Developmental and environmental regulation of pea legumin genes in transgenic tobacco

Abstract

Two distinct legumin genes (LegA1 and LegA2) which encode a major class of seed storage protein in pea were isolated from a genomic library. The cloned fragments were introduced into tobacco via Agrobacterium-mediated transformation and the regenerated plants were used to study the expression characteristics of the genes in a heterologous host. It was found that both LegA1 and LegA2 were functional members of the pea legumin gene family and that their expression was similar in both pea and transgenic tobacco. Legumin was detected only in the seed of tobacco where the primary translation products were processed in a manner analogous to that which occurs in pea. Legumin gene expression was also shown to be temporally regulated during seed development. Legumin polypeptides and mRNA began to accumulate 16 days after flowering (DAF), in contrast to the endogenous tobacco storage proteins which were apparent at 13 DAF. It was also demonstrated that the legumin genes in tobacco were environmentally regulated to the nutritional status of the plant. As has been previously shown in pea, legumin accumulation in transgenic tobacco seed was progressively reduced when the plants were grown under conditions of increasing severity of sulphur-nutrient stress. The reduced accumulation of protein was correlated with lower levels of legumin mRNA in the developing seed. Despite encoding nearly identical subunits, nucleotide sequence data for LegA1 and LegA2 showed that the similarity of their respective 5'-flanking regions was restricted to several short elements mostly within 240 bp from the start of transcription. However, a deletion series using the LegA1 gene demonstrated that 237 bp of 5'-flanking sequence was insufficient to permit the expression of the legumin gene in tobacco. The data indicated that an as yet unidentified sequence element(s) located between positions -668 and -237 was essential in re-establishing the high level of regulated gene expression observed with the full-length LegA1 gene.

Genes Encoding Proteins of the Cation Diffusion Facilitator Family That Confer Manganese Tolerance

“…Denaturing buffer was added to a sample that contained 100 g of protein, and the sample was heated at 95ЊC for 5 min and then separated by SDS-denaturing protein electrophoresis. The proteins were transferred to nitrocellulose, and antigens were detected with the purified anti-ShMTP1 and a secondary antibody conjugated to alkaline phosphatase using methods described by Rerie et al (1991).…”

Isolation of the Ornithine-δ-Aminotransferase cDNA and Effect of Salt Stress on Its Expression inArabidopsis thaliana1

“…Total RNA was isolated from 12-d-old plantlets and 4-week-old plants incubated 0, 6, 24, and 72 h under normal and salt-stress conditions (200 mm NaCl) according to the method described by Rerie et al (1991). Twenty micrograms of RNA samples was subjected to electrophoresis in 1.5% agarose gel containing 6% formaldehyde (37%).…”

Cloning of Higher Plant [omega]-3 Fatty Acid Desaturases

“…RNA was isolated as described in Rerie et al (1991). Poly(A)+ mRNA was isolated using the PolyATtract mRNA isolation system (Promega Corp.).…”