Dennis W. Sutton

Phaseolin gene from bean is expressed after transfer to sunflower via tumor-inducing plasmid vectors.

Sequences coding for the bean seed protein phaseolin were inserted into transferred DNA regions of tumor-inducing plasmids. Constructions were devised in which the coding region of phaseolin was fused in the correct reading frame with the coding region of octopine synthase and placed under the transcriptional control of the octopine synthase promoter. Other plasmids were prepared to permit expression of the phaseolin-encoding sequences from the flanking phaseolin promoter region. The RNA transcribed in sunflower cells transformed with these constructions was characterized by hybridization procedures, SI nuclease mapping, and by translation in vitro of extracted RNA. These tests showed that the genomic intervening sequences were correctly excised. Immunoreactive phaseolin polypeptides were detected by enzyme-linked immunosorbent assay and by antibody hybridization to electrophoretically separated protein extracts of sunflower tissues isolated from crown gall tumors and of transformed sunflower cells grown in tissue culture. These results demonstrate the expression of a plant gene after transfer to a taxonomically distinct botanical family.

SyntheticcryIIIA gene fromBacillus thuringiensis improved for high expression in plants

A 1974 bp synthetic gene was constructed from chemically synthesized oligonucleotides in order to improve transgenic protein expression of thecryIIIA gene fromBacillus thuringiensis var.tenebrionis in transgenic tobacco.The crystal toxin genes (cry) fromB. thuringiensis are difficult to express in plants even when under the control of efficient plant regulatory sequences. We identified and eliminated five classes of sequence found throughout thecryIIIA gene that mimic eukaryotic processing signals and which may be responsible for the low levels of transcription and translation. Furthermore, the GC content of the gene was raised from 36% to 49% and the codon usage was changed to be more plant-like. When the synthetic gene was placed behind the cauliflower mosaic virus 35S promoter and the alfalfa mosaic virus translational enhancer, up to 0.6% of the total protein in transgenic tobacco plants wascryIIIA as measured from immunoblot analysis. Bioassay data using potato beetle larvae confirmed this estimate.

Constitutive expression of the β-phaseolin gene in different tissues of transgenic alfalfa does not ensure phaseolin accumulation in non-seed tissue

Phaseolin is a glycoprotein that constitutes the major storage protein in bean seeds. The phaseolin gene promoters function in a seed-specific manner. In an attempt to understand if events following transcription of the gene also contribute to the seed-specific accumulation of the phaseolin protein, we studied the effect of substituting the constitutive CaMV-35S promoter for the β-phaseolin gene promoter on expression of the phaseolin gene in different plant organs. A chimeric gene consisting of the 35S promoter, the coding sequence of the β-phaseolin gene (all five introns and six exons) and the 3′-flanking region of the β-phaseolin gene, was introduced into alfalfa via Agrobacterium tumefaciens. While all organs examined shared high levels of phaseolin transcripts, the only organ that showed significant accumulation of the phaseolin protein were the mature seeds. Co-migration of the major immunoreactive polypeptides from the non-seed organs with the authentic β-phaseolin polypeptides on SDS-PAGE indicates that the protein in non-seed organs undergoes correct post-translational processing and modification, but are more unstable in a non-seed environment.

BiP and zein binding domains within the delta zein protein

Zeins are alcohol soluble seed storage proteins synthesized within the endosperm of maize and subsequently deposited into endoplasmic reticulum (ER) derived protein bodies. The genes encoding the beta and delta zeins were previously introduced into tobacco with the expectation of improving the nutritional quality of plants (Bagga et al. in Plant Physiol 107:13, 1997). Novel protein bodies are produced in the leaves of transgenic plants accumulating the beta or delta zein proteins. The mechanism of protein body formation within leaves is unknown. It is also unknown how zeins are retained in the ER since they do not contain known ER retention motifs. Retention may be due to an interaction of zeins with an ER chaperone such as binding luminal protein (BiP). We have demonstrated protein–protein interactions with the delta zeins, beta zeins, and BiP proteins using an E. coli two-hybrid system. In this study, four putative BiP binding motifs were identified within the delta zein protein using a BiP scoring program (Blond-Elguindi et al. in Cell 75:717, 1993). These putative binding motifs were mutated and their effects on protein interactions were analyzed in both a prokaryotic two-hybrid system and in plants. These mutations resulted in reduced BiP–zein protein interaction and also altered zein–zein interactions. Our results indicate that specific motifs are necessary for BiP–delta zein protein interactions and that there are specific motifs which are necessary for zein–zein interactions. Furthermore, our data demonstrates that zein proteins must be able to interact with BiP and zeins for their stability and ability to form protein bodies.