Peculiarities [correction of Pfculiarities] of lipid accumulation in Brassica rapa L. embryos on different stages development under altered gravity

Accumulation of lipid inclusions in Brassica rapa embryos generated under slow horizontal clinorotation and in the laboratory control were analyzed by histochemical methods. The research of lipid accumulation was carried out on consecutive stages of the embryo development, from the moment of two-cellular proembryo formation up to the stages of their full differentiation (21-22-day-old embryos). Accumulation of lipid drops was revealed for the first time at early stages of embryogenesis in this species, beginning from 3-day-old embryos (ball-like stage of embryo development) under clinorotation and in the laboratory control. The quantity of lipid inclusion was estimated by morphometrical analysis. Statistically significant differences between the clinorotation and laboratory control variants in quantity of lipid drops per cell were revealed from 6-day-old embryos (heart-shaped stage). Especially pronounced differences were noted in differentiated embryos (beginning from 12-day-old embryos) under horizontal clinorotation in comparison with the laboratory control. The registered differences testify about influence of altered gravity conditions on lipid accumulation in Brassica rapa embryos.     

津田芜菁MYB77蛋白的原核表达及纯化

目的:建立津田芜菁转录因子MYB77的原核表达系统,并在大肠杆菌中获得表达。方法:RT-PCR获得MYB77的编码序列,将其克隆至pGEM-T载体中,在上下游引物中分别引入HindⅢ和EcoRⅠ酶切位点,PCR获得带酶切位点的目的片段并将其连接到重组表达载体pGEX-KG中,转化大肠杆菌DE3工程菌株,IPTG诱导重组质粒pGEX-KG-MYB77在大肠杆菌DE3中表达带有GST标签的融合蛋白,超声裂解大肠杆菌,用MagneGST ProteinPurification System纯化目的蛋白,通过SDS-PAGE和Western印迹验证GST-MYB77融合蛋白的表达。结果:重组菌株可以表达GST-MYB77融合蛋白,用Western印迹鉴定纯化的融合蛋白,在相对分子质量为55.56×103处检测到目的条带。结论:利用大肠杆菌表达系统获得了较高纯度的GST-MYB77融合蛋白,为进一步研究津田芜菁MYB77蛋白的功能奠定了基础。     

Bacteria within ovules and seeds.

Surface-sterilized ovules and seeds of 27 species of plants were cultured in the water of syneresis of a nutrient medium low in agar content. Bacteria were obtained from 30% of the ovules, 15% of the seeds of herbaceous plants, 16% of the seeds of woody plants, 5.4% of the overwintered noncereal seeds, and 13.5% of overwintered cereal seeds. In no instance did every ovule or seed of a plant species contain bacteria. No bacteria were obtained from the hard, waxy seeds of mimosa or yellowwood. They were not obtained from ovules with unbroken coats or from seeds with coats that were not ruptured during the swelling of the seed. Only one species of bacteria was recovered in 93% of the instances in which bacteria were obtained. Bacteria were obtained from seeds that were embedded in the acidic parenchyma of the lemon or surrounded by the thickened flesh of the cucurbits. The bacteria were distributed among 19 genera and 46 species. The species isolated in greatest numbers were Bacillus megaterium, B. cereus, Erwinia herbicola, Flavobacterium devorans, and Pseudomonas fluorescens. Bacteria recovered less frequently were in the genera Achromobacter, Acinetobacter, Alcaligenes, Brevibacterium, Corynebacterium, Cytophaga, Leuconostoc, Micrococcus, Nocardia, Proteus, Streptococcus, Streptomyces, and Xanthomonas. Members of 11 genera and 15 species of bacteria were isolated once.     

