The cDNA cloning of a pea (Pisum sativum) seed lipoxygenase. Sequence comparisons of the two major pea seed lipoxygenase isoforms.
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ABSTRACT:
SUBMITTER: Ealing PM
PROVIDER: S-EPMC1133675 | biostudies-other | 1989 Dec
REPOSITORIES: biostudies-other
Similar Datasets
Project description:A lipoxygenase cDNA clone, pCD45, was identified in a Pisum sativum L. (pea) seed mRNA cDNA library by hybrid-release/translation followed by immunoprecipitation with antiserum raised against lipoxygenase from Glycine max L. (soya bean). pCD45 hybrid-selected an mRNA encoding the larger of the two polypeptides of Mr approximately 95 000 that were immunoprecipitated from cell-free translation products of pea seed poly(A)-containing RNA by the G. max anti-lipoxygenase. 'Northern'-blot analysis showed the mRNA that hybridized to pCD45 to be approximately 3000 nucleotides in length. Three to five copies of the lipoxygenase gene corresponding to pCD45 were estimated to be present per haploid Pisum genome; hybridization of the cDNA insert from pCD45 to G. max DNA was also detected.
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Project description:A near full-length cDNA for a pea (Pisum sativum) seed lipoxygenase was isolated and sequenced. It has a protein coding sequence (2583 bp), 5' (59 bp) and 3' (191 bp) non-coding regions, and a poly(A) tail (20 bp). The predicted amino acid sequence indicates a polypeptide of Mr 97,628 that shows about 86% amino acid identity with a soya-bean lipoxygenase 3 protein sequence [Yenofsky, Fine & Liu (1988) Mol. Gen. Genet. 211, 215-222]. The cDNA directs the transcription of mRNA that can be translated to give an anti-lipoxygenase-precipitable polypeptide in vitro.
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Project description:The existing variation among pea protein isolates' functionality limits their application in food formulations. The source and extent of variations among yellow pea protein profiles was assessed in 10 single seeds of two varieties with different size and weight. A new approach was developed to analyze proteins of yellow pea combining three analytical methods of size exclusion chromatography (SEC), reverse phase high performance liquid chromatography (RP-HPLC), and microfluidic SDS-PAGE, to achieve the highest separation resolution. A high variation of protein concentration was observed not only between varieties, but also among seeds of the same variety. Vicilin to legumin ratio was between 2.72-4.19, and 1.70-2.22 among the individual seeds of AC Agassiz and CDC Saffron varieties, respectively. V/L ratio was significantly different among the individual seeds for both varieties. The amount of some protein fractions/subunits were correlated with seeds' size and weight for AC Agassiz, while such correlations were not observed for CDC Saffron.
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Project description:Pea (Pisum. sativum L.) is a traditional and important edible legume that can be sorted into grain pea and vegetable pea according to their harvested maturely or not. Vegetable pea by eating the fresh seed is becoming more and more popular in recent years. These two type peas display huge variations of the taste and nutrition, but how seed development and nutrition accumulation of grain pea and vegetable pea and their differences at the molecular level remains poorly understood. To understand the genes and gene networks regulate seed development in grain pea and vegetable pea, high throughput RNA-Seq and bioinformatics analysis were used to compare the transcriptomes of vegetable pea and grain pea developing seed. RNA-Seq generated 18.7 G raw data, which was then de novo assembled into 77,273 unigenes with a mean length of 930 bp. Functional annotation of the unigenes was carried out using the nr, Swiss-Prot, COG, GO and KEGG databases. There were 459 and 801 genes showing differentially expressed between vegetable pea and grain pea at early and late seed maturation phases, respectively. Sugar and starch metabolism related genes were dramatically activated during pea seed development. The up-regulated of starch biosynthesis genes could explain the increment of starch content in grain pea then vegetable pea; while up-regulation of sugar metabolism related genes in vegetable pea then grain pea should participate in sugar accumulation and associated with the increase in sweetness of vegetable pea then grain pea. Furthermore, transcription factors were implicated in the seed development regulation in grain pea and vegetable pea. Thus, our results constitute a foundation in support of future efforts for understanding the underlying mechanism that control pea seed development and also serve as a valuable resource for improved pea breeding.
