三疣梭子蟹Pt-ddit4l基因的克隆及表达分析

CLONING AND EXPRESSION ANALYSIS OF Pt-ditt4l IN SWIMMING CRAB (PORTUNUS TRITUBERCULATUS)

  • 摘要: 为明确三疣梭子蟹(Portunus trituberculatus)DNA损伤诱导蛋白因子-4(DNA Damage Inducible Transcript like 4, DDIT4L)基因的结构和功能。利用RACE方法克隆获得三疣梭子蟹Pt-ditt4l基因全长。该基因序列全长2727 bp, 其中ORF为507 bp, 编码了一个168个氨基酸的蛋白, 预测分子质量为19.14 kD, 且在C端有一个高度保守的RTP801_C结构域。Blast相似性比对和系统进化树分析发现, Pt-ddit4l与凡纳滨对虾(Litopenaeus vannamei)ddit4l基因的相似性最高为60%, 且与凡纳滨对虾先聚为一支。梭子蟹不同发育阶段表达分析表明, Pt-ddit4l基因在发育早期发挥了一定的作用; 组织表达分析显示, Pt-ddit4l基因在肝胰腺中的表达量最高, 在血细胞中表达量最低。注射副溶血弧菌(Vibrio parahaemolyticus)后的感染实验表明, 血细胞中Pt-ddit4l的表达量先上升后下降, 在24h达到峰值, 为对照组的8.85倍(P<0.05); 而肝胰腺中则是整体呈下调的趋势, 在72h时达到最低值, 仅为对照组的1/40(P<0.05)。注射dsddit4l 后该基因12—24h表达量显著下调, 且注射dsddit4l后再进行弧菌感染的螃蟹的死亡率要显著远高于对照组。研究丰富了三疣梭子蟹DNA损伤诱导蛋白相关研究资料。

     

    Abstract: The DNA Damage Inducible Transcript like 4 cDNA of swimming crab Portunus trituberculatus was cloned by RACE method to clarify the structure and function of Pt-ditt4l gene. The gene sequence of Pt-ddit4l was 2727 bp long, containing an ORF of 507 bp to encode 168 amino acids. The molecular mass of Pt-ddit4l was 19.14 kD, and has a highly conserved RTP801_C domain at the C-terminal. Blast similarly alignment and phylogentic tree analysis showed that Pt-ddit4l gene had a 60% similarity with Litopenaeus vannamei ddit4l gene, and first clustered with Litopenaeus vannamei. The expression of Pt-ddit4l at different developmental stages of swimming crab showed that it played a role in early development. The Pt-ddit4l was expressed in all tested tissues with the highest expression in hepatopancreas and the lowest in hemocytes. The Vibrio parahaemolyticus infection experiment showed that the expression of Pt-ddit4l in hemocytes increased gradually and then decreased, reaching a peak at 24h, which was 8.85-fold that of the control group (P<0.05). However, the trend was down-regulated with a minimum at 72h in hepatopancreas, which was only 1/40 of the control group (P<0.05). After RNA interference, the expression level of Pt-ddit4l was down-regulated within 6−24h, and the mortality rate of the infected swimming crab with dsddit4l injection was much higher than that of the control group. This study provides a reference for further understanding the physiological function of ddit4l gene in P. trituberculatus.

     

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