凡纳滨对虾输入蛋白α4基因的克隆及表达分析

Cloning and Expression Analysis of the Importin α4 Gene from Litopenaeus vannamei

输入蛋白α(importinα)是核转运蛋白家族中的重要成员,在帮助有核定位信号的蛋白质的入核中起到了至关重要的作用,然而在对虾中,还没有输入蛋白α家族的成员被发现。本研究首次克隆并鉴定了一种凡纳滨对虾中的importinα基因,命名为Lv IMα4,其基因全长c DNA为2 147 bp,包含长度为1 572 bp的开放阅读框(open reading frame,ORF),编码524个氨基酸,包括一个N端的输入蛋白β结合(importin-β-binding,IBB)域和一个包含8个串联重复序列(tandem armadillo,ARM)的核定位信号(nuclear location signal,NLS)中央结合结构域。实时荧光定量PCR(RT-PCR)分析表明Lv IMα4在被测试的所有组织中都有表达,其中在表皮、胃、神经、肌肉和肠中的表达量较高。白斑综合征病毒(white spot syndrome virus,WSSV)感染后,凡纳滨对虾血液中Lv IMα4基因的表达量呈先下降后上升趋势,在感染后12 h降至最低,随后逐渐升高,在感染后72 h达到最高水平。副溶血弧菌(Vibrio parahaemolyticus)感染后凡纳滨对虾血细胞中Lv IMα4基因表达量都低于对照组,24 h达到最低水平,为对照组的0.34倍。所有实验结果表明,Lv IMα4基因可能参与凡纳滨对虾抗病免疫应答途径。

Importin a, an essential member of karyopherins family, plays important roles in the transport of proteins with nuclear location signal （NLS） into the nucleus through the nuclear pore complex. However, no impo- rtin a member has been reported yet in Litopenaeus vannamei. In this study, an importin gene in L. vannamei cloned and identified for the first timeand named as Lvl Ma4. The full-length cDNA of LvlMa4 was 2 147 bp with an open reading flame of 1 572 bp, which encoded a putative protein of 524 amino acids including a importin- β-binding （IBB） domain at the N-terminal and a highly structured domain comprised of eight tandem armadillo （ARM） repeats. Real-time PCR （RT-PCR） analysis showed that LvlMa4 was expressed in all tested issues and was abundant in epithelium, stomach, nerve, muscle and intestine. Upon WSSV challenge, the expression levels of Lvl Ma4 in hemocyte reduced to the lowest level at 12 h and then increased, reaching the peak at 72 h. By Vibrio parahaemolyticus challenging, the level of Lvl Ma4 expression were lower than the control in all the detected time points, with the lowest level at 24 h which is 0.34 times of the control. All the experimental results showed that Lvl Ma4 gene may be involved in antiviral immune response ofLitopenaeus vannamei.