小麦无机氮转运蛋白原核表达与纯化特点

Prokaryotic expression and purification of inorganic nitrogen transporters in wheat

摘要:

硝酸转运蛋白(nitrate transporter,NRT)与铵转运蛋白(ammonium transporter,AMT)是小麦无机氮素吸收、转运、分配的一类重要跨膜蛋白，获得NRT/AMT蛋白并制备其抗体有助于了解其组织定位特点，理解小麦的氮素利用过程。本研究从前期鉴定到的405个TaNRT/TaNPF基因和23个TaAMT基因中，筛选并克隆到了4个表达量较高的基因：TaNPF4.5、TaNPF8.3、TaNRT3.1、TaAMT1.2。利用HMMER软件对4个转运蛋白进行跨膜结构域预测，确定拟表达区段，并进行原核表达和纯化。在37℃、1 mmol/L IPTG诱导条件下，非跨膜区段TaNPF4.5、TaNPF8.3、TaNRT3.1诱导4 h后表达量最大且为包涵体形式，TaAMT1.2诱导3 h后表达量最大，主要是可溶性表达。非跨膜区段TaNPF4.5、TaNPF8.3、TaNRT3.1采用pH梯度分离纯化，TaNPF4.5在pH 2.0时蛋白纯度约为87%，TaNPF8.3在pH 3.0时纯化蛋白纯度约为85%，均可用于抗体制备，TaNRT3.1的蛋白纯度未达到85%；TaAMT1.2采用咪唑梯度分离纯化，在咪唑浓度为20 mmol/L时纯度约95%，并成功制备了抗体。TaAMT1.2的表达纯化与抗体制备，为TaNPF4.5、TaNPF8.3等膜蛋白表达纯化及抗体制备提供了思路，为研究小麦膜蛋白表达与定位特点奠定了基础。

Abstract:

The nitrate transporter (NRT) and ammonium transporter (AMT) are crucial transmembrane proteins involved in the absorption, transport, and distribution of inorganic nitrogen in wheat. Obtaining NRT/AMT and preparing corresponding antibodies are conducive to probing into their tissue localization and comprehending the nitrogen utilization process in wheat. In this study, four genes (TaNPF4.5, TaNPF8.3, TaNRT3.1, TaAMT1.2) with high expression levels were chosen and cloned from 405 genes of the TaNRT/TaNPF family and 23 genes of the TaAMT family identified previously. The transmembrane domains of the four transporters were predicted by HMMER to determine the putative expression segments, followed by prokaryotic expression and purification. Under the induction with 1 mmol/L IPTG at 37 ℃, the non-transmembrane segments of TaNPF4.5, TaNPF8.3, and TaNRT3.1 reached the highest expression levels (as inclusion bodies) after 4 h, while TaAMT1.2 was expressed at the highest level (as a soluble protein) after 3 h. TaNPF4.5, TaNPF8.3, and TaNRT3.1 were purified by a pH gradient. The purity of TaNPF4.5 and TaNPF8.3 reached about 87% and 85% at pH 2.0 and pH 3.0, respectively, both of which were suitable for antibody preparation. However, the purity of TaNRT3.1 did not reach 85%. TaAMT1.2 was purified by an imidazole gradient, reaching the purity of about 95% at 20 mmol/L imidazole, and the antibody was prepared successfully. The expression, purification, and antibody preparation of TaAMT1.2 not only provides insights into the expression, purification, and antibody preparation of membrane proteins including TaNPF4.5 and TaNPF8.3 but also lays a foundation for studying the expression and localization of membrane proteins in wheat.