拟南芥中Fibrillarin基因的克隆·表达·纯化及与ak6蛋白相互作用研究

Cloning,Expression,Purification and Interaction with ak6 Research of Fibrillarin in Arabidops thaliana

[目的]研究拟南芥中Fibdllarin基因的克隆、表达及纯化，并探索其与ak6蛋白的相互作用。[方法]利用pGEX-6P-1与Fibrilla—r流基因构建重组表达质粒，将重组质粒转化到大肠杆菌BL21（DE3）中进行诱导表达，运用同样方法构建PET-28a—ak6原核表达质粒，获得His—ak6融合蛋白，并运用体外pulldown技术验证二者的相互作用。f结果l在温度为37℃、诱导时间为16～20h、IPTG浓度为0．5mmol／L时，fibrillarin蛋白的纯化浓度最高。试验成功获得了符合标准的RNA，并获得了与预期片段大小相符的清晰的目的条带；pull—down试验结果表明，拟南芥中fibrillarin蛋白与ak6蛋白具有相互作用。[结论]拟南芥ak6突变体植株可能由于ak6的缺失影响了rRNA前体的加工，从而抑制核糖体的合成，进而阻碍拟南芥茎的生长，使植株表现明显的矮小症状。

[ Objective] To study cloning, expression and purification of Fibrillarin in Arabidopsis thaliana, and explore interaction with ak6. [ Method] By using pGEX-6P-1 and Fibrillarin to construct recombinant expression plasmid, and then transferred into E. coli B[21 （DE3） to conduct induction expression. The same method was used to construct prokaryotic expression plasmid to obtain His - ak6, in vitro pull down was used to verify the interaction. [ Result ] The purification concentration of Fibrillarin is the highest under the condition of 37 ~C, induction time 16 - 20 h, IPTG concentration 0.5 mmol/L. The experiment successfully obtained RNA in accordance with the standard. The pull - down test re- sult showed that Fibrillarin and ak6 has interaction effect in Arabidopsis thaliana. [ Conclusion] It was concluded that due to the lack of ak6 in Arabidopsis thaliana mutant plant, the processing of rRNA precursors was affected, thereby inhibited the synthesis of ribosomes, hindered the growth of Arabidopsis thaliana, thus the plants showed an obvious short symptoms.