杜氏盐藻寡糖基转移酶亚基STT3a功能结构域的克隆与表达分析

Characterization of the functional domain of STT3a of oligosaccharyltransferase from Dunaliella salina

摘要:

为了研究STT3a基因在杜氏盐藻耐盐及鞭毛再生方面的作用，根据衣藻、拟南芥等STT3a蛋白的氨基酸高度保守序列VCVFTA、DVDYVL设计一对简并引物，采用RT-PCR及3'RACE的方法扩增杜氏盐藻STT3a蛋白功能结构域的cDNA序列。序列分析显示克隆的cDNA全长1 650 bp，具有一定保守性，与衣藻、拟南芥和人的相似性分别为48％、50％和46％。实时荧光定量PCR结果显示杜氏盐藻STT3a mRNA水平随着盐浓度的升高而逐渐增加，其水平在3.5 mol/L NaCl浓度时比在1.5 mol/L NaCl浓度时升高了11倍(P＜0.01)。另外，与没有脱鞭毛的杜氏盐藻相比，STT3a mRNA在鞭毛再生过程中持续高表达。本研究显示杜氏盐藻STT3a基因的高表达可以增强其盐适应和鞭毛再生能力。

Abstract:

To investigate the function of STT3a gene in salt adaptation and flagellar regeneration of Dunaliella salina (D. salina), a pair of degenerate primers was designed according to conserved homologous amino acid sequences of VCVFTA and DVDYVL of STT3a from Chlamydomonas, Arabidopsis thaliana and other organisms. A cDNA sequence of 1 650 bp encoding a whole functional domain of STT3a was amplified from D. salina by RT-PCR and 3￠ Rapid Amplification of cDNA Ends (RACE), which shared homology with Chlamydomonas (48%), Arabidopsis thaliana (50%), Homo sapiens (46%), etc. Real-time fluorescence quantitative PCR (real-time Q-PCR) demonstrated that the STT3a mRNAs from D. salina were induced by increased concentration of NaCl, and increased to 11-fold higher by 3.5 mol/L NaCl than that by 1.5 mol/L NaCl (P<0.01). Also, STT3a mRNA of D. salina maintained at a higher level in the process of flagellar regeneration with than without experiencing deflagellar treatment. In conclusion, the findings of this study demonstrate that the high expression of the STT3a gene enhances the capability of salt adaptation and flagellar regeneration in D. salina.