Comparative structure of ovules and seeds inRafflesiaceae

The genera of theRafflesiaceae show a marked diversity in the structure of their ovules and seeds. Evolutionary trends are recognizable in ovule orientation and number of integuments. A change from anatropous ovules inApodantheae andMitrastemoideae towards incomplete anatropy inRafflesieae and orthotropy inCytineae occurs, next to a change from bitegmic ovules inApodantheae towards unitegmy with rudimentary outer integuments inRafflesieae andCytineae and full unitegmy inMitrastemoideae.—The differences in ovule structure are clearly reflected in the seeds. The seeds are essentially exotegmic, have very small embryos and an oily endosperm.—Seed structure strongly confirms the existing subfamilial classification and supports additional arguments for the generic status ofApodanthes. It does not support a separate status of the genusBerlinianche. InRafflesiaceae, seed micromorphology is only of limited use at the species level. As far as known seed dispersal is endo- or exozoochorous in all genera.     

Influence of microgravity on ultrastructure and storage reserves in seeds of Brassica rapa L.

Successful plant reproduction under spaceflight conditions has been problematic in the past. During a 122 d opportunity on the Mir space station, full life cycles of Brassica rapa L. were completed in microgravity in a series of three experiments in the Svet greenhouse. Ultrastructural and cytochemical analyses of storage reserves in mature dry seeds produced in these experiments were compared with those of seeds produced during a high-fidelity ground control. Additional analyses were performed on developing Brassica embryos, 15 d post pollination, which were produced during a separate experiment on the Shuttle (STS-87). Seeds produced on Mir had less than 20% of the cotyledon cell number found in seeds harvested from the ground control. Cytochemical localization of storage reserves in mature cotyledons showed that starch was retained in the spaceflight material, whereas protein and lipid were the primary storage reserves in ground control seeds. Protein bodies in mature cotyledons produced in space were 44% smaller than those in the ground control seeds. Fifteen days after pollination, cotyledon cells from mature embryos formed in space had large numbers of starch grains, and protein bodies were absent, while in developing ground control seeds at the same stage, protein bodies had already formed and fewer starch grains were evident. These data suggest that both the late stage of seed development and maturation are changed in Brassica by growth in a microgravity environment. While gravity is not absolutely required for any step in the plant life cycle, seed quality in Brassica is compromised by development in microgravity.     

Altered patterns of fractionation and exon deletions in Brassica rapa support a two-step model of paleohexaploidy

The genome sequence of the paleohexaploid Brassica rapa shows that fractionation is biased among the three subgenomes and that the least fractionated subgenome has approximately twice as many orthologs as its close (and relatively unduplicated) relative Arabidopsis than had either of the other two subgenomes. One evolutionary scenario is that the two subgenomes with heavy gene losses (I and II) were in the same nucleus for a longer period of time than the third subgenome (III) with the fewest gene losses. This "two-step" hypothesis is essentially the same as that proposed previously for the eudicot paleohexaploidy; however, the more recent nature of the B. rapa paleohexaploidy makes this model more testable. We found that subgenome II suffered recent small deletions within exons more frequently than subgenome I, as would be expected if the genes in subgenome I had already been near maximally fractionated before subgenome III was introduced. We observed that some sequences, before these deletions, were flanked by short direct repeats, a unique signature of intrachromosomal illegitimate recombination. We also found, through simulations, that short--single or two-gene--deletions appear to dominate the fractionation patterns in B. rapa. We conclude that the observed patterns of the triplicated regions in the Brassica genome are best explained by a two-step fractionation model. The triplication and subsequent mode of fractionation could influence the potential to generate morphological diversity--a hallmark of the Brassica genus.     

Fatty acid content and the functional state of pea chloroplasts under altered gravity

The results of the study of the fatty acid content and the functional state of chloroplasts isolated from leaves of pea plants grown during 7 and 14 days in the stationary conditions and under clinorotation (2 rpm) are presented. An increase in the unsaturated fatty acid content occurred after 7-day clinorotation while it insignificantly decreased after more prolonged 14-day clinorotation. A study of the functional state of chloroplasts (the rate of electron transport in photosystems II [PSII] and in photosystem I [PSI] and in the whole photosynthetic electron transport chain) showed its decrease under both terms of clinorotation in comparison with control ones. In addition, 14-day clinorotation caused more significant lowering of the electron transport rate, particularly in PSI. Changes in both the fatty acid content and the electron transport rate are discussed in relation to the activation of lipid peroxidation and the increased production of activated oxygen species in chloroplasts under clinorotation.     

