凡纳滨对虾输入蛋白α1基因的克隆与表达分析

Cloning and Expression of the Importin α1 Gene from Litopenaeus vannamei

输入蛋白α(importinα)是核转运蛋白家族的重要成员,可通过帮助具有核定位信号(Nuclear location signal,NLS)蛋白入核而参与免疫过程。克隆并鉴定凡纳滨对虾(Litopenaeus vannamei)的importinα1基因,命名为Lv IMα1,基因全长c DNA为2 181 bp,包括1 575 bp的开放阅读框,编码525个氨基酸,5′非编码区长110 bp,3′非编码区长496 bp。Smart分析显示,Lv IMα1蛋白包含一个N端的输入蛋白β结合域和一个包含8个串联重复序列的NLS中央结合结构域。同源性分析发现,Lv IMα1与原鸡(Gallus gallus)IMα1的相似度最高,为73%;与水蚤(Daphnia magna)IMα1相似度最低,为61%。系统进化分析显示,Lv IMα1和多种无脊椎动物IMα1聚为一类。实时荧光定量PCR(QRT-PCR)分析表明,Lv IMα1在所测试组织中均有表达,在神经组织中表达量最高。白斑综合征病毒感染后,凡纳滨对虾血液中Lv IMα1基因的表达量呈先下降后上升趋势,除感染后4 h外,其余时间点的表达量均低于对照组(P<0.05),12 h最低,为对照组的0.22倍,随后逐渐升高。副溶血弧菌(V.parahaemolyticus)感染后,凡纳滨对虾血细胞中Lv IMα1基因表达量低于对照组,在感染后4、24、48、72 h尤为显著,24 h时最低,为对照组的0.38倍。Lv IMα1基因可能参与凡纳滨对虾抗病免疫应答途径。

Importin α, an essential member of karyopherins family, function as a receptor protein which plays important role in the transport of proteins with nuclear location signal（NLS） into the nucleus through the nuclear pore complex. An importin gene in L. vannamei designated as Lv IMα1 is cloned and identified. The full-length c DNA of Lv IMα1 is 2181 bp with 5＇ UTR of 110 bp, 3＇ UTR of 496 bp and an open reading frame of 1 575 bp, which encod a putative protein of 525 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. Multiple alignment analysis shows that Lv IMα1 shares highest amino acid identity with Gallus gallus（73%） while the lowest with Daphnia magna（61%）. Phylogenetic analysis shows that Lv IMα1 is clustered into invertebrates group. QRT-PCR analysis shows that Lv IMα1 is constitutively expressed in all the examined tissues with the highest expression in nerve. Upon WSSV challenge, the expression of Lv IMα1 is significantly lower than the control at all the detected time points except 4 hour past infection（hpi）. At 12 hpi, the level of Lv IMα1 expression reduced into the lowest, which is 0.22-fold of the control. By V. parahaemolyticus challenging, the expression of Lv IMα1 is lower than the control at all the detected time points, especially at 4 hpi, 24 hpi, 48 hpi and 72 hpi. At 24 hpi, the Lv IMα1 expression reduces into the lowest level, which is 0.38-fold of the control. All the result suggests that Lv IMα1 might take part in the innate immune response of L. vannamei triggered by pathogens.