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Gene-Deletor: A New Tool to Address Concerns over GE Crops

Yi Li, Hui Duan, and William Smith

The gene-deletor technology

Traditionally, site-specific DNA recombination systems, such as bacterial phage Cre/loxP and Saccharomyces cerevisiae FLP/FRT systems, have been used to excise marker genes or short spacer sequences in higher plants1. FLP/FRT and Cre/loxP function through interactions of a recombinase with its specific recognition sites. Recombinase recognizes these flanking recognition sites and excises any intervening DNA.

The laboratory of Dr. Yi Li, University of Connecticut, has recently developed a new technology called “gene-deletor” or “GM-gene-deletor”3 (see: http://gene-deletor.ne t). The principle of the technology is shown in Figure 1. As shown in tobacco, the gene-deletor technology, with a loxP-FRT hybrid as the recognition sequences for FLP or Cre recombinase, is 100% efficient in deleting all functional transgenes from both pollen and seeds, based on examination of more than 25,000 progeny per transgenic event.

The flow of transgenes into the wild via pollen and seeds is a serious concern for both the public and the scientific communities1. There are also concerns that novel proteins in certain transgenic crops could cause adverse reactions in some individuals. The gene-deletor technology could be used to remove all transgenes from any organs of a transgenic plant when the functions of transgenes are no longer needed or their presence may cause concerns. The technology may provide a useful tool to address transgene flow problems and also food safety concerns over transgenic crops.

Prevention of gene flow from asexually propagated transgenic crops

The current version of the gene-deletor technology (Fig. 1) is readily applicable to address the pollen- and seed-mediated transgene flow problems of vegetatively propagated plants that are important to the agricultural, forestry, bio-energy, ornamental, and paper/pulping industries. This technology can also be used in corn, soybean, cotton, or rice if artificial seeds (i.e., no pollination or fertilization is needed) are used for the propagation of these crops. Artificial seeds have been produced from many major agricultural crops.

Figure 1. A schematic illustration of the principle of the gene-deletor technology for excision of all transgenes from pollen and seeds. L represents the LoxP recognition sequence from the phage Cre/loxP system and F represents the FRT recognition sequence of the yeast FLP/FRT system. PAB5 is a pollen- and seed-specific gene promoter from Arabidopsis and FLP is a DNA recombinase from the FRT/FLP system. Expression of recombinase under the control of PAB5 leads to deletion of all functional transgenes between the two LF (LoxP-FRT fusion) sites, including the recombinase gene in pollen and seeds specifically. The deleted gene sequences will be destroyed by non-specific nucleases in the cell. Because of the use of the LoxP-FRT fusion as the recognition sequence for FLP, the

progeny examined per event.

deletion efficiency observed is as high as 100%, based on more than 25,000 T

1

Prevention of gene flow from sexually propagated transgenic crops

A modified version of the current gene-deletor technology could also be used in sexually propagated crops such as

RNAi-FLP gene cassette into the gene-deletor system. Application of a chemical inducer at the correct time would prevent the deletion of transgenes in pollen and seeds (Fig. 2). For instance, the ethanol inducible ALCR/alcA (alc) two-component system4 could be used to control RNAi-FLP gene expression. With the ALCR/alcA (alc) system, application of ethanol during pollen/seed development would activate the RNAi-FLP gene. RNAi-FLP represses the pollen/seed specific expression of FLP recombinase. As a result, all transgenes will remain in the pollen or seeds during that generation. However, if the inducer is not applied in a subsequent generation, FLP will be expressed. Consequently, FLP expression will lead to deletion of all functional transgenes in pollen and seeds.

One important feature of the inducible RNAi-FLP gene is that it does not need to be 100% efficient if used in the gene-deletor system. Non-perfect suppression of FLP by RNAi-FLP could cause an undesirable deletion of transgenes in a small number of pollen and seeds. However, non-perfect suppression of FLP expression will not present a problem because seed companies can easily eliminate these “non-transgenic” seeds when producing certified seeds. Seed companies may apply a selection agent such as herbicide at early stages of seed development to kill non-transgenic seeds. Also, if a small percentage of certified seeds are non-transgenic, it should not cause a significant yield loss to farmers. For crops that are engineered for insect resistance, some researchers believe that planting 1% to 3% non-transgenic plants in an insect resistant field may reduce the potential risks for developing resistant insects. Furthermore, because application of the chemical inducer is performed by seed companies, and acreages for certified seed production are generally small, it is relatively easy to achieve uniform and effective applications of the inducer.

