百脉根半胱氨酸蛋白酶基因在根瘤衰老过程中的表达分析
Analysis of cysteine protease expression during nodule senescence in Lotus japonicus

为明确半胱氨酸蛋白酶与定型根瘤衰老的相关性，通过与已鉴定的紫云英半胱氨酸蛋白酶AsNODf32进行序列比对分析，从百脉根中鉴定出2个与根瘤衰老相关的蛋白酶基因LjCyp1和LjCyp5。蛋白序列及结构分析表明，LjCyp1和LjCyp5同源性高达96.2%，均含有2个典型的半胱氨酸蛋白酶结构域和液泡定位信号肽，属于木瓜蛋白酶家族。体外酶活检测显示，2个蛋白都具有半胱氨酸蛋白酶活性。通过Real time PCR和组织化学定位分析表明，2个蛋白酶基因主要在成熟和衰老的根瘤中表达。结果表明LjCyp1和LjCyp5可能在百脉根根瘤衰老过程中发挥重要作用。
In order to explore the correlation between cysteine proteases and the senescence of determinate nodules,LjCyp1 and LjCyp5,associated with root nodule senescence were identified from Lotus japonicus by sequence alignment analysis with cysteine protease AsNODf32.The protein sequence and structure showed that the homology of LjCyp1 and LjCyp5 was as high as 96.2%,both of which contained two typical cysteine protease domains and vacuolar localization signal peptides,belonging to the papain family. In vitro enzyme activity assays showed that both proteins had cysteine protease activity. Real time PCR and histochemical localization analysis showed that two protease genes were mainly expressed in mature and aged root nodules. The results indicated that LjCyp1 and LjCyp5 cysteine proteases may play an important role during nodule senescence in Lotus japonicus