Donald Spencer

Bean [alpha]-Amylase Inhibitor Confers Resistance to the Pea Weevil (Bruchus pisorum) in Transgenic Peas (Pisum sativum L.)

Bruchid larvae cause major losses of grain legume crops through-out the world. Some bruchid species, such as the cowpea weevil and the azuki bean weevil, are pests that damage stored seeds. Others, such as the pea weevil (Bruchus pisorum), attack the crop growing in the field. We transferred the cDNA encoding the [alpha]-amylase inhibitor ([alpha]-AI) found in the seeds of the common bean (Phaseolus vulgaris) into pea (Pisum sativum) using Agrobacterium-mediated transformation. Expression was driven by the promoter of phytohemagglutinin, another bean seed protein. The [alpha]-amylase inhibitor gene was stably expressed in the transgenic pea seeds at least to the T5 seed generation, and [alpha]-AI accumulated in the seeds up to 3% of soluble protein. This level is somewhat higher than that normally found in beans, which contain 1 to 2% [alpha]-AI. In the T5 seed generation the development of pea weevil larvae was blocked at an early stage. Seed damage was minimal and seed yield was not significantly reduced in the transgenic plants. These results confirm the feasibility of protecting other grain legumes such as lentils, mungbean, groundnuts, and chickpeas against a variety of bruchids using the same approach. Although [alpha]-AI also inhibits human [alpha]-amylase, cooked peas should not have a negative impact on human energy metabolism.

Transformation and Regeneration of Two Cultivars of Pea (Pisum sativum L.)

A reproducible transformation system was developed for pea (Pisum sativum L.) using as explants sections from the embryonic axis of immature seeds. A construct containing two chimeric genes, nopaline synthase-phosphinothricin acetyl transferase (bar) and cauliflower mosaic virus 35S-neomycin phosphotransferase (nptII), was introduced into two pea cultivars using Agrobacterium tumefaciens-mediated transformation procedures. Regeneration was via organogenesis, and transformed plants were selected on medium containing 15 mg/L of phosphinothricin. Transgenic peas were raised in the glasshouse to produce flowers and viable seeds. The bar and nptII genes were expressed in both the primary transgenic pea plants and in the next generation progeny, in which they showed a typical 3:1 Mendelian inheritance pattern. Transformation of regenerated plants was confirmed by assays for neomycin phosphotransferase and phosphinothricin acetyl transferase activity and by northern blot analyses. Transformed plants were resistant to the herbicide Basta when sprayed at rates used in field practice.

The sequence of a pea vicilin gene and its expression in transgenic tobacco plants

A 5.5 kb Eco RI fragment containing a vicilin gene was selected from a Pisum sativum genomic library, and the protein-coding region and adjacent 5′ and 3′ regions were sequenced. A DNA construction comprising this 5.5 kb fragment together with a gene for neomycin phosphotransferase II was stably introduced into tobacco using an Agrobacterium tumefaciens binary vector, and the fidelity of expression of the pea vicilin gene in its new host was studied. The seeds of eight transgenic tobacco plants showed a sixteen-fold range in the level of accumulated pea vicilin. The level of accumulation of vicilin protein and mRNA correlated with the number of integrated copies of the vicilin gene. Pea vicilin was confined to the seeds of transgenic tobacco. Using immunogold labelling, vicilin was detected in protein bodies of eight out of ten embryos (axes plus cotyledons) and, at a much lower level, in two out of eleven endosperms. Pea vicilin was synthesized early in tobacco seed development; some molecules were cleaved as is the case in pea seeds, yielding a major parental component of Mr∼50000 together with a range of smaller polypeptides.

Monitoring the fate of dietary proteins in rumen fluid using gel electrophoresis.

