Sequence Characterization And Activity Of The IL-4/13B Promoter In Large Yellow Croaker (Larimichthys Crocea)

The large yellow croaker(Larimichthys crocea)is an economically important fish in China,with the highest annual production of all marine fish species cultured in China.However,infectious diseases have become increasingly severe as large yellow croaker aquaculture has expanded,resulting in major economic losses.Chemical drugs and antibiotics remain the major methods that prevent fish diseases,although their extensive application has also caused resistance,drug residues and destruction of aquatic microecosystems.Therefore,immunological control is considered to be a good method for control of the large yellow croaker diseases.IL-4/13 B is a pleiotropic cytokine that plays an important role in promoting T cell proliferation,development and differentiation,B cell proliferation and antibody production,and regulating immune response against pathogenic infection.However,the regulatory mechanism on IL-4/13 B transcription is still unclear in large yellow croaker.The aim of this thesis is to investigate the characteristics,activity and regulatory mechanism of IL-4/13 B promoter in large yellow croaker.The promoter fragment of large yellow croaker IL-4/13B(Lc IL-4/13B)was 1995 bp.Bioinformatic analysis showed that the promoter of Lc IL-4/13 B had the core promoter region,a TATA box and several transcription factors(GATA3,MAF,AP-1,STAT6 and IRF-4)binding sites,but Cp G island and CTAA box were not found.Comparison of the promoter sequences of IL-4 genes from different species revealed that the binding sites for STAT6 and GATA3 were conserved among the selected species.Dual luciferase assays showed that the obtained promoter of Lc IL-4/13 B had basal promoter activity and its activity could be increased by PHA-M and ploy(I:C)induction,suggesting Lc IL-4/13 B may be activated during T cell response and antiviral response.In order to find the key transcription factors for Lc IL-4/13 B promoter,we constructed a series of deletion mutations(IL-4/13B-P1-P5)and investigated the activities of these mutations.Analysis of changes in promoter activity using a dual luciferase reporter assay showed a trend of increasing and then decreasing promoter activity as the rhododendron IL-4/13 B promoter was gradually truncated,suggesting the possible existence of a negative regulator between P and P3(-1826 to-230),with activity falling to a minimum at interception to P5(-17 to +168),indicating the existence of this promoter core region between P4 and P5(-149 to +57),suggesting the presence of this promoter core region.Based on the predicted transcription factor binding sites and deletion mutation data,we selected and mutated the possible binding sites,and found that the mutations of IRF-4,STAT6,and GATA3 binding sites significantly decreased the promoter activity of Lc IL-4/13 B.These results suggest that the transcription factors IRF-4,STAT6 and GATA3 may play key roles in regulating Lc IL-4/13 B transcription.To confirm this hypothesis,we cloned and inserted the coding sequences of IRF-4,STAT6 and GATA3 into pc DNA3.1 plasmids,and co-transfected with luciferase report plasmid containing Lc IL-4/13 B promoter.The results showed that overexpression of IRF-4,STAT6 and GATA3 all could increase the Lc IL-4/13 B promoter activity,supporting that IRF-4,STAT6 and GATA3 are key transcription factors for Lc IL-4/13 B.The promoter sequence of Lc IL-4/13 B was cloned and identified in large yellow croaker.The obtained promoter of Lc IL-4/13 B had basal promoter activity and its activity could be increased by PHA-M and ploy(I:C)induction.IRF-4,GATA3 and STAT6 were further confirmed to play key roles in regulating Lc IL-4/13 B transcription.These results revealed the regulatory mechanism of IL-4/13 B in large yellow croaker and provide some new insights into transcriptional control of fish cytokines.