中华根瘤菌NP1氨单加氧酶基因的克隆与功能鉴定

以中华根瘤菌NP1(Sinorhizobium sp.NP1)为原始菌株,通过同源克隆与Tail-PCR方法,获得1089bp的氨单加氧酶基因(amo)全长序列.该基因编码362个氨基酸,其二级结构与Sinorhizobium meliloti1021AMO的二级结构相似,该蛋白有9个跨膜区段.以自杀穿梭质粒pJQ200SK为原始载体,构建NP1amo基因敲除质粒pJQ200SK-amo-Tc.采用三亲本杂交的方法将该质粒转入原始菌株NP1中,获得amo基因敲除菌株NP1∷amo.通过本贝洛氏(Berthelot)法对氨氮进行测定,发现NP1∷amo的脱氮效率比原始菌株NP1下降约35%.该结果表明,本实验中所克隆的氨单加氧酶基因为脱氮关键酶基因.

Cloning and Identification of Ammonia Monooxygenase Gene of Sinorhizobium sp. NP1

The ammonia monooxygenase (AMO) gene of 1 089 bp in Sinorhizobium sp. NP1, a strain established by our laboratory, was cloned by homology method and Tail-PCR. The gene encodes 362 amino acids and the predicted secondary structure with nine transmembrane regions is similar to that of Sinorhizobium meliloti. We have constructed a NP1 amo knockout plasmid named pJQ200SK-amo-Tc and transformed into the original NP1 strain using three parent hybridization and obtained a amo knockdown strain. The enzymatic activities in NP1∶∶ amo decreases about 35% compared to original NP1 strains by Berthelot test. The results showed that amo encoded a key enzyme for denitrification.