半滑舌鳎(Cynoglossus semilaevis)雌性特异基因CSW3重组蛋白的表达与应用

The Expression and Application of the Recombinant Protein of the Female Specific Gene CSW3 in Half-Smooth Tongue Sole (Cynoglossus semilaevis)

中文关键词: 半滑舌鳎  重组蛋白  CSW3  蛋白转染  性别相关基因

英文关键词: Half-smooth tongue sole (Cynoglossus semilaevis)  Recombinant protein  CSW3  Protein transfection  Sex-related gene

中文摘要:

      CSW3基因序列来源于半滑舌鳎(Cynoglossus semilaevis)全基因组测序项目，是经过筛选得到的雌性特异候选基因之一。本研究探索了半滑舌鳎雌性相关基因CSW3的表达与应用，构建了半滑舌鳎雌性特异CSW3基因重组表达载体，通过大肠杆菌进行了体外重组表达，对基因表达产物进行分离纯化，并通过蛋白转染的方法研究了纯化的蛋白对几个性别相关基因的影响。结果显示，雌性特异CSW3重组蛋白注射鱼体后对半滑舌鳎性腺foxl2、sox9a、amh这3种性别相关基因的表达水平具有显著影响，能够引起雌性相关基因foxl2表达上调、雄性相关基因sox9a和amh表达下降。从蛋白水平对雌性相关基因CSW3的基因功能进行初步研究，为半滑舌鳎性别控制及全雌苗育种提供基因资源及技术方法。

英文摘要:

      The half-smooth tongue sole (Cynoglossus semilaevis) is a mariculture fish species in China that has high economic values. The growth of this fish is sexually dimorphic, and females grow 23 times faster than males. The tongue sole exhibits the phenomenon of sex reversal during the growth and has a ZZ-ZW sex-determining system, thus it is a valuable animal model in the study of the mechanisms of fish sexual growth dimorphism. Several sex-specific genes have been cloned from half-smooth tongue sole, but the functions of these genes remain unknown. This research aimed to preliminarily study the function of the female-related gene CSW3. CSW3 was one of the female specific genes in half-smooth tongue sole which was identified from the screening in the whole genome sequencing project of half-smooth tongue sole. The full length of CSW3 cDNA was 1677 bp and contained a 414 bp open reading frame which coded 137 amino acids. The sequence also included a 173 bp 5 noncoding region and a 1090 bp 3 noncoding region that possessed a polyadenylation signal and a poly A tail. We constructed a recombinant expression vector for CSW3 and the plasmid was transformed into E. coli. IPTG was used to induce the expression of CSW3 protein. The proteins were isolated and purified with His-tag purification of affinity chromatography. The western-blot analysis showed that the recombinant protein was 33 kDa, which was consistent with the bioinformatics prediction. We then investigated the function of CSW3 protein using protein transfection. The liposomes encapsulating CSW3 protein were injected into the gonad of male half-smooth tongue sole. The experimental group was injected with bioactive CSW3 protein, and the control group was injected with heat-inactivated CSW3 protein. Real time quantitative PCR analysis showed that there were changes in the expression of three sex-related genes including one female-related gene and two male-related genes. The results suggested that the female specific CSW3 protein may cause the up-regulation of the female-related gene foxl2, and the down-regulation of the male-related genes sox9a and amh.