谷丙转氨酶基因OsAlaAT4调控水稻氮素吸收和产量

doi:10.16819/j.1001-7216.2026.250206

摘要/Abstract

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

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

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.

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.

图/表 6

图1 进化树、表达量分析及保守结构域分析 A: 进化树分析，红色字体表示在水稻中的AlaAT家族基因；B: OsAlaAT4不同组织部位的相对表达量；DAF: 受精后天数；C: 保守结构域分析，蓝色线条下为保守氨基酸序列。

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.

图2 OsAlaAT4的突变系鉴定 A: 2个突变系的碱基类型，---表示缺失的碱基序列，红色字体表示插入的碱基；B: 2个突变系的测序结果；C: 野生型与突变系OsAlaAT4表达量。

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.

图3 突变系幼苗表型及氮含量 A：野生型和OsAlaAT4突变系水培14 d的地上部，标尺=10 cm；B：野生型和OsAlaAT4突变系水培14 d的根长，标尺=10 cm；C：野生型和OsAlaAT4突变系的苗高；D：野生型和OsAlaAT4突变系的干质量；E：野生型和OsAlaAT4突变系的鲜质量；F：野生型和OsAlaAT4突变系的根长；G：野生型和OsAlaAT4突变系氮含量。N0: 无氮; N1/2: 1/2倍标准氮; N4: 4倍标准氮; N8: 8倍标准氮。

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.

图4 关键酶活性及相关基因表达量测定 A：谷丙转氨酶活性；B：α-酮戊二酸酶活性；C：GOGAT酶活性；D-G：野生型与突变系中OsAlaAT1、OsAlaAT2、OsAlaAT3、OsAlaAT5的表达量；H为野生型与突变系中氮转运相关基因的表达量。

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.

图5 突变系田间表型、氮含量及产量 A：成熟期野生型和OsAlaAT4突变系的表型；B：野生型和OsAlaAT4突变系的株高；C：野生型和OsAlaAT4突变系的分蘖数，n=12；D：成熟期野生型和OsAlaAT4突变系的单株产量；E：低氮条件下野生型和OsAlaAT4突变系的叶、茎和穗的氮含量；F：高氮条件下野生型和OsAlaAT4突变系的叶、茎和穗的氮含量；G：野生型和OsAlaAT4突变系的氮素利用率。LN：低氮; HN：高氮。

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.

图6 OsAlaAT4突变对蛋白质和粒型的影响 A：蛋白质含量；B：千粒重；C-E：粒长、粒宽、粒厚。

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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