杜氏盐藻糖原合成酶激酶3cDNA片段的克隆及其在鞭毛再生中的功能

Cloning and function analysis of glycogen synthase kinase 3 cDNA fragment of Dunaliella salina during flagellar regeneration

目的:克隆杜氏盐藻糖原合成酶激酶3(GSK3)基因序列并探讨它在鞭毛再生中的作用。方法:根据杜氏盐藻转录组测序得到的GSK3的2个片段设计上下游引物,提取盐藻总RNA进行RT-PCR,以此为模板扩增2个片段之间的序列。采用3'RACE方法扩增杜氏盐藻GSK3基因3'端序列,通过杜氏盐藻消减杂交模板扩增得到5'端序列。采用pH休克法去除杜氏盐藻鞭毛,通过实时荧光定量PCR观察鞭毛再生过程中GSK3基因在转录水平上的变化。结果:获得一段长为2116bp的GSK3 cDNA片段,其中包含737bp的3'UTR和1158bp的开放阅读框。实时荧光定量PCR结果表明,GSK3的mRNA转录水平在鞭毛再生过程中明显升高。结论:杜氏盐藻GSK3基因与鞭毛再生密切相关。

Aim： To investigate the function of glycogen synthase kinase 3（ GSK3） during flagellar regeneration in Du-naliella salina. Methods： According to the two GSK3 gene fragments of transcriptome sequencing results,a pair of primers was designed for amplifying the sequence between the two fragments. Nested PCR was performed with 3＇RACE and subtrac-tive hybridization template to amplify 3＇ and 5＇ fragments of the GSK3 gene. The flagella of Dunaliella salina were got rid of by pH shock method and the transcription level of the GSK3 gene during flagellar regeneration was investigated with real-time fluorescence quantitative PCR. Results： The results showed that a 2 116 bp GSK3 gene fragment containing 737 bp3＇UTR and 1 158 bp ORF was cloned. And GSK3 mRNA transcription level was obviously increased dur-ing flagellar regeneration compared with control group. Conclusion： The GSK3 gene of Dunaliella salina is relat-ed to regeneration of the flagella.