过表达AngdhA对水稻生长与氮素利用效率的影响

Influence of over-expression of AngdhA gene on growth and nitrogen use efficiency of transgenic rice lines

【目的】研究过表达黑曲霉AngdhA基因对水稻生长、氮代谢关键酶基因表达、碳氮含量、氮素利用效率、产量及耐盐性的影响,为水稻节本增效、高产和高抗多目标育种提供科学参考。【方法】以水稻转基因株系TG3、TG13与野生型(wild-type,WT)为材料进行水培试验。常规生长试验在水稻幼苗3叶期取样,分析谷氨酰胺合成酶、谷氨酸合酶编码基因的表达及碳、氮含量;成熟期取样测定水稻产量、碳和氮含量及氮素利用效率。抗盐胁迫试验以3叶期幼苗为试材,设置营养液盐胁迫浓度分别为0、50、100 mmol/L NaCl,在胁迫开始后0、2、4天,取样分析水稻幼苗相对生长速率和脯氨酸含量,调查叶片萎蔫率和各部位生物量。【结果】PCR扩增和电泳检测证实黑曲霉AngdhA基因成功导入水稻TG3和TG13株系,qRT-PCR荧光定量分析发现,TG3和TG13株系中黑曲霉AngdhA基因表达量分别为内参UBI基因的248和41倍。但TG3和TG13株系中氮代谢关键酶基因OsGS1;1、OsGS1;2和OsGOGAT表达量与WT差异不显著,仅TG13的OsGS2表达量极显著低于WT。同时,导入黑曲霉AngdhA基因并未明显影响转基因株系TG3和TG13苗期叶片、根和成熟期籽粒、茎叶、根中氮含量和碳/氮值,但TG3和TG13植株体内碳和氮的积累量增加,其中籽粒中碳和氮的积累量均显著高于WT。另外,与WT相比,转基因株系TG3和TG13的氮素利用率分别提高了14.67%和44.12%,氮素籽粒生产效率分别提高了26.96%和39.83%,氮素偏生产力分别提高了24.53%和39.67%。转基因株系TG3和TG13的穗粒数、每株穗数和单株产量显著或极显著提高,其中单株产量较WT分别提高29.18%和43.95%。不同盐浓度胁迫下转基因株系TG3和TG13的相对生长速率极显著高于WT,尤其是在50 mmol/L NaCl条件下,TG3和TG13相对生长速率甚至高于对照条件(0 mmol/L)。盐胁迫下水稻脯氨酸含量明显提高,其中TG3和TG13脯氨酸含量在50 mmol/L NaCl条件下显著高于WT,但在100 mmol/L条件下极显著低于WT。【结论】水稻TG3和TG13株系中的外源AngdhA基因不扰乱转基因水稻体内的碳氮平衡,能促进水稻对氮素的吸收、利用和再活化,提高水稻氮素利用效率、产量和耐盐能力,但TG3中AngdhA基因的超量表达可能增加谷氨酸合成与分解途径的底物循环,使转基因水稻对氮素的吸收、利用和产量增幅下降。盐胁迫下脯氨酸的差异积累可能与水稻对胁迫程度的响应与耐受度存在差异有关。

【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 overexpression 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.