A role of phosphorylase in the potato minituber function under altered gravity

The goal of our work was a role of phosphorylase (EC. 2.4.1.1) in starch accumulation in plastids of storage parenchyma cells in potato minitubers forming under clinorotation. An increased enzyme activity under the influence of simulated microgravity has been revealed by using the biochemical and electron cytochemical methods. The obtained results suggest the correlation between an increase in phosphorylase activity and acceleration growth rate and senescence of plant storage organs in microgravity.     

Pre-fertilization degeneration of both synergids in Brassica campestris ovules

Summary In Brassica campestris, both synergids of the ovule degenerate before the arrival of the pollen tube. Synergid degeneration does not depend on pollination. At the non-degenerated stage, the synergids are completely filled with a complexly organized cytoplasm containing numerous mitochondria with many cristae, a large number of dictyosomes with many associated vesicles, and a very extensive rough endoplasmic reticulum. The degenerative changes that occur in the cytoplasm of the synergids are characterized by a loss of visibility of the membranes of the endoplasmic reticulum and the simultaneous formation of dense deposits on the surrounding membranes of the mitochondria. Locally, the plasma membranes of the synergids disappear, and some ground plasma of the synergids penetrates into the space between the plasma membranes of the egg cell and the central cell.     

A sequence-tagged linkage map of Brassica rapa

A detailed genetic linkage map of Brassica rapa has been constructed containing 545 sequence-tagged loci covering 1287 cM, with an average mapping interval of 2.4 cM. The loci were identified using a combination of 520 RFLP and 25 PCR-based markers. RFLP probes were derived from 359 B. rapa EST clones and amplification products of 11 B. rapa and 26 Arabidopsis. Including 21 SSR markers provided anchors to previously published linkage maps for B. rapa and B. napus and is followed as the referenced mapping of R1-R10. The sequence-tagged markers allowed interpretation of the pattern of chromosome duplications within the B. rapa genome and comparison with Arabidopsis. A total of 62 EST markers showing a single RFLP band were mapped through 10 linkage groups, indicating that these can be valuable anchoring markers for chromosome-based genome sequencing of B. rapa. Other RFLP probes gave rise to 2-5 loci, inferring that B. rapa genome duplication is a general phenomenon through 10 chromosomes. The map includes five loci of FLC paralogues, which represent the previously reported BrFLC-1, -2, -3, and -5 and additionally identified BrFLC3 paralogues derived from local segmental duplication on R3.     

Destiny of a transgene escape from Brassica napus into Brassica rapa

Transgenic Brassica napus can be easily crossed with wild Brassica rapa. The spread of the transgene to wild species has aroused the general concern about its effect on ecological and agricultural systems. This paper was designated, by means of population genetics, to study the fate of a transgene escape from B. napus to B. rapa. Three models were proposed to survey the change in gene frequency during successive backcross processes by considering selection pressures against aneuploids, against herbicide-susceptible individuals, and by considering A-C intergenomic recombination and the effect of genetic drift. The transmission rate of an A-chromosome gene through an individual to the next generation was 50%, irrespective of the chromosome number; while that of a C-chromosome transgene varied from 8.7% to 39.9%, depending on the chromosome number of the individual used in the backcross. Without spraying herbicide, the frequency of an A-chromosome gene was 50% in the BC(1) generation, and decreased by 50% with the advance of each backcross generation; that of a C-chromosome gene was around 39.9% in BC(1), 7.7% in BC(2), 1.2% in BC(3) and 0.1% in the BC(4) generation. Under the selection pressure against herbicide-susceptible individuals, the frequency of a transgene reached a stable value of about 5.5% within six generations of successive backcrossings. The effect of genetic drift and intergenomic exchange on gene transmission rate was discussed. It is suggested that the transgene integrated on a C-chromosome (or better on a cytoplasm genome) is safer than that on an A-chromosome. The transgenic cultivars should be cultivated rotationally by year(s) with other non-transgenic varieties in order to reduce the transfer of the transgene to wild B. rapa species.     

