【Objective】The investigation of OsAlaAT4-mediated nitrogen utilization, contributes to an insight into the rice nitrogen regulatory network and supports the breeding of high nitrogen use efficiency rice varieties.【Method】Based on the amino acid sequence of OsAlaAT1, homologous genes were searched in the NCBI database. A knockout vector was constructed and transformed into Nipponbare (NIP) callus via Agrobacterium-mediated genetic transformation to obtain transgenic rice plants. Homozygous mutants were subsequently obtained. Wild-type Nipponbare (NIP) and mutant lines were grown in hydroponic systems at different nitrogen concentrations and in field trials to examine the relationship between OsAlaAT4 and nitrogen uptake and utilization.【Result】The homologous gene OsAlaAT4 was identified, and the knockout vector was successfully constructed and introduced into Nipponbare (NIP), resulting in two homozygous mutant lines. Hydroponic experiments at different nitrogen concentrations showed that the plant height, dry weight, and fresh weight of the mutant lines were lower than those of the wild-type under the same treatment, while the root length of mutant lines was higher. Under 1/2 standard nitrogen (N1/2), 4 times standard nitrogen (N4), and 8 times standard nitrogen (N8) treatments, the total nitrogen content of the mutant lines (alaat4-1 and alaat4-2) increased by 27.7%, 6.6%, 7.7% and 26.0%, 7.8%, 4.5%, respectively. The grain protein content of the mutant lines decreased by 3.0% and 3.3% under standard nitrogen conditions, respectively. Under low nitrogen field conditions, the plant height of the mutant lines was significantly lower than that of the wild-type. Compared to the wild-type, the nitrogen content in the leaves and stems of the two mutant lines increased by 12.1%, 13.5% and 14.4%, 6.9%, respectively, while the nitrogen content in the panicles decreased by 2.6% and 4.1%, and the yield decreased by 7.2% and 7.6%. Under high nitrogen field conditions, the number of tillers in the mutant lines was significantly higher than that of the wild-type. Compared to the wild-type, the nitrogen content in the leaves and stems of the mutant lines increased by 7.9%, 6.7% and 16.6%, 16.1%, respectively, while the nitrogen content in the panicles decreased by 4.9% and 4.5%, and the yield decreased by 6.5% and 5.4%. Under both low and high nitrogen conditions, the nitrogen use efficiency (NUE) of the mutant lines was lower than that of the wild-type.【Conclusion】Knockout of OsAlaAT4 resulted in increased nitrogen content in the leaves and stems of rice, but reduced nitrogen content in the panicles, protein content, and yield, resulting in reduced nitrogen use efficiency (NUE).
