Cloning Of The New Lysine-Rich Protein Genes And Application In Maize Genetic Improvement

Maize(Zea mays L.) is a major source of food and animal feed worldwide, especially in developing and underdeveloped countries. However, maize seeds are deficient in two essential amino acids:lysine and tryptophan. Consuming a protein-deficient diet over the long term may lead to poor growth, disease and, in severe cases, death. Therefore, improving the composition of maize proteins is highly desirable.In this study, a lysine-rich protein(Gossypium hirsutum L. lysine rich protein, GhLRP) gene, was cloned from the cotton(Gossypium hirsutum L.) genome. It consists of only one exon and encodes a protein with195amino acids. The lysine content of GhLRP is18.97%(w/w). So far, very little is known about its function. A construct containing the GhLRP gene under the control of the seed-specific promoter F128was introduced into the immature embryos of the hybrid08x178mediated by Agrobacterium tumefaciens. The156positive regenerated plants from88independent transformation events were identified. In the Ti generation, the lysine contents of40transgenic lines among88transgenic lines were increased relative to WT by levels ranging from16.2to65.0%, and that of26transgenic lines increased significantly. The total protein content was not distinctly different except in the six transgenic lines. Further analysis of3transgenic lines showed that the high lysine content was heritable in the T2, T3, and T4generations. Moreover, there were no significant drawbacks in terms of quality (lipid and starch content) and agronomic traits in most of the transgenic maize. These results demonstrate that GhLRP is a high-lysine protein candidate gene to increase the lysine content of maize.Another lysine-rich protein (Setaria italica Remorin6, SiREM6) gene, was cloned from the foxtail millet(Setaria italica). The open reading frame of SiREM6is639bp, which encodes a Remorin family protein with212amino acids, and the lysine content is18.99%(w/w). SiREM6could oligomerize into filamentous structures. SiREM6expressed in root, stem, leaf and inflorescences of foxtail millet, and the transcript level of SiREM6was increased by high salt stress, low temperature stress and ABA treatment, but not by drought. Further analysis showed that overexpression of SIREM6improved the high salt stress tolerance in transgenic Arabidopsis at the germination and seedling stages. The SiREM6promoter contains two dehydration responsive elements (DREs) and one ABA responsive element (ABRE). Electrophoresis mobility shift assay (EMSA) analysis showed that Setaria italica ABA-responsive DRE-binding protein (SiARDP) could physically bind to the DREs, but Setaria italica ABRE-binding protein1(SiAREB1) could not. These results indicated that SiREM6might be regulated by SiARDP in foxtail millet under salt stress. Further, in order to evaluate the application value in maize genetic improvement, the SiREM6gene under the control of the CaMV35S promoter and the seed-specific promoter19-kDa a-zein were introduced into the maize callus of the inbred178and the hybrid08×178mediated by Agrobacterium tumefaciens, respectively. Now, the resistant calli are at the stage of differentiation.In this study, we cloned two candidate genes encoding lysine-rich proteins, and primary functions of that were analyzed. These will provide the gene resources for maize genetic improvement.