The Transcription Regulation Of ChHsc70 By Gnaq And The Expression Patterns Of The Two Genes Of Crassostrea Hongkongensis In Response To Physico-chemical Stressors

Heat shock proteins 70 are divided into two isoforms including stress-inducible?Hsp70?and constitutive?Hsc70?forms.The proteins play an important role in normal cells.Crassostrea hongkongensis is an important economic bivalve species.The researches showed that when C.hongkongensis were stressed by physical or chemical factors,ChHsc70 expression changed,and was regulated positively or negatively by transcription factors.So far,no positive transcription factors have been reported for ChHsc70 transcription in molluscs.In this study,Gnaq,one of Guanine nucleotide-binding protein?subunits,was isolated from cellular nucleus extracts of C.hongkongensis gills with biotin-labeled ChHsc70 promoter by means of DNA-affinity purification,and preliminarily identified with mass spectrometry analysis.The full-length cDNA and the protein three-dimensional structure of ChGnaq were obtained by RACE and the three-dimensional modeling of a protein.Finally,a positively regulatory role of ChGnaq in controlling ChHsc70 transcription of C.hongkongensis was discovered using RNAi,over-expression and qRT-PCR methods.The full length of ChGnaq cDNA was 1476 bp,consisting of a 5'-terminal untranslated region?UTR?of 211 bp,and a 3'-terminal UTR of 203 bp,as well as an opening reading frame of 1062 bp.ChGnaq cDNA sequence was deposited in the GenBank with the nucleotide sequence accession number of MG944312.Gnaq protein contains a GTPase region and a hellical region.ChGnaq mRNA depletion by RNAi technique led to a clear reduction of ChHsc70 mRNA expression in C.hongkongensis hemocytes for all sampling points in the test trial when compared with those in the control.This result demonstrated a positive regulatory role of ChGnaq on the ChHsc70 mRNA expression in native C.hongkongensis host.Upon ChGnaq over-expression,the relative luciferase activities driven by the ChHsc70 promoter were significantly enhanced at all sampling points of the test trial when compared with those of the control.This result demonstrated that ChGnaq positively regulated ChHsc70 transcription in heterologous HEK293T cells.The ChGnaq had similar transcription expression pattern?increased first,and peaked,then returned back to normal level?to that of ChHsc70 when C.hongkongensis were treated by heat shock at 37 ⁰C for 1 h or pollutants.But the ChGnaq transcription was earlier than the ChHsc70at the first significant increament of mRNA level when the oysters were continuously treated by either CdCl₂ or NP,respectively.This suggested that ChGnaq played an important role in positive transcription regulation of ChHsc70.Meanwhile,ChGnaq expression is more persistently induced by pollutants compared with that instantaneously induced by heat shock.This suggested that ChGnaq was able to be taken as a supplementary warning molecule of pollution to wipe off the influence of temperature change on expression level of ChHsc70.The analysis indicates that the relative expression ratio?at>4?of ChHsc70/ChGnaq suggests non-pollution occurrence,but is just caused by temperature changes,however,the ratio at<3suggests the pollution occurrence.This study discovered a positively regulatory role of ChGnaq in controlling ChHsc70transcription of C.hongkongensis,and conduced to a better understanding of the regulatory mechanisms in control of Hsc70 transcription.Meanwhile,a suitable supplementary warning molecule of pollution to eliminate the influence of temperature changes on expression level of ChHsc70 was discovered,and laid a strong foundation of ChHsc70 used as a reliable warning biomarker of pollution.