21 and TV19 cultivars of Camellia ssp., obtained on embryo induction medium (EIM), Murashige and Skoog medium...     

Effect of γFe2O3 nanoparticles on photosynthetic characteristic of soybean (Glycine max (L.) Merr.): foliar spray versus soil amendment

There is an urgent need to address comprehensive biosafety issues associated with the use of Fe₂O₃ nanoparticles (IONPs). The present study was performed to investigate the effect of 6-nm IONPs and citrate-coated IONPs (IONPs-Cit) on photosynthetic characteristics and root elongation during germination of Glycine max (L) Merr. Plant physiological performance was assessed after foliar and soil IONPs fertilization. No adverse impacts at any growth stage of the soybeans were observed after application of IONPs. The Fe₂O₃ nanoparticles produced a significant positive effect on root elongation, particularly when compared to the bulk counterpart (IOBKs) suspensions of concentrations greater than 500 mg L^?1. Furthermore, IONPs-Cit significantly enhanced photosynthetic parameters when sprayed foliarly at the eight-trifoliate leaf stage (P < 0.05). The increases in photosynthetic rates following spraying were attributed to increases in stomatal opening rather than increased CO₂ uptake activity at the chloroplast level. We observed more pronounced positive effects of IONPs via foliar application than by soil treatment. This study concluded that IONPs coated with citric acid at IONPs to citrate molar ratio of 1:3 can markedly improve the effectiveness of insoluble iron oxide for Fe foliar fertilization.     

Agrobacterium tumefaciens– mediated transformation of soybean [Glycine max (L.) Merrill.] using immature zygotic cotyledon explants

Agrobacterium tumefaciens -mediated transformation of soybean [Glycine max (L.) Merrill. cv. Jack] using immature zygotic cotyledons was investigated to identify important factors that affected transformation efficiency and resulted in the production of transgenic soybean somatic embryos. The factors evaluated were initial immature zygotic cotyledon size, Agrobacterium concentration during inoculation and co-culture and the selection regime. Our results showed that 8- to 10-mm zygotic cotyledons exhibited a higher transformation rate, as indicated by transient GUS gene expression, whereas the smaller zygotic cotyledons, at less than 5 mm, died shortly after co-cultivation. However, the smaller zygotic cotyledon explants were found to have a higher embryogenic potential. Analysis of Agrobacterium and immature cotyledon explant interactions involved two Agrobacterium concentrations for the inoculation phase and three co-culture regimes. No differences in explant survival or somatic embyogenic potential were observed between the two Agrobacterium concentrations tested. Analysis of co-culture regimes revealed that the shorter co-culture times resulted in higher explant survival and higher somatic embryo production on the explants, whereas the co-culture time of 4 days severely reduced survival of the cotyledon explants and lowered their embryogenic potential. Analysis of selection regimes revealed that direct placement of cotyledon explants on hygromycin 25 mg/l was detrimental to explant survival, whereas 10 mg/l gave continued growth and subsequent somatic embryo development and plant regeneration. The overall transformation frequency in these experiments, from initial explant to whole plant, was 0.03 %. Three fertile soybean plants were obtained during the course of these experiments. Enzymatic GUS assays and Southern blot hybridizations confirmed the integration of T-DNA and expression of the GUS-intron gene in the three primary transformants. Analysis of 48 progeny revealed that three copies of the transgene were inherited as a single Mendelian locus. Received: 6 December 1999 / Revised: 11 February 2000 / Accepted: 14 March 2000     

Chromatographic profiling of Pancharishta at different stages of its development using HPTLC,HPLC, GC–MS and UPLC–MS

Pancharishta is the traditional Ayurvedic polyherbal formulation prepared by decoction of plant materials followed by fermentation for preservation and facilitation of extraction due to the production of alcohol. Since the preparation of pancharishta involves various steps. The aim of the current investigation was to carry out comparative metabolomics profiling at different stages of preparation for the understanding impact of different steps and ingredients. A decoction of 21 plant materials are main components in pancharishta formulations followed by fermentation and addition of other ingredients with or without fermentation yielded eight different formulations. The vacuum concentration of pancharishta samples yielded a semisolid mass of different formulations ranging from 8 to 37% w/v. The HPTLC fingerprinting analysis of samples was carried out in butanol: ethanol: 0.5% v/v ammonia (5:4:0.5, v/v/v). Derivatization with anisaldehyde-sulphuric acid showed the presence of two major peaks at R_f 0.29 and 0.35. The peak at R_f 0.29 is intense in a formulation containing 12 extra plant materials. Quantification of gallic acid, ellagic acid, tannic acid, kaemferol and quercetin were carried out on newly developed HPLC method using acetonitrile and 0.5% v/v formic acid with a gradient elution. A significant difference in their content was found in different formulations. Further, polar and nonpolar metabolites of pancharishtha were analyzed using UPLC–MS and GC–MS, respectively. GC–MS profiling results in the identification of 144 metabolites among them 26 are common metabolites at different stages. The UPLC–MS analysis resulted in the tentative identification of 43 metabolites. The results of UPLC–MS and GC–MS analysis were used for multivariate analysis using XLSTAT. Principal Component Analysis plot distributed all samples into four different clusters with two formulations each.     

