Saverio Carl Falco

A Cre/loxP-mediated self-activating gene excision system to produce marker gene free transgenic soybean plants

Marker-gene-free transgenic soybean plants were produced by isolating a developmentally regulated embryo-specific gene promoter, app1, from Arabidopsis and developing a self-activating gene excision system using the P1 bacteriophage Cre/loxP recombination system. To accomplish this, the Cre recombinase gene was placed under control of the app1 promoter and, together with a selectable marker gene (hygromycin phosphotransferase), were cloned between two loxP recombination sites. This entire sequence was then placed between a constitutive promoter and a coding region for either β-glucuronidase (Gus) or glyphosate acetyltransferase (Gat). Gene excision would remove the entire sequence between the two loxP sites and bring the coding region to the constitutive promoter for expression. Using this system marker gene excision occurred in over 30% of the stable transgenic events as indicated by the activation of the gus reporter gene or the gat gene in separate experiments. Transgenic plants with 1 or 2 copies of a functional excision-activated gat transgene and without any marker gene were obtained in T0 or T1 generation. This demonstrates the feasibility of using developmentally controlled promoters to mediate marker excision in soybean.

Expression of de novo high-lysine alpha-helical coiled-coil proteins may significantly increase the accumulated levels of lysine in mature seeds of transgenic tobacco plants.

We have designed protein molecules based on an α-helical coiled-coil structure. These proteins can be tailored to complement nutritionally unbalanced seed meals. In particular, these proteins may contain up to 43% mol/mol of the essential amino acid lysine. Genes encoding such proteins were constructed using synthetic oligonucleotides and the protein stability was tested for in vivo by expression in an Escherichia coli model system. A protein containing 31% lysine and 20% methionine (CP 3-5) was expressed in transgenic tobacco seeds utilizing the seed specific bean phaseolin and soybean β-conglycinin promoters. Both promoters provided a level of expression in the mature transgenic tobacco seeds which resulted in a significant increase in the total lysine content of the seeds. Several of these transgenic lines were analyzed for three generations to determine the stability of gene expression. Plants transformed with the soybean β-conglycinin promoter/CP 3-5 gene consistently expressed the high-lysine phenotype through three generations. However, expression of the high-lysine phenotype in plants transformed with the bean phaseolin/CP 3-5 was variable. This is the first report of a significant increase in seed lysine content due to the seed-specific expression of a de novo protein sequence.

MOLECULAR ANALYSIS OF SULFONYLUREA HERBICIDE RESISTANT ALS GENES

The enzyme acetolactate synthase (ALS) is the target of several classes of herbicides, including the sulfonylureas, the imidazolinones, and the triazolopyrimidines. ALS is required for the biosynthesis of isoleucine, leucine and valine. We have cloned ALS genes from a variety of monocotyledonous and dicotyledonous plants. The number of ALS genes in these species varies from one, as in Arabidopsis and sugar beets, to two or more, as in corn and soybeans. The deduced amino acid sequences of the mature plant ALS genes which have been sequenced are closely related. Three domains of the plant ALS proteins are also conserved relative to bacterial and yeast ALS proteins. ALS genes have been isolated and sequenced from sulfonylurea herbicide resistant plants and the resistance trait shown to result from single or double mutations in the conserved domains of the genes. To produce additional genes able to confer sulfonylurea herbicide resistance in transgenic crops, in vitro-generated mutations were introduced into the coding regions of cloned plant ALS genes. A number of single and double mutations were shown to be effective in conferring resistance, upon subsequent introduction of the altered genes into plants. The mutations conferred varying degrees of resistance, depending upon the site of the resulting amino acid substitution and upon the amino acid substituted at that site. Replacement of ALS regulatory sequences led to increases in the proportion of herbicide resistant enzyme such that up to 90% of the total ALS activity was herbicide resistant. The altered genes conferred sulfonylurea herbicide resistance in glasshouse trials of a wide variety of transgenic crop species. Additionally, transgenic tobacco, tomato and oilseed rape were shown to be herbicide resistant in field trials.