大豆半胱氨酸蛋白酶抑制剂基因GmCYS2的功能鉴定

半胱氨酸蛋白酶抑制剂(cystatin,CYS)在结瘤,根瘤发育和衰老过程中起重要作用。本研究克隆了1个大豆CYS家族基因GmCYS2,氨基酸序列比对及进化树分析表明,GmCYS2与木豆(Cajanus cajan)CYS相似性最高。在体外对该基因编码蛋白进行了表达和纯化,重组蛋白GmCYS2的酶活性抑制实验显示,该蛋白对L类和B类组织蛋白酶的抑制活性明显高于对H类组织蛋白酶,并且在30 d根瘤提取物中的抑制活性高于在60 d根瘤提取物中。此外,构建GmCYS2的植物表达载体,通过百脉根毛根转化法获得转GmCYS2基因的超表达复合体植株。GmCYS2的超量表达增加了百脉根的结瘤数目,并且上调共生标记基因的表达。结果说明GmCYS2蛋白具有一定的酶活性抑制作用,并且正调控百脉根共生结瘤过程。

Functional Characterization of Soybean Cystatins Gene GmCYS2

CYS (cystatin) plays an important role in nodulation, nodule development and senescence. In this study, we cloned a soybean CYS family gene GmCYS2, which had the highest similarity with pigeonpea (Cajanus cajan) CYS, shown by amino acid sequence alignment and phylogenetic tree analysis. The gene encoding protein was expressed and purified in vitro. Enzyme activity inhibition experiment of recombinant protein GmCYS2 showed that the cathepsin inhibitory activity of GmCYS2 protein on L and B classes was higher than that on H class, and cathepsin inhibitory activity in nodule extract from nodule at 30 d was generally higher than that at the 60 d. In addition, the plant overexpression vector was constructed and the GmCYS2-overexpression composite plants were obtained by Lotus japonicus hairy root transformation method. Overexpression of GmCYS2 increased the number of nodules in Lotus japonicus plants and up regulated the expression levels of symbiotic marker genes. These results suggest that GmCYS2 protein can inhibit the enzyme activity and positively regulate the symbiotic nodulation of Lotus japonicus.