Seed-specific Expression Research Articles

A novel seed protein gene from Vicia faba is developmentally regulated in transgenic tobacco and Arabidopsis plants

We have isolated a novel gene, denoted USP, from Vicia faba var. minor, which corresponds to the most abundant mRNA present in cotyledons during early seed development; however, the corresponding protein does not accumulate in cotyledons. The characterized USP gene with its two introns is 1 of about 15 members of a gene family. A fragment comprising 637 bp of 5' flanking sequence and the total 5' untranslated region was shown to be sufficient to drive the mainly seed-specific expression of two reporter genes, coding for neomycin phosphotransferase II and beta-glucuronidase, in transgenic Arabidopsis thaliana and Nicotiana tabacum plants. We showed that the USP promoter becomes active in transgenic tobacco seeds in both the embryo and the endosperm, whereas its activity in Arabidopsis is detectable only in the embryo. Moreover, we demonstrated a transient activity pattern of the USP promoter in root tips of both transgenic host species.

High-Level, Seed-Specific Expression of Foreign Coding Sequences in Brassica napus

The napin seed storage proteins of oilseed rape (Brassica napus) are encoded by a multigene family of at least 16 members. We have isolated five independent napin genes and five napin cDNAs from a rapid cycling line of B. napus (CrGC45). Two of the isolated genomic fragments, G1 and G2, were introduced into tobacco, and napin gene expression was analysed in the developing seeds. Both genes are expressed during the maturation phase of transformed tobacco seeds as judged by the accumulation of napin RNA in immature embryos. These genes appear to utilise the same transcription start site in tobacco as in Brassica, but are expressed at 20-50-fold lower levels in the heterologous host plant. The G1 and G2 promoter regions and 3' flanking regions were also fused to two foreign coding sequences: the chloramphenicol acetyl transferase (CAT) coding sequence and the pea seed 2S albumin (PSA) coding sequence, and these chimeric genes were introduced into B. napus and tobacco. Upon introduction into tobacco, only the PSA mRNA accumulated to a measurable extent. In transgenic rapeseed plants, PSA RNA accumulated to high levels, but CAT RNA was present only at low concentrations. The results demonstrate that high level expression of foreign coding sequences is possible in developing seeds of B. napus and that differential mRNA stability may contribute significantly to the level of foreign transcripts which can be observed in genetically engineered rapeseed.

Upstream sequences regulating legumin gene expression in heterologous transgenic plants

We have previously isolated a legumin gene LeB4 from Vicia faba and shown that a 4.7 kb DNA fragment containing the gene leads to seed-specific expression in transgenic tobacco plants. Here we report that the 2.4 kb upstream sequence alone, when fused to either the neomycin phosphotransferase II (nptII) gene or the beta-glucuronidase (uidA) gene, leads to high enzyme levels in transgenic seeds of both tobacco and Arabidopsis. beta-Glucuronidase (GUS) activity is especially intense in the cotyledons fading out towards the embryonal root tip, a result confirmed by in situ hybridization. Staining of endosperm cells is consistent in both species. Analysis of a series of promoter deletion mutants fused to the nptII gene and introduced into tobacco plants revealed that about 1 kb of 5'-flanking sequence is sufficient for high-level expression but indirect evidence suggests the presence of weak positive regulatory elements further upstream. Deletions leaving only 0.2 kb of upstream sequence reduce enzyme levels to less than 10%. A deletion which destroys the legumin box with its seed protein gene-specific CATGCATG motif has no obvious effects on expression levels.

Manipulation of methionine-rich protein genes in plant seeds

Animal feeds based on soybean and corn meals are often deficient in the sulfur-containing amino acids, methionine and cysteine. At present, this deficiency is remedied by amino acid supplementation of the diet. A number of methionine-rich seed proteins have been identified; seed-specific expression of genes encoding these proteins is expected to improve the protein quality in feed seed and thereby eliminate the need for supplementation.

The bifunctional ?-amylase/subtilisin inhibitor of barley: nucleotide sequence and patterns of seed-specific expression

We have cloned and sequenced a full-length cDNA from barley (Hordeum vulgare L.) seeds encoding the bifunctional α-amylase/subtilisin inhibitor (BASI). The nucleotide sequence predicts an open reading frame coding for a protein of 203 amino acids. The first 22 amino acids exhibit the sequence characteristic of a signal peptide, as found in several other plant protease inhibitors. Northern blot hybridization experiments indicate that BASI mRNA accumulation is strictly tissue-specific and is developmentally programmed. BASI mRNA transcripts were only identified in 1) developing starchy endosperm tissue from 14 days after flowering and 2) aleurone tissue of germinating seeds. In this latter tissue, BASI mRNA accumulation is enhanced by abscisic acid and abolished by gibberellic acid. Expression of BASI mRNA was also studied in the lys 3a high-lysine barley mutants Risø No. 1508 and Piggy. These high-lysine barleys show 2-4-fold higher levels as well as prolonged accumulation of BASI mRNA compared to the normal motherline Bomi. This correlates with the increased deposition of BASI protein in lys 3a barley mutants. Genomic blot analysis of barley DNA suggests that there are one or two BASI structural genes per haploid genome. Possible roles of BASI as part of a defence mechanism against precocious germination and pathogens are discussed.

Tissue-specific expression of a pea legumin gene in seeds of Nicotiana plumbaginifolia

A 3.4-kilobase genomic DNA fragment from Pisum sativum L. containing the LegA gene, which encodes a major legumin storage protein, was transferred to Nicotiana plumbaginifolia using an Agrobacterium tumefaciens strain containing the Bin 19 binary vector system. Northern hybridisation analysis of legA-transformed plants demonstrated that legumin-specific RNA was present in developing seeds but not in developing leaves. Legumin protein was immunologically detected in the mature seeds of legA-transformed plants, and was present as the correct-size protein composed of disulphide-bonded polypeptides. It is concluded that the transferred pea genomic fragment contains all the information necessary for seed-specific expression of the legA gene, and for correct processing of the primary transcript and the precursor legumin protein.