Production of Marker-free Transgenic Rice (Oryza sativa L.) with Improved Nutritive Quality Expressing AmA1

Rice seed proteins are lacking essential amino acids (EAAs). Genetic engineering offers a fast and sustainable method to solve this problem as it allows the specific expression of heterologous EAA-rich proteins. The use of selectable marker gene is essential for generation of transgenic crops, but might also lead to potential environmental and food safety problems. Therefore, the production of marker-free transgenic crops is becoming an extremely attractive alternative and could contribute to the public acceptance of transgenic crops.
The present study was conducted to examine whether AmA1 can be expressed specifically in rice seeds, and generate marker-free transgenic rice with improved nutritive value.
AmA1 was transferred into rice using Agrobacterium-mediated co-transformation system with a twin T-DNA binary vector and its integration in rice genome was confirmed by southern blot. Transcription of AmA1 was analyzed by Real-Time PCR and its expression was verified by western analysis. Protein and amino acid content were measured by the Kjeldahl method and the high-speed amino acid analyzer, respectively.
Five selectable marker-free homozygous transgenic lines were obtained from the progeny. The expression of recombinant AmA1 was confirmed by the observation of a 35 kDa band in SDS-PAGE and western blot. Compared to the wild-type control, the total protein contents in the seeds of five homozygous lines were increased by 1.06~12.87%. In addition, the content of several EAAs, including lysine, threonine, and valine was increased significantly in the best expressing line.
The results indicated that the amino acid composition of rice grain could be improved by seed-specific expression of AmA1.