凡纳滨对虾CTNNBL1基因克隆及表达分析

Cloning and expression of CTNNBL1 gene from Litopenaeus vannamei

【目的】明确CTNNBL1基因在凡纳滨对虾抗病过程中的功能作用,为深入探讨对虾抗病免疫机制提供参考依据。【方法】采用RACE-PCR克隆凡纳滨对虾CTNNBL1基因(Lv CTNNBL1),利用在线软件进行生物信息学分析,并进一步分析其组织表达特征及应答白斑综合征病毒(WSSV)和副溶血弧菌感染的表达模式。【结果】Lv CTNNBL1基因c DNA全长1906 bp,其中开放阅读框(ORF)长1692 bp,编码563个氨基酸,含有1个CTNNBL结构域。同源性分析发现Lv CTNNBL1蛋白与大型溞(Daphnia magna)同源蛋白的相似度最高,为77%。基于CTNNBL1基因序列的系统发育进化分析结果显示,凡纳滨对虾被聚为无脊椎动物一类,与同为节肢动物的大型溞、西方蜜蜂(Apis mellifera)和美洲鲎(Limulus polyphemus)的亲缘关系较近。Lv CTNNBL1基因在凡纳滨对虾中呈组成型表达,以在肠道中的表达量最高、表皮中的表达量最低。注射感染WSSV后,Lv CTNNBL1基因在凡纳滨对虾血细胞中的表达量呈先下降后上升趋势,在所有检测时间点与对照组间存在显著(P<0.05)或极显著(P<0.01,下同)差异;注射感染副溶血弧菌后,Lv CTNNBL1基因在凡纳滨对虾血细胞中的表达量呈先上升后下降趋势,感染后12 h内较对照组极显著升高。【结论】Lv CTNNBL1基因表达量在病毒和细菌感染时发生明显变化,可能参与了凡纳滨对虾的抗病免疫途径。

[Objective]The function related to disease resistance of β-catenin like protein 1 （CTNNBL1） gene in Litopenaeus vannamei were investigated, in order to provide reference for further studying immune mechanism of disease resistance of shrimp. [Method]CTNNBL1 gene was cloned from L. vannamei by RACE-PCR. Online softwares were used for bioinformatics analysis on sequence of LvCTNNBL1. Then tissue expression characteristics and expression pattern response to infection of white spot syndrome virus（WSSV） and Vibrio parahaemolyticus of LvCTNNBL1 gene were further analyzed. [Result]Full-length eDNA sequence of CTNNBL1 gene from L. vannamei （named LvCTNNBL1） was 1906 bp, containing open reading frame （ORF） of 1692 bp in length, coding 563 amino acids, with a CTNNBL domain. Multiple alignment analysis showed that LvCTNNBL1 shared the highest amino acid identity（77%） with Daphnia magna. The phylogenetic analysis showed that, L. vannamei was clustered into invertebrate group and more closely related to CTNNBL1 of Daphnia magna, Apis mellifera and Limulus polyphemus which were all arthropods. LvCTNNBL1 was constitutively expressed in all the examined tissues, with the highest expression in intestine and lowest expression in epithelium. After WSSV injection, the expression of LvCTNNBL1 first decreased and then increased. In all the detected time points, the expression of LvCTNNBL1 in hemocyte were significantly（P〈0.05） or extremely significant（P〈0.01, the same below） different from the control. After V. parahaemolyticus injection, the level of LvCTNNBL1 gene expression in hemocyte first increased and then decreased, and which was singnificantly increased compared with control within 24 h. [Conclusion]The expression of LvCTNNBL1 gene is significantly changed after virus and bacterial infection, suggesting that LvCTNNBL1 gene is involved in the innate immune response of L vannamei triggered by pathogens.