The Crassostrea Hongkongensis YB-1 Positively Regulating Hsc70 Transcription Expression And The Transcriptional Expression Patterns Of YB-1 And Hsc70 Triggered By Heat Shock Or Chemical Pollutants

The heat shock protein with molecular mass of 70 kDa?Hsp70s?,the most important member of the heat shock proteins family,includes both stress-inducible?Hsp70?and constitutive?Hsc70?proteins.They play essential roles in assisting cells to acclimatize gradually environment changes except the function of molecular chaperones.In general,Hsc70acts as a housekeeper gene,and is responsive to external stressors in specific hosts,as well as is regulated by various transcription factors.However,the transcription factors are unknown for transcription regulation of Hsc70,and need to be clarified.In this study,we aimed to screen and identify transcription factors involved in the transcriptional expression regulation of ChHsc70 in Crassostrea hongkongensis.Furthermore,the transcriptional expression patterns of ChHsc70 and the transcriptional factor gene of ChHsc70 triggered by heat shock or chemical pollutants were investigated.A potential transcription factor bound specifically to the promoter region of ChHsc70 was screened by DNA affinity purification,and then the transcription factor was identified as a Y-box binding protein?ChYB-1?by mass spectrometry.The ChYB-1 cDNA was determined to be 1257 bp in full length by rapid amplification of cDNA ends?RACE?technology,including a coding region of924 bp,as well as 5'and 3'untranslated regions of 107 bp and 226 bp,respectively.The deduced molecular model of ChYB-1 consists of five antiparallel?-strands connected by a few loops at the end,which has been called as cold shock domain.YB-1 is known as a nucleic acid chaperone and participates almost all DNA-and mRNA-dependent activities,including transcription.ChYB-1 was demonstrated to positively regulate ChHsc70 expression by Luciferase Reporter Assay and RNAi.The overexpression of ChYB-1 in heterologous HEK293T cells led to clear enhancement of ChHsc70 promoter expression,while ChYB-1 depletion correlated with significant reduction of ChHsc70 transcription in the hemocytes of C.hongkongensis.Quantitative Real-time PCR analysis revealed both ChHsc70 and ChYB-1 transcriptions were responsive to heat shock or chemical pollutants,while the accumulated peaks of ChYB-1 was slightly earlier than that of ChHsc70.This suggested that ChYB-1 positively regulated ChHsc70transcription expression.The relative gene expression of ChHsc70 in the oyster treated by NP under changing temperature conditions was much higher than that by the same concentration of NP under constant temperature conditions.This indicated a synergistic effect between temperature and pollutants.Besides,in comparison to the long-term induction of ChYB-1 transcription expression caused by pollutants,a rather instant response was presented upon heat treatment.This suggested that ChYB-1 was a transcription factor which was much more sensitive to pollutants than to heat treatment and suitable as a supplementary warning molecule of pollution.The ratio of ChYB-1 to ChHsc70(4^-??Ct)in C.hongkongensis can eliminate the interference of temperature change on expression level of ChHsc70,the early warning molecule of pollution.The research work is the basis of establishing the early warning technology of marine environmental pollution based on the ratio of ChYB-1 to ChHsc70(4^-??Ct)in C.hongkongensis.This study demonstrates the positive regulatory function of ChYB-1 on ChHsc70transcription in C.hongkongensis.Besides,it indicates that ChYB-1 is more sensitive to pollutants than to temperature changes and suitable for using as a supplementary warning molecule of pollution.It contributes to a better understanding of the regulatory mechanisms governing Hsc70 expression and to establishing the early warning technology of marine environmental pollution.