Cellular localization of soybean storage protein mRNA in transformed tobacco seeds

Abstract

We transformed tobacco plants with a soybean (3-conglycinin gene that encodes the 1.7-kilobase (3-subunit mRNA. We showed that the (8-conglycinin mRNA accumulates and decays during tobacco seed development and that (3-conglycinin mRNA is undetectable in the tobacco leaf. We utilized in situ hybridization to localize (8- Here, we introduced a p3-conglycinin 8-subunit gene into tobacco plants. Using in situ hybridization, we demonstrate that p-conglycinin mRNA is detectable only within the tobacco embryo and is localized preferentially within cotyledon and upper axis cells. In addition, we show that p-conglycinin mRNA is not detectable in the tobacco leaf, and that 8-conglycinin mRNA appears and disappears at precise stages of tobacco seed development. We conclude that sequences required for both temporal control and cell specificity are linked to the 8-subunit gene and that regulatory molecules that control 8-conglycinin gene expression are spatially distributed within analogous tobacco and soybean seed regions. MATERIALS AND METHODSIsolation of a j-Conglycinin Plasmid. The characteristics of the p-conglycinin cDNA plasmid A-16 have been described (5).DNA Isolation and Labeling. DNAs were isolated as described by Fischer and Goldberg (14) and were labeled by nick-translation (15).Polysomal mRNA Isolation. Soybean and tobacco polysomal mRNAs were isolated as described (16,17).Filter Hybridization Experiments. DNA gel-blot and RNA dot-blot experiments were carried out according to published procedures (18)(19)(20). RNA gel-blot studies were performed as described (5) except that nitrocellulose filters were used.In Situ Hybridization Experiments. In situ hybridization studies were carried out as described by Cox et al. (21) with modification. In brief, seeds 19 days after pollination (DAP) were fixed in 10% formalin/5% acetic acid/50% ethanol (22), embedded in paraffin, and sliced into 10-,um sections. Singlestranded p3-conglycinin 35S-labeled RNA probes were synthesized by using the pGEM transcription system (Promega Biotec). The 35S-labeled RNA probes were hydrolyzed to approximately 0.2 kb in size and then were hybridized with slides containing fixed seed sections for 14 hr at selection criteria of 420C, 0.3 M Na', and 50% formamide (21). A 35S-labeled anti-mRNA probe was used to localize pconglycinin mRNA within the tobacco seed while a 35s-labeled mRNA probe monitored nonspecific hybridization within the fixed seed section. Following hybridization, the fixed seed sections were incubated with RNase A at 50

Expression and Processing of an Arabidopsis 2S Albumin in Transgenic Tobacco

“…The small variation in the level of Arabidopsis 2S albumin accumulating in different transformants is correlated with the gene copy number. Transformants 8, 9, 10, 14, and 15 contained three copies; transformants 3,6, and 12 had two copies, whereas transformant 2 contained only one copy of the A T2SM gene (data not shown).…”

Kunitz trypsin inhibitor genes are differentially expressed during the soybean life cycle and in transformed tobacco plants.

“…Tobacco leaf discs and protoplasts were transformed with the KTil/2, KTi2, and KTi3/4 gene regions (Figure 9) according to the procedures of Horsch et al (1985), Okamuro et al (1986), and Barker, Harada, and Goldberg (1988).…”

Soybean Seed Protein Genes Are Regulated Spatially during Embryogenesis

“…In this study, we investigated the cellular expression programs of two Kunitz trypsin inhibitor gene classes, designated as KTi1/2 and KTi3 Jofuku, Schipper, and Goldberg, 1989), and compared these programs with those of the @-conglycinin storage protein gene family (Barker, Harada, and Goldberg, 1988;Chen et al, 1988;Chen et al, 1989;Harada, Barker, and Goldberg, 1989). Our results show that seed protein genes are highly regulated with respect to cell type in both zygotic and somatic embryos, that seed protein mRNAs accumulate in a "wave-like" pattern during embry-ogenesis, and that similar events occur during the development of transformed tobacco seeds.…”