1. When fractionated by sodium dodecylsulphate-polyacrylamide gel electrophoresis (SDS-PAGE), strained rumen fluid from sheep fed on pelleted lucerne (Medicago sativa) hay showed no major protein components that stain with Coomassie Blue. This feature made it possible to monitor the fate of individual polypeptides within a protein mixture incubated in rumen fluid in vitro. 2. Extracts from a number of seed meals (sunflower (Helianthus annuus), lupin (Lupinus angustifolius), rape (Brassica napus) and pea (Pisum sativum L.)), as well as casein and bovine serum albumin, were examined in this system. The protein components of each seed type showed a wide range of resistances to degradation. One protein in pea seeds (pea albumin 1), which is particularly rich in cysteine, was almost as resistant to rumen degradation as bovine serum albumin. 3. Analysis of synthetic-fibre-bag experiments by SDS-PAGE showed that the rate of loss of total protein from solid meal residues does not provide an index of the resistance of individual protein components of the meal to rumen degradation. While there was no qualitative change in the protein profile of residual pea-seed meal inside a synthetic-fibre bag, there was considerable variation in the rate at which individual, solubilized protein components were degraded in the surrounding rumen fluid.

Accumulation of a sulphur-rich seed albumin from sunflower in the leaves of transgenic subterranean clover (Trifolium subterraneum L.)

A gene encoding a sulphur-rich, sunflower seed albumin (23% cysteine plus methionine) was modified to contain the promoter for the 35S RNA of cauliflower mosaic virus, in order to obtain leaf expression in transgenic plants. In addition, a sequence encoding an endoplasmic reticulum-retention signal was added to the 3′ end of the coding region so as to stabilize the protein by diverting it away from the vacuole. The modified gene was introduced into subterranean clover (T. subterraneum L.) and its expression was detected by northern and western blots and by immunogold localization. The albumin was accumulated in the lumen of the endoplasmic reticulum, and, among six independent, transformed lines, it accumulated in the leaves of T0 transgenic plants at varying levels up to 0.3% of the total extractable protein. The level of accumulation of the sunflower albumin increased with increasing leaf age, and in the older leaves of the most highly expressing plants of the T1 generation it reached 1.3% of total extractable protein. Expression of the SSA gene was stable in the first and second generation progeny. These results indicate that there is potential for significantly improving the nutritional value of subterranean clover for ruminant animals such as sheep by expressing genes that code for sulphur-rich, rumen-stable proteins in leaves.

Sequence interrelationships of the subunits of vicilin from pea seeds.

Serological studies and comparison of N-terminal amino acid sequences with the amino acid sequence deduced from a cDNA clone have been used to establish the sequence relationships between the subunits of the pea seed storage protein, vicilin. Subunits smaller than Mr∼50 000 (i.e., Mr 34 000, 30 000, 25 000, 18 000, 14 000, 13 000 and 12 000) show extensive homology with molecules within Mr∼50 000 group. Both the sequencing and serological data confirm earlier evidence from studies on vicilin synthesisin vivo andin vitro which indicated that the vicilin subunits smaller than Mr∼50 000 arose by endoproteolytic cleavage of parent molecules within the Mr∼50 000 group. Cleavage in different Mr 50 000 parent molecules containing either one or both of two susceptible processing sites accounts for the formation of all the vicilin subunits smaller than Mr∼50 000, with the possible exception of the Mr34 000 polypeptide. The position of these sites in the putative parents were defined by reference to a complete amino acid sequence deduced from the sequence of DNA complementary to mRNA for one member of the Mr∼50 000 group.

The role of glycosylation in storage-protein synthesis in developing pea seeds.

Intact pea (Pisum sativum L.) cotyledons were incubated with [14C]glucosamine at several stages of seed development and the resultant radioactive proteins were analysed by gel electrophoresis combined with immunoaffinity chromatography and sucrose gradient fractionation. Glucosamine was incorporated into at least five vicilin polypeptides (approx. molecular weight 70,000; 50,000, two components; 14,000, two components). No incorporation was detected into the subunits of legumin. Tunicamycin at 50 μg/ml largely inhibited glucosamine incorporation but had little effect on the incorporation of 14C-labelled amino acids into cotyledon proteins, including vicilin. The assembly of vicilin polypeptides into full-sized protein oligomers (7–9 S) was also unaffected by tunicamycin. Chromatography on concanavalin A confirmed that glycosylation of cotyledon proteins was inhibited by tunicamycin. It is concluded that glycosylation of most cotyledonary proteins involves lipid-linked sugar intermediates, but that glycosylation itself is not an essential step in the synthesis of vicilin polypeptides nor in their assembly into oligomers.