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Project description:Understanding the molecular mechanisms regulating pea seed developmental process is extremely important for pea breeding. In this study, we used high-throughput RNA-Seq and bioinformatics analyses to examine the changes in gene expression during seed development in vegetable pea and grain pea, and compare the gene expression profiles of these two pea types. RNA-Seq generated 18.7 G of raw data, which were then de novo assembled into 77,273 unigenes with a mean length of 930 bp. Our results illustrate that transcriptional control during pea seed development is a highly coordinated process. There were 459 and 801 genes differentially expressed at early and late seed maturation stages between vegetable pea and grain pea, respectively. Soluble sugar and starch metabolism related genes were significantly activated during the development of pea seeds coinciding with the onset of accumulation of sugar and starch in the seeds. A comparative analysis of genes involved in sugar and starch biosynthesis in vegetable pea (high seed soluble sugar and low starch) and grain pea (high seed starch and low soluble sugar) revealed that differential expression of related genes at late development stages results in a negative correlation between soluble sugar and starch biosynthetic flux in vegetable and grain pea seeds. RNA-Seq data was validated by using real-time quantitative RT-PCR analysis for 30 randomly selected genes. To our knowledge, this work represents the first report of seed development transcriptomics in pea. The obtained results provide a foundation to support future efforts to unravel the underlying mechanisms that control the developmental biology of pea seeds, and serve as a valuable resource for improving pea breeding.
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Project description:Pea seeds are widely consumed in their immature form, known as garden peas and petit pois, mostly after preservation by freezing or canning. Mature dry peas are rich in iron in the form of ferritin, but little is known about the content, form or bioavailability of iron in immature stages of seed development. Using specific antibodies and in-gel iron staining, we show that ferritin loaded with iron accumulated gradually during seed development. Immunolocalization and high-resolution secondary ion mass spectrometry (NanoSIMS) revealed that iron-loaded ferritin was located at the surface of starch-containing plastids. Standard cooking procedures destabilized monomeric ferritin and the iron-loaded form. Iron uptake studies using Caco-2 cells showed that the iron in microwaved immature peas was more bioavailable than in boiled mature peas, despite similar levels of soluble iron in the digestates. By manipulating the levels of phytic acid in the digestates we demonstrate that phytic acid is the main inhibitor of iron uptake from mature peas in vitro. Taken together, our data show that immature peas and mature dry peas contain similar levels of ferritin-iron, which is destabilized during cooking. However, iron from immature peas is more bioavailable because of lower phytic acid levels compared to mature peas.
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Project description:Aimed at gene-based markers design, we generated and analyzed transcriptome sequencing datasets for six pea (Pisum sativum L.) genetic lines that have not previously been massively genotyped. Five cDNA libraries obtained from nodules or nodulated roots of genetic lines Finale, Frisson, Sparkle, Sprint-2 and NGB1238 were sequenced using a versatile 3'-RNA-seq protocol called MACE (Massive Analysis of cDNA Ends). MACE delivers a single next-generation sequence from the 3'-end of each individual cDNA molecule that precisely quantifies the respective transcripts. Since the contig generated from the 3'-end of the cDNA by assembling all sequences encompasses the highly polymorphic 3'-untranslated region (3'-UTR), MACE efficiently detects single nucleotide variants (SNVs). Mapping MACE reads to the reference nodule transcriptome assembly of the pea line SGE (Transcriptome Shotgun Assembly GDTM00000000.1) resulted in characterization of over 34,000 polymorphic sites in more than 9700 contigs. Several of these SNVs were located within recognition sequences of restriction endonucleases which allowed the design of co-dominant CAPS markers for the particular transcript. Cleaned reads of sequenced libraries are available from European Nucleotide Archive (http://www.ebi.ac.uk/) under accessions PRJEB18101, PRJEB18102, PRJEB18103, PRJEB18104, PRJEB17691.
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