Alleviation of the safety concerns over transgenic foo ds

The gene-deletor technology can be extended to generate non-transgenic edible parts from transgenic plants to mitigate consumer concerns over the presence of transgenes in food products. As shown in Figure 3, if one uses a pollen- and seed-specific gene promoter to control recombinase expression, all transgenes will be deleted from pollen and seeds specifically. This is particularly useful for herbicide resistant crops like soybean. Similarly, with a fruit- or tuber-specific gene promoter to control FLP expression, one can produce non-transgenic fruits or tubers from transgenic crops. The technology could also be used for post-harvest removal of transgenes in crops like lettuce and broccoli with a conditionally active (e.g., post-harvesting active, high- or low- temperature inducible, or ethanol-inducible) gene promoter to control FLP expression. In all of these cases, the transgenic protein products will be destroyed in a matter of hours or days after the transgenes are deleted.

Figure 2. The principle of a modified version

of the current gene-deletor system for its

applications in sexually propagated crops.

FLP is a DNA recombinase that recognizes the

FRT sequences and excises the intervening

DNA sequences. PAB5 is a pollen- and seed-

specific gene promoter. If an inducible RNAi-FLP

gene cassette is inserted in the gene-deletor

system, application of the inducer will activate

RNAi-FLP expression and therefore suppress

FLP expression. If the expression of FLP is

suppressed, deletion of transgenes will not

occur. During the sexual production of certified

seeds, seed companies could apply the inducer

to prevent the deletion of transgenes from

pollen and seeds as shown. In a subsequent

generation, all transgenes will be deleted from

pollen and seeds as shown in Fig. 1 if the

inducer is not applied.

Prevention of transgenic volunteer crops

V olunteer crops are plants growing from seeds which fall out in harvesting from a previous planting. V olunteer

transgenic plants are a source of many environmental risks and have resulted in lawsuits from neighboring farms or seed companies. Because the gene-deletor technology can remove all functional transgenes from pollen and seeds, there will be no transgenic volunteer plants in subsequent seasons.

A better technology than sterile seed technologies

Compared to sterile seed technologies like the terminator technology 5, the gene-deletor technology offers several advantages. Similar to the sterile technologies, the gene-deletor technology can address the problem of pollen- and seed-

mediated gene flow. However, the gene-deletor technology is superior to sterile seed technologies because

it can be used to produce non-transgenic seeds, fruits, tubers, flowers, leaves, or stems to alleviate the concerns over transgenic food safety. Furthermore, unlike the sterile seed technologies that may pose potential problems for farmers in developing countries, the gene-deletor technology allows the production of viable seeds and the yield of the subsequent generation crops is the same as their non-transgenic counterparts.

The UConn team started the development of the gene-deletor technology in 2000 with funding from Connecticut Innovation Inc (CII) and from the Consortium for Plant Biotechnology Research (CPBR) and UConn. The team and their collaborators at Chinese Southwest University and the University of Tennessee published the technology in the March issue of Plant Biotechnology Journal in 2007.

References and additional readings:

1. Srivastava V, Ow DW. (2004) Marker-free site-specific gene integration in plants. Trends Biotechnol . 22(12), 627-9

2. Stewart CN Jr, Halfhill MD, Warwick SI. (2003) Transgene introgression from genetically modified crops to their wild relatives. Nat Rev Genet . 4(10), 806-17

3. Luo K, et al. (2007) ‘GM-gene-deletor’: Fused loxP-FRT recognition sequences dramatically improve the efficiency of FLP or CRE recombinase on transgene excision from pollen and seeds of tobacco plants. Plant Biotech. J . 5(2), 263-274

4. Roberts GR, et al. (2005) The alc-GR system: A modified alc gene switch designed for use in plant tissue culture. Plant Physiol . 138, 1259-1267

Figure 3. The gene-deletor technology can be

used to produce non-transgenic organs from transgenic plants (Transgenic is shown in red; non-transgenic is shown in green). Transgenic plants produced from the current technologies contain transgenes in all organs and cells (all transgenic). With the gene-deletor technology, non-transgenic organs could be produced if an appropriate gene promoter is used to control FLP expression.

5. Daniell H. (2002) Molecular strategies for gene containment in transgenic crops. Nat Biotechnol . 20(6).581-586

Gene-Deletor: For additional information, news reports and commentaries about the gene-deletor technology, please visit: https://www.360docs.net/doc/1515142830.html,

Yi Li

Department of Plant Science, U-5082

University of Connecticut

Storrs, CT 06268yi.li@https://www.360docs.net/doc/1515142830.html,

William Smith

Department of Plant science

University of Connecticut

Storrs, CT 06268

Hui Duan

Department of Cell & Structural Biology University of Illinois,Urbana, IL 61801