拟南芥RPS14基因的克隆表达及功能初步研究

Cloning,Expression and Function Preliminary Research of RPS14 in Arabidopsis thaliana

拟南芥RPS14(Ribosomal protein S14)是从拟南芥中分离出的一种核糖体蛋白,是核糖体40S亚基的组成成分,属于S11P核糖体蛋白家族,主要负责RNA转录后加工。提取拟南芥总RNA,用RT-PCR技术得到RPS14全长基因,经T-A克隆插入到pEASY-T3载体上。利用亚克隆法成功构建pGEX-6P-1-RPS14原核表达质粒。GST-RPS14融合蛋白在大肠杆菌BL21(DE3)中表达,分子量约为43 kD。运用同样方法构建pET-28a-AK6原核表达质粒,获得大小约为26 kD的HIS-AK6融合蛋白。运用体外pull-down技术,并用SDS-PAGE和Western blot进行验证,证明拟南芥RPS14蛋白与AK6蛋白之间存在相互作用。因此,推测拟南芥的AK6与RPS14蛋白共同参与核酸代谢过程,影响RNA转录后加工,进而抑制拟南芥茎细胞的生长,使植株表现矮小特征。

RPS14 （ Ribosomal protein S14 ） is a ribosomal protein ofArabidopsis thaliana. It is a structural component of 40S ribosomal subunit and belongs to the S11P ribosomal protein family. It is responsible for RNA post-transcriptional processing. We extracted the total RNA ofArabidopsis thaliana. The RPS14 full-length gene was obtained by RT-PCR. Through inserting the gene fragment into pEASY-T3 vector by T-A cloning, we successfully constructed a recombinant vector pGEX-6P-1-RPS14. The GST-RPS14 protein was expressed in BL21 （ DE3 ） strain and molecular weight was approximately 43 kD. We constructed the recombinant vector pET-28a-AK6 in the same way and obtained the approximately 26 kD HIS-AK6 protein. We demonstrated the interaction between RPS14 and AK6 using pull-down, SDS-PAGE and Western blot. Therefore, we speculate that they together participate in the nucleic acid metabolism and play a role in the RNA post-transcriptional processing, thereby inhibiting growth of stem cells and making plants show dwarf phenotype.