Epigenetics’ Role in the Common Bean (Phaseolus vulgaris L.) and Soybean (Glycine max (L.) Merr.) Nodulation: a Review

Plant Molecular Biology Reporter - Nitrogen-fixing bacteria establish symbiosis with legumes for the biological nitrogen fixation process in specific root structures called nodules. Among these...     

The isolation,characterization and sequence of two divergent β-tubulin genes from soybean (Glycine max L.)

Two divergent -tubulin genes (designated S-1 and S-2) were isolated by screening a soybean genomic library with a Chlamydomonas reinhardtii -tubulin cDNA probe. Restriction fragment analysis of the clones recovered, and of soybean genomic DNA, indicated that these represent two unique classes of structurally different -tubulin genes in the soybean genome. However, it is possible that unidentified members of these classes or additional highly divergent classes of -tubulin genes (thus far undetected) exist in the soybean genome. The S-1 and S-2 genomic clones were sequenced, revealing that both are potentially functional genes which would encode -tubulins of 445 and 449 amino acids, respectively. A comparison of their derived amino acid sequences with -tubulins from several organisms showed that they are most homologous to Chlamydomonas -tubulin (85–87%), with lesser degrees of homology to -tubulins of vertebrate species (79–83%), Trypanosoma brucei (80–81%) and Saccharomyces cerevisiae (66–68%). The amino acid sequences of S-1 and S-2 are as divergent from each other as they are from the Chlamydomonas -tubulin. The amino acids at the diverged positions in S-2 are nearly all conservative substitutions while in S-1, 18 of the 69 substitutions were non-conservative. Both soybean -tubulin genes contain two introns in exactly the same positions. The first soybean intron is located in the same position as the third intron of the Chlamydomonas -tubulin genes. Codon usage in the two soybean -tubulins is remarkably similar (D ²=0.87), but differs from codon usage in other soybean genes.     

A genetic map of soybean (Glycine max L.) using an intraspecific cross of two cultivars: ‘Minosy’ and ‘Noir 1’

Genetic markers were mapped in segregating progeny from a cross between two soybean (Glycine max (L.) Merr.) cultivars: Minsoy (PI 27.890) and Noir 1 (PI 290.136). A genetic linkage map was constructed (LOD 3), consisting of 132 RFLP, isozyme, morphological, and biochemical markers. The map defined 1550cM of the soybean genome comprising 31 linkage groups. An additional 24 polymorphic markers remained unlinked. A family of RFLP markers, identified by a single probe (hybridizing to an interspersed repeated DNA sequence), extended the map, linking other markers and defining regions for which other markers were not available.     

Comparative metabolite profiling and fingerprinting of medicinal licorice roots using a multiplex approach of GC–MS,LC–MS and 1D NMR techniques

Glycyrrhiza glabra, commonly known as licorice, is a popular herbal supplement used for the treatment of chronic inflammatory conditions and possesses anticancer and antiviral activities. This species contains a plethora of phytochemicals including terpenoids, saponins, flavonoids, polyamines and polysaccharides. The full complement of bioactive compounds has yet to be elucidated, a step necessary in order to explain its medicinal use. There are over 30 species in the Glycyrrhiza genus world-wide, most of which have been little characterized in terms of phytochemical or pharmacological properties. Here, large scale multi-targeted metabolic profiling and fingerprinting techniques were utilized to help gain a broader insight into Glycyrrhiza species chemical composition. UV, MS and NMR spectra of extracted components were connected with NMR, MS, and multivariate analyses data from Glycyrrhiza glabra, Glycyrrhiza uralensis, Glycyrrhiza inflata and Glycyrrhiza echinata. Major peaks in ¹H NMR and MS spectra contributing to the discrimination among species were assigned as those of glycyrrhizin, 4-hydroxyphenyl acetic acid, and glycosidic conjugates of liquiritigenin/isoliquiritigenin. Primary metabolites profiling using GC–MS revealed the presence of cadaverine, an amino acid, exclusively found in G. inflata roots. Both LC–MS and NMR were found effective techniques in sample classification based on genetic and or geographical origin as revealed from derived PCA analysis.     

