异源表达芸薹生链格孢谷氨酸脱氢酶基因AbGDH提高水稻氮素利用率的研究

摘要

氮元素是植物生长发育所必需的重要元素之一.高等植物中的NADP(H)型谷氨酸脱氢酶(glutamate dehydrogenase,GDH)对NH4+亲和力较低,因此植物主要通过谷氨酰胺合成酶(GS)/谷氨酸合酶(GOGAT)途径吸收NH4+.真菌等低等生物中的GDH对NH4+亲和力较高,所以它们在对NH4+的利用途径中起着重要作用.本研究克隆了来自芸薹生链格孢(Alternaria brassicicola)的谷氨酸脱氢酶基因(AbGDH),并在水稻(Oryza sativa L.cv.Kitaake)中成功表达.体外酶活性分析表明,AbGDH对NH4+、α-酮戊二酸和谷氨酸的Km值分别为2.144±0.141 mmol/L、2.690±0.233 mmol/L和96.772±0.542 mmol/L.体内酶活性测定显示,与野生型相比,过表达AbGDH的水稻有更高的NH4+亲和力和氨同化能力.此外,水培实验表明,与对照植物相比,转基因幼苗在低氮条件下植物高度和干重显著增加.这些结果说明,在水稻中异源表达AbGDH能促进α-酮戊二酸转化为谷氨酸,并在低氮条件下促进水稻的氨同化,从而提高氮素利用效率.%Nitrogen is one of the most important elements in agriculture industry.NADP(H)-dependent glutamate dehydrogenase (GDH) has a lower affinity for ammonium in higher plants,whose nitrogen is thus mainly assimilated by the glutamine synthetase (GS)/glutamate synthase (GOGAT) pathway.But,in lower organ isms such as fungi,GDHs play an important role in nitrogen assimilation due to their higher affinity for ammonium.In this study,a glutamate dehydrogenase gene AbGDH from Alternaria brassicicola was cloned and successfully over-expressed in rice (Oryza sativa L.cv.Kitaake).Enzyme activity assay in vitro demonstrated that the Km values of AbGDH for NH4+,2-oxoglutarate and glutamate were 2.144±0.141 mmol/L,2.690± 0.233 mmol/L and 96.772±0.542 mmol/L,respectively.Meanwhile,the enzyme activity assay in vivo revealed that,compared with the nontransgenic plants,the AbGDH over-expressing plants showed a higher aminating activity and a lower Km value for NH4+.Additionally,the hydroponic experiments revealed that,compared with control plants,the transgenic seedlings exhibited a significant increase in plant height and dry weight at lower nitrogen conditions.These results suggested that ectopic expression of AbGDH was inclined to convert 2-oxoglutarate to glutamate,which subsequently improved the ammonium assimilation and enhanced the nitrogen use efficiency particularly under low nitrogen fertility.