红螯螯虾冷休克蛋白Y-box编码基因的克隆及表达分析

Molecular cloning and expression analysis of the cold shock protein Y-box coding gene of red claw crayfish,Cherax quadricarinatus

采用RT-PCR和RACE技术,从红螯螯虾(Cherax quadricarinatus)血细胞中分离到冷休克蛋白Y-box编码基因的cDNA序列。结果显示,冷休克蛋白Y-box编码基因的长度为1 733 bp,其中包括82 bp的5'非翻译区、676 bp的3'非翻译区和975 bp的编码序列,可编码324个氨基酸。理论相对分子质量和等电点分别为35 800和9.81。结构域分析和序列比对显示,红螯螯虾冷休克Y-box蛋白由3部分组成:富含丙氨酸或脯氨酸区域、冷休克结构域和带电荷区域,其中冷休克结构域高度保守。系统进化树分析表明,红螯螯虾冷休克Y-box蛋白与水蚤等节肢动物冷休克Y-box蛋白聚类,且三级结构非常相似。最适温度养殖条件下,冷休克蛋白Y-box的虾组织分布特异性研究表明,红螯螯虾神经组织的冷休克蛋白Y-box转录活性最强,而后依次为肝脏、血淋巴和鳃,在肠和心脏中也有活性,在肌肉组织中转录保持较低水平,揭示神经组织是重要的冷休克Y-box蛋白表达区。

A cold shock protein Y - box gene was cloned from hemocytes of Cherax quadricarinatus by reverse transcription polymerase chain reaction （ RT - PCR） and rapid amplification of cDNA ends （RACE）. The cold shock Y - box protein gene cDNA consisted of 1733 bp with 82 bp UTR, 676 bp UTR and an open reading frame （ORF） of 975 bp that encoded 324 amino acids （aa）. The predicted relative molecular mass of the protein was 35 800 with an estimated PI of 9. 81. The protein may be divided into three distinct domains： the alanine/prolinerich amino -terminal part, the centrally located cold shock domain （ CSD）, and a carboxy - terminal region consisting of alternating regions with predominant basic or acidic amino acid residues by functional domain analysis. The CSD was highly conserved for all eukaryotes. The cold shock Y - box protein of C. quadricarinatus showed similarity with those of Daphnia pulex and other arthropod. The predicted spatial structures of cold shock Y - box protein between C. quadricarinatus and D. pulex were similar. The tissue specific expression of cold shock Y - box protein gene of C. quadricarinatus was explored using Realtime - RT - PCR. The mRNA of cold shock Y - box protein gene was expressed in all tissues examined. The results demonstrated that the highest expression level of cold shock Y - box protein was detected in nerve, followed by hepatopancreas, hemocytes and gill, and lower levels were seen in other tissues （intestine, heart and muscle）.