橡胶树HbJAZ3突变体蛋白的原核表达及纯化

Prokaryotic Expression and Purification of Mutant HbJAZ3 Protein of Hevea brasiliensis

植物JAZ蛋白是茉莉酸信号调控途径的关键环节之一。HbJAZ3基因是橡胶树乳管细胞中编码JAZ蛋白的基因家族成员之一。本文采用原核表达和定点突变技术,构建了pET28a(+)-JAZ3、pET28a(+)-JAZ3-ZIM-mut(缺失ZIM结构域中TIFY基序)和pET28a(+)-JAZ3-Jas-mut(缺失Jas结构域的保守氨基酸FLEKRK)的His标签融合蛋白表达载体,并成功转化大肠杆菌Rosetta(DE3)。在37℃条件下,用1 mmol/L IPTG诱导2 h能够诱导目的蛋白以包涵体的形式大量表达。通过镍柱纯化了目的蛋白,获得了HbJAZ3及其ZIM结构域和Jas结构域的突变体蛋白,为进步一鉴定乳管细胞茉莉酸信号途径的关键环节打下了良好基础。

Jasmonate-ZIM Domain （JAZ） proteins are one of the core components of jasmonate signaling pathway in plant.Recently,we cloned the gene HbJAZ3 from laticifer cells in rubber tree.In this study,the prokaryotic expression vector pET28a （＋）-JAZ3,pET28a （＋）-JAZ3-ZIM-mut and pET28a （＋）-JAZ3-Jas-mut were successfully constructed and transformed into E.coli Rosetta （DE3）.The expression of fusion protein could be induced by 1 mmol/L IPTG,in the form of inclusion body at 37 ℃ for 2 hours.Finally,we have got the JAZ3 and mutant fusion proteins by His tag purification.The mutant proteins were introduced deletions of TIFY motif for the ZIM domain and the conserved amino acid FLEKRK for the Jas domain.