The Function Of GATA Transcription Factor(GLP) In Hematopoiesis Of Eriocheir Sinensis

Hematopoiesis is a series of complex processes that hemocytes occur,develop and mature from specific hematopoietic sites.The different forms of blood cells（immune cells）produced during hematopoiesis are the core and foundation of cellular and humoral immunity in many organisms.Therefore,understanding the occurrence,differentiation,and immune process of blood cells is the prerequisite to explore the immune defense mechanism of the organisms.Eriocheir sinensis is an important economic crustacean,and mainly cultured in southern Asia.E.sinensis depends on the innate immune defense system to cope with the pathogen invasion.during the process of immune response,the blood cells are constantly consumed,and the body needs a continuous supply of new blood cells.Therefore,the hematopoiesis plays a vital role in the E.sinensis.In this study,the E.sinensis GATA-like protein（EsGLP）was cloned,structural and functional analyzed by the means of molecular biology and histochemistry and so on.In the present study,a GATA-like protein（named as EsGLP）was characterized from E.sinensis,including an 834 bp full length open reading frame of EsGLP,encoding a polypeptide of 277 amino acids.The deduced amino acid sequence of EsGLP contained one conserved GATA-type zinc finger of the form Cys-X₂-Cys-X₁₇-Cys-X₂-Cys,which is used for the identification and combination of GATA transcription factors to target genes.The EsGLP mRNA was constitutively expressed in the hematopoietic tissue（HPT）,hemocytes,hepatopancreas,gonad,heart and gills,and the highest expression level was in HPT.The mRNA transcripts of EsGLP in hemocytes were significantly decreased at 24 h（0.39-fold and 0.27-fold,p<0.05）and48 h（0.35-fold and 0.16-fold,p<0.05）after LPS and Aeromonas hydrophila stimulation,respectively.The EsGLP mRNA transcripts were significantly up-regulated at 24 h（8.71-fold,p<0.05）in HPT after A.hydrophila stimulation.The expression level of EsGLP mRNA in HPT was significantly up-regulated at 2 h,2.5 h and 9 h（41.74-fold,45.38-fold and 26.07-fold,p<0.05）after exsanguination stimulation.Tissue localization and subcellular localization of EsGLP were carried out by using anti-rEsGLP specific polyclonal antibody,and the EsGLP protein was mainly distributed in the edge of lobules in the HPT and the cytoplasm of hemocytes.The expression level of EsGLP mRNA in HPT was significantly decreased（0.21-fold,p<0.05）at 48h after EsGLP gene expression inhibited by the injection of double-stranded RNA.When the expression of EsGLP was interfered,the total hemocytes counts and the rate of EdU-positive hemocytes were significantly decreased（0.32-fold and 0.56-fold compared to that in control group,p<0.05）.All these results suggest that EsGLP is similar to the GATA transcription factors of vertebrate in molecular structure and function,but there are also some differences.The expression of EsGLP was significantly increased after immune stimulation and exsanguination stimulation,which suggested that EsGLP played an important role in the neonatal hemocytes generation in E.sinensis,which would greatly enrich our understanding of invertebrate immunomodulatory mechanism,and provide theoretical guidance and technical support for the prevention and control of animal diseases in aquaculture.