Objectives We studied the effect of over-expressing Aspergillus niger AngdhA gene on the growth, expression of key enzyme genes related to nitrogen metabolism, carbon and nitrogen contents, nitrogen use efficiency, yield, and salt tolerance of transgenic rice lines, aiming to provide scientific support for breeding rice with high efficiency, yield, and tolerance.
Methods Transgenic rice lines TG3 and TG13 (expressing Aspergillus niger AngdhA gene), and wild-type (WT) were used as materials in hydroponic experiments. Seedling samples at the 3-leaf stage under normal cultural condition were used to analyze gene expression of glutamine synthetase and glutamate synthase, carbon and nitrogen contents, and samples at harvest were used for investigation of yield traits and nitrogen use efficiency. Salt stress treatment was simulated by adding 0, 50, and 100 mmol/L NaCl to the nutrient solution, and the relative growth rate, proline content, leaf wilting rate, and organ biomass of seedlings at the 3-leaf stage were analyzed after subjected to salt stress for 0, 2, and 4 days.
Results PCR amplification and electrophoresis analysis confirmed that Aspergillus niger AngdhA gene was successfully introduced into TG3 and TG13 lines of rice. The expression levels of AngdhA gene in TG3 and TG13 lines were 248-fold and 41-fold to the reference gene UBI, respectively. However, the expression of key nitrogen metabolism enzyme genes , including OsGS1;1, OsGS1;2 and OsGOGAT, was slightly affected in both TG3 and TG13 lines. Only OsGS2 in TG13 was significantly down-expressed, compared to WT (P<0.01). The N contents and C/N ratio in leaf and root at seedling stage, and in grain, shoot and root at harvest were not significantly different among the three rice lines. However, TG3 and TG13 had significantly higher C and N accumulation than WT in plants, particularly in grains. TG3 and TG13 increased nitrogen utilization efficiency by 14.67% and 44.12%, nitrogen use efficiency for grain production (NUEg) by 26.96% and 39.83%, and nitrogen partial productivity (NPFP) by 24.53% and 39.67% over WT, respectively, with significant or highly significant increase in panicle number per plant, grain number per plant, and yield per plant. The relative growth rates of TG3 and TG13 under 50 mmol/L NaCl were higher than that under the control (0 mmol/L NaCl). The proline content in rice was sharply increased under salt stress. Compared to WT, TG3 and TG13 elicited significantly higher proline content under 50 mmol/L NaCl, but lower proline content under 100 mmol/L NaCl (P<0.01).
Conclusions Aspergillus niger AngdhA gene was successfully introduced into TG3 and TG13 lines of rice without apparent disturbance of carbon-nitrogen balance. The exogenous AngdhA gene promoted nitrogen uptake, utilization, and remobilization, thus improved nitrogen use efficiency, yield, and salt tolerance of rice. However, the over-expression of AngdhA gene in TG3 might have increased substrate cycle in glutamate synthesis and catabolic pathway and bring down the increment of nitrogen uptake and utilization and yield. The differential accumulation of proline might be related to the different response and tolerance of rice to salt stress.
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