叶锈菌侵染诱导产生的小麦Wrab17蛋白的原核表达、纯化及其抗体制备

Prokaryotic expression,protein purification and antiserum preparation of wheat Wrab17 induced by leaf rust

叶锈菌(Puccinia triticina)引起的小麦叶锈病是影响世界小麦生产的重要病害之一。研究小麦抗叶锈相关防卫蛋白的表达,对于深入研究小麦对叶锈菌的抗病机制,更好地为农业生产服务,具有重要意义。Wrab17(Wheat response to ABA)是一个冷胁迫诱导蛋白,前期工作通过构建叶锈菌侵染早期的小麦SSH文库及ESTs序列分析,筛选到Wrab17基因在不亲和组合接种后早期显著上调表达,初步表明Wrab17基因可能参与了小麦抗叶锈菌侵染过程,这是首次发现Wrab17参与抗病反应。本研究进一步利用RT-PCR技术从接种叶锈菌的小麦叶片中克隆获得了Wrab17基因的CDS区,构建了Wrab17蛋白原核表达载体,并在大肠杆菌中高效表达Wrab17蛋白。利用镍柱亲和层析方法获得了纯度较高的重组蛋白,将融合蛋白纯化后制备其兔抗血清,得到特异性较好的多克隆抗体。为进一步通过Western Blotting技术检测Wrab17在小麦被叶锈菌侵染后不同时间点表达量的变化,明确Wrab17参与小麦抵抗叶锈菌侵染的防卫反应奠定基础。

Wheat leaf rust is one of the important diseases caused by Puccinia triticina affect- ing the world＇s wheat production. It is important to study the expression of defense related proteins and the resistant mechanism to wheat leaf rust, which will better serve for agricultur- al production. Wrabl7（Wheat response to ABA）protein was reported to be related to cold stress. A SSH library of the wheat infected by leaf rust was established and EST sequences were analyzed, from which the Wrab17 EST had a high level of expression in the wheat leaves that inoculated by incompatible leaf rust. This result implied that Wrab17 might be involved in the infection process of leaf rust. This is the first discovery that Wrabl7 might be involved in disease resistance. In this study, the CDS of Wrab17 was cloned from wheat leaves infected by leaf rust. Prokaryotic expression vectors for Wrabl7 were constructed and transformed in- to E. coli. The results showed that Wrab17 could express efficiently. Recombinant proteins were further purified by the Ni-column affinity chromatography. The purified fusion protein was prepared for the rabbit antiserum and got specific polyclonal antibody. The study will contribute to the wheat Wrabl7 expression at different time points after infection by leaf rust through Western Blotting. It is also helpful to the clearing of the association between Wrab17 gene and leaf rust resistance.