Agrobacterium-Mediated Transformation of Subterranean Clover (Trifolium subterraneum L.).

We have developed a rapid and reproducible transformation system for subterranean clover (Trifolium subterraneum L.) using Agrobacterium tumefaciens-mediated gene delivery. Hypocotyl segments from seeds that had been allowed to imbibe were used as explants, and regeneration was achieved via organogenesis. Glucose and acetosyringone were required in the co-cultivation medium for efficient gene transfer. DNA constructs containing four genes encoding the enzymes phosphinothricin acetyl transferase, [beta]-glucuronidase (GUS), neomycin phosphotransferase, and an [alpha]-amylase inhibitor were used to transform subterranean clover. Transgenic shoots were selected on a medium containing 50 mg/L of phosphinothricin. Four commercial cultivars of subterranean clover (representing all three subspecies) have been successfully transformed. Southern analysis revealed the integration of T-DNA into the subterranean clover genome. The expression of the introduced genes has been confirmed by enzyme assays and northern blot analyses. Transformed plants grown in the glasshouse showed resistance to the herbicide Basta at applications equal to or higher than rates recommended for killing subterranean clover in field conditions. In plants grown from the selfed seeds of the primary transformants, the newly acquired gene encoding GUS segregated as a dominant Mendelian trait.

The expression of a chimeric cauliflower mosaic virus (CaMV-35S) — pea vicilin gene in tobacco

A gene encoding the 7S pea seed protein vicilin was modified for general expression in transgenic tobacco by fusion of the cauliflower mosaic virus 35S enhancer to the promoter region of the gene. The 7S seed protein was accumulated in leaves, stems, roots, seeds and callus but the level of expression varied widely in the different organs. Vicilin per unit of total soluble protein was higher in younger (upper) leaves (0.01%) than in older (lower) leaves (0.001%) indicating that vicilin was less stable than the average leaf protein. The vicilin in tobacco leaves was cleaved preferentially at or near one of the two post-translational cleavage sites used in pea seeds and in a manner very similar to that which occurs in tobacco seeds with either the unmodified vicilin gene or the 35S-vicilin construct. Leaf vicilin was also assembled into a normal 7S oligomer. These results indicate that a seed protein gene can be modified for expression in most organs of a transgenic host and that expression proceeds with a high level of fidelity. Minor variations in expression were related to selective cleavage of the polypeptide chain and to the production of two mRNA size classes instead of one. The fact that the seed protein was moderately stable in the heterologous environment augurs well for the engineered expression of other seed protein genes in transgenic hosts.

cDNA and protein sequence of a major pea seed albumin (PA 2 : Mr≈26 000).

SummaryPea albumin 2 (PA2:Mr≈26000) is a major component of the albumin fraction derived from aqueous salt extracts of pea seed. Sodium dodecylsulfate-polyacrylamide gel electrophoresis and chromatography on DEAE-Sephacel resolve PA2 into two closely related components (PA2a and PA2b). A cDNA clone coding for one of these components has been sequenced and the deduced amino acid sequence compared with partial, chemically-determined sequences for cyanogen bromide peptides from both PA2 components. Complete amino acid sequences were obtained for the C-terminal peptides. The PA2 molecule of 230 amino acids contains four imperfect repeat sequences each of approximately 57 amino acids in length.The combined sequence data, together with a comparison of PA2-related polypeptides produced in vitro and in vivo, indicate that PA2 is synthesized without a signal sequence and does not undergo significant post-translational modification. Although both forms of PA2 contain Asn-X-Thr consensus sequences, neither form is glycosylated. Accumulation of PA2 contributes approximately 11% of the sulfur-amino acids in pea seeds (cysteine plus methionine equals 2.6 residues percent). Suppression of levels of PA2 polypeptides and their mRNAs in developing seeds of sulfur-deficient plants is less marked than that for legumin, in spite of the lower content of sulfur-amino acids in legumin.