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

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

摘要:

〗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GSTABRv1、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.

Abstract:

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.