Identification and characterisation of seed storage protein transcripts from Lupinus angustifolius

Abstract

Background: In legumes, seed storage proteins are important for the developing seedling and are an important source of protein for humans and animals. Lupinus angustifolius (L.), also known as narrow-leaf lupin (NLL) is a grain legume crop that is gaining recognition as a potential human health food as the grain is high in protein and dietary fibre, gluten-free and low in fat and starch.

Results: Genes encoding the seed storage proteins of NLL were characterised by sequencing cDNA clones derived from developing seeds. Four families of seed storage proteins were identified and comprised three unique α, seven β, two γ and four δ conglutins. This study added eleven new expressed storage protein genes for the species. A comparison of the deduced amino acid sequences of NLL conglutins with those available for the storage proteins of Lupinus albus (L.), Pisum sativum (L.), Medicago truncatula (L.), Arachis hypogaea (L.) and Glycine max (L.) permitted the analysis of a phylogenetic relationships between proteins and demonstrated, in general, that the strongest conservation occurred within species. In the case of 7S globulin (β conglutins) and 2S sulphur-rich albumin (δ conglutins), the analysis suggests that gene duplication occurred after legume speciation. This contrasted with 11S globulin (α conglutin) and basic 7S (γ conglutin) sequences where some of these sequences appear to have diverged prior to speciation. The most abundant NLL conglutin family was β (56%), followed by α (24%), δ (15%) and γ (6%) and the transcript levels of these genes increased 103 to 106 fold during seed development. We used the 16 NLL conglutin sequences identified here to determine that for individuals specifically allergic to lupin, all seven members of the β conglutin family were potential allergens.

Conclusion: This study has characterised 16 seed storage protein genes in NLL including 11 newly-identified members. It has helped lay the foundation for efforts to use molecular breeding approaches to improve lupins, for example by reducing allergens or increasing the expression of specific seed storage protein(s) with desirable nutritional properties.

Figure 1

NLL Conglutin Sequence Alignment. Deduced amino acid alignment using CLC Genomics Workbench 3 software [20] of L. angustifolius (A) ALPHA [ALPHA1 Genbank:HQ670406; ALPHA2:Genbank:HQ670407; ALPHA3:Genbank:HQ670408] and (B) BETA [BETA1 Genbank:HQ670409, BETA2 Genbank:HQ670410, BETA3 Genbank:HQ670411, BETA4 Genbank:HQ670412, BETA5 Genbank:HQ670413, BETA6 Genbank:HQ670414, BETA7 Genbank:HQ670415].

Figure 2

NLL Gamma and Delta Conglutin Sequence Alignment. Deduced amino acid alignment using CLC Genomics Workbench 3 software [20] of L. angustifolius (A) GAMMA [GAMMA1 Genbank:HQ670416, GAMMA2 Genbank:HQ670417] and (B) DELTA [DELTA1 Genbank:HQ670418, DELTA2 Genbank:HQ670419, DELTA3 Genbank:HQ670420, DELTA4 Genbank:HQ670421] conglutins. Amino acids labelled blue represent those with the highest conservation among NLL congluting sequences, while those labelled red represent those with the least conservation. Dashes have been inserted to optimize alignment.

Figure 3

Seed Storage protein Phylogenetic Relationships. Phylogenetic relationships between Arachis hypogaea (Ah), Glycine max (Gm), Medicago truncatula (Mt), Lupinus albus (La), and Pisum sativum (Ps) conglutin-like sequences and L. angustifolius (A) 11S globulin (α conglutin), (B) 7S globulin (β conglutin), (C) basic 7S (γ conglutin) and (D) 2S sulphur-rich albumin (δ conglutin) deduced amino acid sequences. L. angustifolius conglutins are boxed for easy recognition. Identification and accession number for each protein are listed in Table 1.

Figure 4

EST Conglutin Expression. Pie chart of relative numbers of specific members in each L. angustifolius conglutin family. (A) ALPHA, (B) BETA, (C) GAMMA and (D) DELTA conglutin ESTs. Total number of ESTs of each member is listed in parenthesis, followed by percentage of each total conglutin number.

Figure 5

qRT-PCR Conglutin Expression. Relative expression of conglutin genes determined by qRT-PCR using specific primers for (A) ALPHA, (B) BETA, (C) GAMMA and (D) DELTA conglutin sequences normalised to β-tubulin. RNA was extracted from L. angustifolius seeds collected from different stages of development. The average and standard error of three biological replicates are plotted against the log of the relative transcript expression.

Figure 6

Identification of L. angustifolius seed storage proteins. Lupin flour proteins were separated by 2D-PAGE and (A) stained with Coomassie-blue stained or (B) blotted onto a membrane, which was probed with serum from lupin-allergic individuals, to identify potentially allergenic IgE-binding proteins. Protein spots that were either IgE-binding (spots 3 - 59, 94) or non-IgE-binding (spots 87 - 89, 97-114) were analysed by mass spectrometry, and those for which identifications were made are enclosed by ovals, with different colours corresponding to different proteins as shown in the figure. Sections of the gel and blot (boxes i, ii and iii) have been enlarged to show more detail, with Coomassie-blue stained gels on the top and IgE-binding proteins on the bottom panel for each section. In the enlarged boxes i, ii and iii spots that bind IgE are shown in black and those that do not in red. IgE binding was determined by aligning the original film and the Coomassie-blue stained gel but for 4 spots (37, 38, 51, 57) the resolution of the gel does not give a clear image of this binding. 'Beta?' indicates spots for which the form of conglutin β could not be determined.