单环刺螠GATA B2的基因克隆及表达分析

Gene Cloning and Expression Analysis of GATA B2 in Urechis unicinctus

GATA家族是经典的转录因子家族,因其能特异性的与核苷酸序列T/A(GATA)A/G结合而得名。前期研究在单环刺螠(Urechis unicinctus)中筛选到GATA家族成员GATA B2与硫代谢关键酶硫醌氧化还原酶(Sulfide quinone oxidoreductase,sqr)启动子高转录活性区相互作用。为了进一步研究其是否参与硫化物代谢过程,本文鉴定了单环刺螠GATA B2基因(UuGATA B2),并分析其在成体各组织和硫化物暴露后的表达特征。结果显示:单环刺螠GATA B2的ORF全长为1788 bp,编码585个氨基酸;该氨基酸序列包含2个GATA家族保守的锌指结构域;系统进化分析显示UuGATA B2先与小头虫的GATA B2聚类,再与其它GATA4/5/6亚家族成员聚类。随后制备UuGATA B2多克隆抗体,利用Western blotting检测其在单环刺螠成体不同组织中的表达水平,结果显示其在体壁中的蛋白表达水平最高,中肠次之,后肠和肛门囊表达量较低,体腔液最低。由于sqr在硫化物暴露后的后肠中表达量最高,进一步对单环刺螠暴露在150μmol/L硫化物环境时后肠中UuGATA B2的表达进行了检测,发现其显著的响应硫化物胁迫,并在暴露12 h时含量达到最高,是对照组的2.32倍,表明UuGATA B2参与了单环刺螠硫化物应激过程。本文首次报道了GATA B2在硫化物应激中的表达特征,为后续深入探讨GATA家族在硫化物应激中的作用提供了基础数据。

The GATA gene family is a type of transcription factor,named after the consensus DNA-binding sequence T/A(GATA)A/G,which is recognized by the zinc-finger domains common to all family members.In the previous study,GATA B2 was screened by the yeast one-hybrid assay using the high transcriptional activity region of the sqr promoter of Urechis unicinctus,suggesting that it may be involved in sulfide metabolism.To further study its function,we identified the UuGATA B2 gene and analyzed its expression in different tissues and the hindgut after sulfide exposure.The results showed that the full length of ORF was 1788 bp,encoding 585 amino acids.Its amino acid sequence contained two conserved GATA-type zinc finger domains.Phylogenetic analysis showed that UuGATA B2 was closely related to the GATAB2 of Capitella teleta,then clustered with other GATA4/5/6 subfamily members.Subsequently,the UuGATA B2 polyclonal antibody was obtained to detect its content in different tissues by Western blotting.The result showed that UuGATA B2 expressed the highest in body wall tissue,followed by the midgut,lower in the hindgut and anal sac,and the lowest in coelom fluid.Moreover,the hindgut showed the highest sqr mRNA expression level compared to other tissues after sulfide exposure,the expression of UuGATA B2 in the hindgut after 150μmol/L sulfide exposure were characterized.The results showed that UuGATA B2 expression significantly responded to sulfide stress in the hindgut,reaching the highest at 12 h,which was 2.32 times that of the control group,suggesting that UuGATA B2 is involved in sulfide stress in U.unicinctus.This research reveals the expression characteristic of GATA B2 in sulfide stress for the first time,which provides basic data for exploring the role of GATA family in sulfide stress.