Regulation of Nitrogen Uptake and Yield in Rice by the Alanine Aminotransferase Gene OsAlaAT4

doi:10.16819/j.1001-7216.2026.250206

Abstract

Abstract:

【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).

Key words: rice, nitrogen content, OsAlaAT4, nitrogen use efficiency, yield

摘要：

【目的】研究OsAlaAT4在调控水稻氮素利用中的功能，有助于完善水稻氮调控网络，同时为水稻高氮利用率提供理论依据。【方法】根据OsAlaAT1的氨基酸序列在NCBI数据库找同源基因，构建敲除载体，通过农杆菌介导的遗传转化方法转化日本晴愈伤组织，从而获得水稻转基因植株并筛选纯合突变体，将野生型日本晴与突变系通过不同氮浓度的水培试验和田间试验来验证OsAlaAT4与氮吸收利用相关。【结果】查找到同源基因OsAlaAT4，成功构建敲除载体并转入日本晴(NIP)，获得2个纯合突变系。不同氮浓度水培结果显示，突变系株高、干质量和鲜质量低于相同处理的野生型，根长长于相同处理的野生型，在1/2倍标准氮(N_1/2)、4倍标准氮(N₄)、8倍标准氮(N₈)处理下突变系alaat4-1和alaat4-2的整株氮含量分别提高了27.7%、6.6%、7.7%和26.0%、7.8%、4.5%。正常施氮条件下突变系籽粒的蛋白质含量分别降低了3.0%和3.3%。在低氮田条件下，突变系的株高显著低于野生型。与野生型相比，两个突变系的叶和茎氮含量分别提高了12.1%、13.5%和14.4%、6.9%，穗部氮含量则分别降低了2.6%和4.1%，产量分别降低了7.2%和7.6%；高氮田条件下，突变系的分蘖数显著高于野生型，与野生型相比，突变系的叶和茎氮含量分别提高了7.9%、6.7%和16.6%、16.1%，穗部氮含量则降低了4.9%、4.5%，产量下降了6.5%和5.4%。无论低氮还是高氮处理下，突变系的氮素利用率(NUE)均显著低于野生型。【结论】敲除OsAlaAT4导致水稻叶、茎中的氮含量增加，但穗部氮含量、蛋白质含量和产量下降，最终导致氮素利用率(NUE)下降。

关键词: 水稻, 氮含量, OsAlaAT4, 氮素利用率, 产量

WANG Yixin, LIN Shen, MA Liuyang, CHEN Long, FENG Baohua, NI Shen, WEI Xiangjin, HE Jiwai, CHEN Tianxiao. Regulation of Nitrogen Uptake and Yield in Rice by the Alanine Aminotransferase Gene OsAlaAT4[J]. Chinese Journal OF Rice Science, 2026, 40(1): 51-60.

王轶欣, 林参, 马刘洋, 陈龙, 奉保华, 倪深, 魏祥进, 贺记外, 陈天晓. 谷丙转氨酶基因OsAlaAT4调控水稻氮素吸收和产量[J]. 中国水稻科学, 2026, 40(1): 51-60.

Figures/Tables 6

Fig. 1. Phylogenetic tree, expression analysis, and conserved domain analysis A, Phylogenetic tree analysis, with red font indicating the AlaAT family genes in rice; B, Relative expression levels of OsAlaAT4 in different tissues; DAF, Days after fertilization; C, Conserved domain analysis, with conserved amino acid sequences under the blue lines.

Fig. 2. Identification of OsAlaAT4 mutant lines A, Base types of the two mutant lines, --- indicates the deleted base sequence, and red font indicates the inserted bases; B, Sequencing results of the two mutant lines; C, Expression levels of OsAlaAT4 in the wild-type and mutant lines.

Fig. 3. Phenotype and nitrogen content of mutant seedlings A, Above-ground part of wild-type and OsAlaAT4 mutant lines after 14 days of hydroponic cultivation, bar=10 cm; B, Root length of wild-type and OsAlaAT4 mutant lines after 14 days of hydroponic cultivation, bar=10 cm; C, Seedling height of wild-type and OsAlaAT4 mutant lines; D, Dry weight of wild-type and OsAlaAT4 mutant lines; E, Fresh weight of wild-type and OsAlaAT4 mutant lines; F, Root length of wild-type and OsAlaAT4 mutant lines; G, Nitrogen content of wild-type and OsAlaAT4 mutant lines. N0, No nitrogen; N1/2, 1/2 standard nitrogen; N4, 4 times standard nitrogen; N8, 8 times standard nitrogen.

Fig. 4. Enzyme activity and related gene expression level measurement A, Alanine aminotransferase (ALT) enzyme activity; B, α-Ketoglutarate enzyme activity; C, Glutamate synthase (GOGAT) enzyme activity; D-G, Expression levels of OsAlaAT1, OsAlaAT2, OsAlaAT3, OsAlaAT5 in wild-type and mutant lines; H, Expression levels of nitrogen transport-related genes in wild-type and mutant lines.

Fig. 5. Field phenotype, nitrogen content, and yield of mutant lines A, Phenotype of wild-type and OsAlaAT4 mutant lines at maturity; B, Plant height of wild-type and OsAlaAT4 mutant lines; C, Number of tillers in wild-type and OsAlaAT4 mutant lines, n=12; D, Single-plant yield of wild-type and OsAlaAT4 mutant lines at maturity; E, Nitrogen content in leaves, stems, and panicles of wild-type and OsAlaAT4 mutant lines under low nitrogen conditions; F, Nitrogen content in leaves, stems, and panicles of wild-type and OsAlaAT4 mutant lines under high nitrogen conditions; G, Nitrogen use efficiency (NUE) of wild-type and OsAlaAT4 mutant lines. LN, Low nitrogen; HN, High nitrogen.

Fig. 6. Impact of OsAlaAT4 mutation on protein and grain shape A, Protein content; B, Thousand-grain weight; C-E, Grain length, grain width, grain thickness.

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