Biofortification of rice with lysine using endogenous histones.

Rice is the most consumed cereal grain in the world, but deficient in the essential amino acid lysine. Therefore, people in developing countries with limited food diversity who rely on rice as their major food source may suffer from malnutrition. Biofortification of stable crops by genetic engineering provides a fast and sustainable method to solve this problem. In this study, two endogenous rice lysine-rich histone proteins, RLRH1 and RLRH2, were over-expressed in rice seeds to achieve lysine biofortification. Their protein sequences passed an allergic sequence-based homology test. Their accumulations in rice seeds were raised to a moderate level by the use of a modified rice glutelin 1 promoter with lowered expression strength to avoid the occurrence of physiological abnormalities like unfolded protein response. The expressed proteins were further targeted to protein storage vacuoles for stable storage using a glutelin 1 signal peptide. The lysine content in the transgenic rice seeds was enhanced by up to 35 %, while other essential amino acids remained balanced, meeting the nutritional standards of the World Health Organization. No obvious unfolded protein response was detected. Different degrees of chalkiness, however, were detected in the transgenic seeds, and were positively correlated with both the levels of accumulated protein and lysine enhancement. This study offered a solution to the lysine deficiency in rice, while at the same time addressing concerns about food safety and physiological abnormalities in biofortified crops.

Transformation of LRP gene into Brassica napus mediated by Agrobacterium tumefaciens to enhance lysine content in seeds.

Lysine rich protein (LRP) gene derived from the seed of Psophocarpus tetragonolobus was transformed into Brassica napus, employing cotyledon petiole as explants and by using the Agrobacterium tumefaciens strain LBA4404. Transformation efficiency was found to be closely related with phytohormone concentration, infection incubation, and co-cultured time. A medium containing 4 mg/l 6-benzyladenine (6-BA) and 0.3 mg/l naphthalene acetic acid (NAA) was used for plant regeneration. With infection incubation of A. tumefaciens (OD600 = 0.4) for 20 min and co-culture of infected cotyledon petiole for 3 days, the highest transformation efficiency of 8.5% was obtained. To confirm LRP gene expression, PCR and Southern blot analysis were performed on leaf-isolated DNA from regenerated plants resistant to kanamycin. All transgenic plants of the generation T0 formed fertile seeds, which were sowed for the inheritance study of generational T1 and amino acid analysis. It was found that the lysine content of seeds from T1 generation increased by 16.7% compared with non-transgenic lines.