Primary structure and sequence organization of the 16S–23S spacer in the ribosomal operon of soybean (Glycine max L.) chloroplast DNA

Summary The nucleotide sequence of a spacer region between 16S and 23S rRNA genes from soybean chloroplasts has been determined. The spacer region is over 3000 bp long and contains two tRNA genes, coding for rRNA^Ile and tRNA^Ala which contain intervening sequences of 953 and 811 base pairs respectively. There is a strong homology between the two introns suggesting that they have a common origin. These spacer tRNAs are synthesized as part of a kb precursor molecule containing 16S and 23S rRNA sequences.     

大豆(Glycine max(L.)Merr.)致敏蛋白Gly m Bd 30K检测方法及低致敏优异种质鉴定

Gly m Bd 30K蛋白是大豆中主要的免疫显性过敏原之一,会引起人和牲畜腹泻和肠道炎症等过敏反应.因此,发掘低Gly m Bd 30K蛋白含量优异种质对于培育优质大豆品种具有重要意义.为了获得致敏蛋白Gly m Bd 30K低含量的优异种质,根据Gly m Bd 30K蛋白的190-379aa多肽序列制备多克隆抗体...     

Use of chemical ionization for GC–MS metabolite profiling

Metabolite profiling is commonly performed by GC–MS of methoximated trimethylsilyl derivatives. The popularity of this technique owes much to the robust, library searchable spectra produced by electron ionization (EI). However, due to extensive fragmentation, EI spectra of trimethylsilyl derivatives are commonly dominated by trimethylsilyl fragments (e.g. m/z 73 and 147) and higher m/z fragment ions with structural information are at low abundance. Consequently different metabolites can have similar EI spectra, and this presents problems for identification of “unknowns” and the detection and deconvolution of overlapping peaks. The aim of this work is to explore use of positive chemical ionization (CI) as an adjunct to EI for GC–MS metabolite profiling. Two reagent gases differing in proton affinity (CH₄ and NH₃) were used to analyse 111 metabolite standards and extracts from plant samples. NH₃-CI mass spectra were simple and generally dominated by [MH]⁺ and/or the adduct [M+NH₄]⁺. For the 111 metabolite standards, m/z 73 and 147 were less than 3% of basepeak in NH₃-CI and less than 30% of basepeak in CH₄-CI. With CH₄-CI, [MH]⁺ was generally present but at lower relative abundance than for NH₃-CI. CH₄-CI spectra were commonly dominated by losses of CH₄ [M+1-16]⁺, 1–3 TMSOH [M+1-nx90]⁺, and combinations of CH₄ and TMSOH losses [M+1-nx90-16]⁺. CH₄-CI and NH₃-CI mass spectra are presented for 111 common metabolites, and CI is used with real samples to help identify overlapping peaks and aid identification via determination of the pseudomolecular ion with NH₃-CI and structural information with CH₄-CI.

     

Use of chemical ionization for GC–MS metabolite profiling

Metabolite profiling is commonly performed by GC–MS of methoximated trimethylsilyl derivatives. The popularity of this technique owes much to the robust, library searchable spectra produced by electron ionization (EI). However, due to extensive fragmentation, EI spectra of trimethylsilyl derivatives are commonly dominated by trimethylsilyl fragments (e.g. m/z 73 and 147) and higher m/z fragment ions with structural information are at low abundance. Consequently different metabolites can have similar EI spectra, and this presents problems for identification of “unknowns” and the detection and deconvolution of overlapping peaks. The aim of this work is to explore use of positive chemical ionization (CI) as an adjunct to EI for GC–MS metabolite profiling. Two reagent gases differing in proton affinity (CH₄ and NH₃) were used to analyse 111 metabolite standards and extracts from plant samples. NH₃-CI mass spectra were simple and generally dominated by [MH]⁺ and/or the adduct [M+NH₄]⁺. For the 111 metabolite standards, m/z 73 and 147 were less than 3% of basepeak in NH₃-CI and less than 30% of basepeak in CH₄-CI. With CH₄-CI, [MH]⁺ was generally present but at lower relative abundance than for NH₃-CI. CH₄-CI spectra were commonly dominated by losses of CH₄ [M+1-16]⁺, 1–3 TMSOH [M+1-nx90]⁺, and combinations of CH₄ and TMSOH losses [M+1-nx90-16]⁺. CH₄-CI and NH₃-CI mass spectra are presented for 111 common metabolites, and CI is used with real samples to help identify overlapping peaks and aid identification via determination of the pseudomolecular ion with NH₃-CI and structural information with CH₄-CI.