拟南芥表皮模式因子EPFs的表达纯化与功能鉴定

Prokaryotic expression, purification and functional identification of epidermal pattern factors in Arabidopsis thaliana

摘要:

气孔密度是影响农作物产量的重要形态学指标。文中以拟南芥气孔发育相关的表皮模式因子 (EPFs) 为研究对象，构建原核表达载体并进行蛋白表达和纯化，并与新型气体信号分子硫化氢 (H2S) 建立联系。首先克隆基因AtEPF1、AtEPF2和AtEPFL9，构建pET28a表达载体；然后对重组质粒pET28a-AtEPF1、pET28a-AtEPF2和pET28a-AtEPFL9进行酶切检测和测序，结果显示重组载体构建成功；分别转入大肠杆菌BL21(DE3) 进行异丙基β-D-硫代半乳糖苷 (IPTG) 诱导表达。优化后的表达条件：IPTG诱导浓度分别为0.5、0.3、0.05 mmol/L；最适诱导温度分别为28 ℃、28 ℃和16 ℃；最适诱导时间分别为16 h、16 h和20 h；经Ni琼脂糖凝胶柱纯化获得融合蛋白，大小分别为18 kDa、19 kDa和14.5 kDa左右。将纯化到的AtEPF2和AtEPFL9蛋白分别处理拟南芥幼苗，与对照相比，其H2S产率均有不同程度的变化，且差异显著。即表皮模式因子AtEPFs影响植物内源H2S信号的产生。为后续深入研究H2S和EPFs对植物气孔发育影响的作用机制奠定基础，对增加作物产量、增强植物抗逆性有重要意义。

Abstract:

Stomatal density is important for crop yield. In this paper, we studied the epidermal pattern factors (EPFs) related to stomatal development. Prokaryotic expression vectors were constructed to obtain EPFs. Then the relationship between EPFs and hydrogen sulfide (H2S) was established. First, AtEPF1, AtEPF2 and AtEPFL9 were cloned and constructed to pET28a vectors. Then recombinant plasmids pET28a-AtEPF1, pET28a-AtEPF2 and pET28a-AtEPFL9 were digested and sequenced, showing successful construction. Finally, they were transformed into E. coli BL21(DE3) separately and induced to express by isopropyl β-D-galactoside (IPTG). The optimized expression conditions including IPTG concentration (0.5, 0.3 and 0.05 mmol/L), temperature (28 °C, 28 °C and 16 °C) and induction time (16 h, 16 h and 20 h) were obtained. The bands of purified proteins were about 18 kDa, 19 kDa and 14.5 kDa, respectively. In order to identify their function, the purified AtEPF2 and AtEPFL9 were presented to Arabidopsis thaliana seedlings. Interestingly, the H2S production rate decreased or increased compared with the control, showing significant differences. That is, EPFs affected the production of endogenous H2S in plants. These results provide a foundation for further study of the relationship between H2S and EPFs on stomatal development, but also a possible way to increase the yield or enhance the stress resistance.