过表达E3泛素连接酶ABRv1通过泛素化CPK3提高拟南芥干旱耐受

拟南芥基因ABRv1编码了一个E3泛素连接酶,其表达量受到干旱胁迫的诱导.为了探究ABRv1功能,我们构建了ABRv1的过表达株系并进行干旱处理,结果显示ABRv1过表达株系OE-3具有90.4%的存活率,ABRv1过表达株系OE-7有88.2%的存活率,野生型Col的存活率则为53.8%,而abrv1突变体仅有7.7%的存活率,说明过表达ABRv1提高了拟南芥对干旱的耐受能力.我们还测定了干旱处理下的失水率,也能得出与上述一致的结论.此外,我们使用纯化的GSTABRv1、His-CPK3蛋白进行了体外pull-down实验和体外泛素化实验,结果证明ABRv1能够和CPK3发生相互作用,ABRv1具有E3泛素连接酶活性并且能够把CPK3单泛素化.结果揭示了ABRv1作为一个正调控因子通过泛素化作用底物CPK3参与了拟南芥的干旱胁迫应答过程.

Over-expressing E3 ligase ABRv1 enhances drought tolerance via ubiquitinating CPK3

Arabidopsis thaliana gene ABRv1 encodes an E3 ligase, its expression is induced by drought stress. To investigate the function of ABRv1, ABRv1 over expressing lines were constructed. Under drought stress, the over expressing lines OE 3 and OE 7 have 90.4% and 88.2% survival rate respectively, and ABRv1 wild type shows a survival rate of 53.8%, but the survival rate of abrv1 mutant is only 7.7%. That is to say, over expressing ABRv1 enhances the ability of drought tolerance in Arabidopsis We also detected the water loss rate of abrv1 mutant, ABRv1 wild type and ABRv1 over expressing lines, which came to an consistent conclusion with mentioned above. Furthermore, we used purified GST ABRv1 and His CPK3 protein to conduct an in vitro pull down assay and an ubiquitination assay. The pull down assay shows a physical interaction between ABRv1 and CPK3, and the ubiquitination assay shows that CPK3 protein as a target is ubiquitinated by ABRv1. In conclusion, our results demonstrate that ABRv1 playing as a positive regulator is involved in the response of drought stress via interacting with and ubiquitinating its substrate CPK3.