Berkeley - Using a form of genetic hitchhiking, researchers have developed a method of placing genes in barley and other cereals in a way that eases safety concerns and minimizes the problem of "gene-silencing."
Developed by scientists at the University of California, Berkeley, the new method uses so-called "jumping genes" to ferry new genes into cereals like barley. The technique will allow plant biologists to boost nutritional content, improve pest resistance, reduce allergenicity and even perhaps speed up beer-making. The process does not use viral particles for transporting genes, nor does it rely on antibiotic-resistance screening, two methods that have raised safety concerns for some individuals.
"Our method is ready for use by plant biologists to create new varieties of cereal crops in a manner that is both very quick and very safe," said Peggy Lemaux, a plant biologist at UC Berkeley's Department of Plant and Microbial Biology, in the College of Natural Resources. Lemaux conducted the work with post-doctoral researcher Thomas Koprek and laboratory assistant Sergio Rangel. The research will be published in the March issue of the journal Plant Physiology.
Until now, a major challenge in genetic engineering of grains has been maintaining activity of the genes over time. Grains are surprisingly adept at stifling the implanted gene's activity, a process known as gene-silencing.
Lemaux and her colleagues devised a way to surmount this hurdle. The trick is to deliver just one copy of the gene and coax it into an area where genes can be expressed, away from dense regions of repeating DNA.
But creating a plant that contains only one gene copy is difficult using some conventional techniques. Instead, Lemaux and her colleagues engineered the genes to hitchhike on mobile pieces of DNA called jumping genes. Also known as transposons, jumping genes hop from place to place inside the genome, causing, for example, the mosaic pattern seen in Indian corn. The idea to use jumping genes to introduce new genes was demonstrated in tomatoes at UC Davis in 1997.
When the jumping gene hops through the plant's genome, it usually lands in low-density DNA territory where conditions are favorable for making the protein that is encoded by the inserted gene.
To harness this system, Lemaux and colleagues hitch their gene of interest to the part of the jumping gene that allows it to jump. The researchers sandwich a gene called bar - which confers resistance to an herbicide called Basta - between the ends of a jumping gene called Ds (for Dissociation), forming a Ds-bar-Ds segment. Using a gene gun, Lemaux and her team then inject this segment into barley cells.
For the gene to jump, it requires help from an enzyme called Ac transposase, or AcTPase. So, the team creates another transgenic plant containing the AcTPase gene and crosses it with the Ds-bar-Ds plant. With both genes on hand, the Ds element can jump to a location away from the original insertion site. "We use traditional breeding to move transponsons into the plant genome," said Lemaux.
In subsequent generations, the AcTPase gene naturally breeds out of the plant, shutting down the jumping ability of the Ds-bar-Ds gene. To verify that the gene remains active, the researchers paint the leaves of the plants with Basta. If the leaves wilt, the bar gene is no longer active. To date, Lemaux and her colleagues have detected active genes in six successive generations with very low rates of gene-silencing, compared to a high rate of silencing in multiple-copy plants and single-copy plants that were produced without the help of transposons.
By hitching their genes to transposons, Lemaux's group can generate hundreds of independent, single-copy plants very rapidly. The plants contain none of the original DNA sequences used to prepare the Ds-bar-Ds and AcTPase genes for insertion into the seed.
Lemaux and UC Berkeley colleague Bob Buchanan hope to use the technique to insert genes like thioredoxin, which can help promote the degradation of certain food allergens. The technique also works in other cereal grains and is being used in a study to help boost the digestible protein content of sorghum, a major animal feed in many parts of the world. And brewers might like to use the technique to produce barley with a shorter malting time.
This work was supported by the German government research agency Deutscheforschungsgemeinschaft, by the U.S. Department of Agriculture Cooperative Extension Service and by the Novartis Agricultural Discovery Institute.