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PROTEOME ANALYSIS OF NODULATION-RELATED PROTEINS IN THE WILD TYPE AND A SUPERNODULATION MUTANT (sunn) OF THE MODEL LEGUME, MEDICAGO TRUNCATULA

U. Mathesius, G. Keijzers, J.J. Weinman, B.G. Rolfe

Research School Biol. Sci., Australian National University, Canberra, Australia

We studied differential protein expression in the wild type and the supernodulating mutant, sunn, of Medicago truncatula in response to Rhizobium infection to elucidate the nature of the sunn mutation. Sunn is a supernodulating mutant with a proposed defect in the regulation of auxin transport and/or sensitivity, suggesting that an auxin defect is underlying its supernodulating phenotype.

We have previously established a proteome reference map for Medicago truncatula using two-dimensional gel electrophoresis combined with peptide mass fingerprinting to separate and identify differentially expressed proteins. The M. truncatula EST database was used as an essential tool for the identification of the proteins because it provided a database for the generation of theoretical peptide mass fingerprints and prediction of protein function using BLAST searching.

Wild type and sunn showed only minor differences in the untreated state, but differential protein expression after Rhizobium inoculation, including auxin-induced and ripening-related proteins, two nodulins, and S-adenosyl methionine synthase, involved in ethylene synthesis. The differential expression of these proteins supports the hypothesis that hormonal regulation is affected in sunn. The number of hormonally regulated proteins changing in amount during nodule development might reflect the importance of plant hormones in nodule organogenesis.

GENETIC TRANSFORMATION OF PHASEOLUS VULGARIS

CIFN/UNAM, Res. Program Plant Mol. Biol., Cuernavaca, Morelos, México

Beans (Phaseolus vulgaris) are the most widely consumed legumes in the world. This crop is the principal protein source for people in Mexico and other American countries. P. vulgaris establishes symbiosis with Rhizobium etli. Both the macro and micro-symbionts originated in America and have co-evolved for a long time. Our main research interest is towards P. vulgaris functional genomics; this effort is part of the global project on Phaseolus genomics from an international consortium recently formed. An efficient and reliable genetic transformation procedure is a critical issue that needs to be satisfied in order to propose a certain legume species for genomic projects. In this regard, we now report a genetic transformation system of P. vulgaris var. Negro Jamapa 81, mediated by Agrobacterium tumefaciens.

First we established a regeneration protocol for three different bean cultivars, widely consumed in Mexico: Negro Jamapa 81, Flor de Mayo and Peruano. The explants used were cotyledonary nodes, from germinated seedling. Shoots were induced, through direct organogenesis, in MS media with BAP (5 mg/L) and sucrose. Around two shoots per explant were formed in the three varieties tested. The use of liquid media for shoot induction and development was a key element for an efficient regeneration.

Based in the efficient regeneration procedure, a reliable protocol for genetic transformation mediated by Agrobacterium tumefaciens has been developed for the Negro Jamapa 81 variety. Cotyledonary nodes were cocultivated with A. tumefaciens LBA4404, bearing pBI121 plasmid, followed by in vitro regeneration using kanamycin (kan) for selection. The kan concentration (50 mg/L) used only for a short period (10 days) were critical factors for efficient regeneration of putative (kanR) tranformants. KanR shoots were generated from the apical and lateral meristematic regions of the explant. Stable integration of the transgenes in the kanR putative primary transformants (To) were evidenced by PCR and gus activity analyses. Near 60% of the plants analyzed were PCR+ or were stably transformed; while 40% were false positives (PCR"). The frequency for obtaining PCR+ plants was reproducible among different experiments. The transformation efficiency was 7%, meaning that 7 transgenic plants are obtained from 100 explants. The To plants were fertile and showed a normal symbiotic phenotype with R. etli. The To plants were self-fertilized and the transfer of the transgenes to the progeny is being confirmed by PCR and Southern analysis. All the Ti plants checked, so far, are PCR+ indicating the stable heredity of the transgenes.

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