Expression of a sulfur-rich maize seed storage protein, delta-zein, in white clover (Trifolium repens) to improve forage quality

A modified gene encoding a sulfur-rich maize seed storage protein, δ-zein, was introduced into white clover plants by Agrobacterium-mediated transformation. Expression of the gene was under the control of the double 35S promoter of cauliflower mosaic virus and the nopaline synthase gene transcription terminator. All of the transgenic plants expressing transgene-specific mRNA also accumulated δ-zein in their leaves. Levels of the HA epitope tagged δ-zein in the first fully expanded young leaves of different transgenic plants varied from 0.06 to 0.3% of total water-soluble protein. Expression of the protein was also detected in petioles, nodes, internodes, roots and seeds of the transgenic plants. N-terminal sequencing of the modified δ-zein from transgenic plants revealed that the protein is processed in white clover leaves as in maize seeds. All the transgenic plants expressing the δ-zein showed monogenic inheritance of the linked nptII gene conferring kanamycin resistance. The epitope tagged δ-zein is relatively stable in white clover leaves and in the highest expressing plants, its accumulation increased with increasing leaf age from 0.3 (youngest leaves) to 1.3% (oldest leaves) of total water-soluble protein. These results open up the possibility of using sulfur-rich and rumen-protected δ-zein to improve white clover forage quality.