The ultrastructure of Chlorella mitochondria and their energetic state under altered gravity

This study looks at the predominance of the participation of the mitochondria in the energy supply of Chlorella cells. The localization of the Mg(2+)-activated ATPases were studied during the culture development under the geterotrophic conditions, and with an investigation of the mitochndria ultrastructure at the various stages of the culture development under clinorotation. It was found that the activity level of this enzyme is one main indice of the energy state of these organelles.     

Reserve nutrient substance accumulation in Brassica rapa L. seeds in microgravity conditions (STS-87)

The results of study of Brassica embryo differentiation and reserve nutrient substance accumulation in the seeds were represented. Near resemblance of the spaceflight and around control embryo development was revealed. Different character of the reserve substance accumulation was noted, despite of the morphologic similarity in seeds produced in spaceflight and on the ground. It allows to consider spaceflight embryos morphologically more younger compared to the ground control.     

Protein sequence similarity searches using patterns as seeds.

Protein families often are characterized by conserved sequence patterns or motifs. A researcher frequently wishes to evaluate the significance of a specific pattern within a protein, or to exploit knowledge of known motifs to aid the recognition of greatly diverged but homologous family members. To assist in these efforts, the pattern-hit initiated BLAST (PHI-BLAST) program described here takes as input both a protein sequence and a pattern of interest that it contains. PHI-BLAST searches a protein database for other instances of the input pattern, and uses those found as seeds for the construction of local alignments to the query sequence. The random distribution of PHI-BLAST alignment scores is studied analytically and empirically. In many instances, the program is able to detect statistically significant similarity between homologous proteins that are not recognizably related using traditional single-pass database search methods. PHI-BLAST is applied to the analysis of CED4-like cell death regulators, HS90-type ATPase domains, archaeal tRNA nucleotidyltransferases and archaeal homologs of DnaG-type DNA primases.     

Phylogenetic Analysis of Brassica rapa MATH-Domain Proteins

The MATH (meprin and TRAF-C homology) domain is a fold of seven anti-parallel β-helices involved in protein-protein interaction. Here, we report the identification and characterization of 90 MATH-domain proteins from the Brassica rapa genome. By sequence analysis together with MATH-domain proteins from other species, the B. rapa MATH-domain proteins can be grouped into 6 classes. Class-I protein has one or several MATH domains without any other recognizable domain; Class-II protein contains a MATH domain together with a conserved BTB (Broad Complex, Tramtrack, and Bric-a-Brac ) domain; Class-III protein belongs to the MATH/Filament domain family; Class-IV protein contains a MATH domain frequently combined with some other domains; Class-V protein has a relative long sequence but contains only one MATH domain; Class-VI protein is characterized by the presence of Peptidase and UBQ (Ubiquitinylation) domains together with one MATH domain. As part of our study regarding seed development of B. rapa, six genes are screened by SSH (Suppression Subtractive Hybridization) and their expression levels are analyzed in combination with seed developmental stages, and expression patterns suggested that Bra001786, Bra03578 and Bra036572 may be seed development specific genes, while Bra001787, Bra020541 and Bra040904 may be involved in seed and flower organ development. This study provides the first characterization of the MATH domain proteins in B. rapa     

Isolation and characterization of microsatellites in Brassica rapa L

We report here the isolation and characterization of microsatellites, or simple sequence repeats (SSRs), in Brassica rapa. The size-fractionated genomic library was screened with (GA)(15) and (GT)(15) oligonucleotide probes. A total of 58 clones were identified as having the microsatellite repeats, and specific primer pairs were designed for 38 microsatellite loci. All primer pairs, except two, amplified fragments having the sizes expected from the sequences. Of the 36 primer pairs, 35 amplified polymorphic loci in 19 cultivars of B. rapa, while monomorphism was observed in only one primer pair. A total of 232 alleles was identified by the 36 primer pairs in 19 cultivars of B. rapa, and these primer pairs were examined also in nine Brassicaceae species. Most of the 36 primer pairs amplified the loci in the Brassicaceae species. Segregation of the microsatellites was studied in an F(2) population from a cross of doubled-haploid lines DH27 x G309. The microsatellites segregated in a co-dominant manner. These results indicate that the microsatellites isolated in this study were highly informative and could be useful tools for genetic analysis in B. rapa and other related species.