Research On The Transcriptional Regulation Mechanism Of ABCA3 Gene Mediated By C-Myc In Cadmium Enrichment Of Blood Clam,Tegillarca Granosa

The blood clam Tegillarca granosa(T.granosa)is an important cultivated economic shellfish in Zhejiang and other Provinces,China.The phenomenon of cadmium superenrichment by blood clams has attracted more and more attention.However,research on the enrichment content of heavy metals in blood clams have mainly focused on the physiological and ecotoxicological effects aspects of its,while the molecular mechanisms of Cd enrichment in blood clams are less reported.Gene transcription level is affected by a variety of endogenous factors,among which transcription factors regulate the expression of related genes by activating or inhibiting promoters,and the final comprehensive reaction is the change of phenotypic traits.c-Myc(cell myelocytomatosis viral oncogene,c-Myc)is an important class of nuclear transcription factor-like proto-oncogene,which is induced in the form of massive gene amplification.It is involved in numerous cellular life activities such as cell growth,proliferation,differentiation and apoptosis,and can directly regulate the transcriptional expression of other genes,playing an indispensable role in the evolutionary process of life.With the completion of whole genome sequencing and heavy metal transcriptome sequencing of blood clam,the preliminary study of our group found that the blood clam ABCA3(ATP-binding cassette transporter sub-family A)gene is involved in the transport of cadmium ions and it’s also a downstream target gene of c-Myc.In this study,we firstly obtained the full-length c DNA sequence of c-Myc(named as Tgc-Myc)of blood clam by RACE technique for bioinformatics analysis,and then analyzed the expression pattern of Tgc-Myc in different tissues by quantitative real-time PCR technique(q RT-PCR)and Western blot,studied the response pattern of this gene in cadmium stress of blood clam and explored its regulatory role mechanism on cadmium stress response-related genes,and preliminarily resolved the regulation of c-Myc involved in cadmium stress of mud clam.Secondly,modern molecular experimental techniques such as Electrophoretic Mobility Shift Assay(EMSA)and Chromatin Immunoprecipitation Assay(CHIP)were used for in vivo and in vitro experiments to deeply investigate the effects of Tgc-Myc on the transcriptional regulation of the transporter ABCA3 gene and its involvement in the regulation of heavy metal stress.The main results of this study are as follows:1.Cloning and bioinformatics analysis of transcription factor c-Myc cDNA of Tegillarca granosa.The c DNA of Tgc-Myc is 3063 bp in length,including a a 129-bp 5′-UTR,1188-bp ORF,and 1746-bp 3′-UTR,encoding 395 amino acids,with a predicted molecular weight of 44.9 k Da and a theoretical isoelectric point of 6.82.The predicted protein contained two functional domains that are typically found in c-Myc proteins: an N-terminal transactivation domain,a C-terminal basic helix-loop-helix leucine zipper(b HLH-LZ)domain binds to E-box mods in downstream gene promoters to initiate transcription.The results of amino acid sequence homology comparison showed that the Tgc-Myc gene showed the highest similarity of 59% to the Pinctada fucata,and also clustered with bivalves first in the phylogenetic tree.2.Analysis of the specific distribution of c-Myc genes in different tissues of Tegillarca granosa and expression changes under cadmium stress.The q RT-PCR and western blot assay techniques were used to detect the distribution of Tgc-Myc in blood clam tissues and the spatio-temporal expression during cadmium stress,and the results showed that:Tgc-Myc showed different levels of expression in all the tissue sites examined in blood clam,with the expression level in the gill showing the highest,significantly higher than the other four tissues(P<0.05);Tgc-Myc transcript and protein expression were significantly higher after 24 h of cadmium stress(P<0.05).Heavy metal cadmium ion stress induced upregulation of Tgc-Myc expression.3.Identification of the Tegillarca granosa Tgc-Myc protein bound to ABCA3 gene.In vitro binding identification of Tgc-Myc nuclear protein gel migration electrophoresis assay showed that the protein was able to recognize and bind to the CACGTG core sequence(E-box)of the promoter sequence of the target gene ABCA3 gene.The binding of Tgc-Myc protein to ABCA3 gene was further verified in vivo using chromosomal immunoprecipitation assay.The complexes of Tgc-Myc protein cross-linked with ABCA3 gene were enriched and purified for specific target proteins and DNA fragments using Tgc-Myc specific antibody immunoprecipitation.q RT-PCR analysis was performed to obtain sequence information of target DNA fragments and c-Myc binding reaction located in the core promoter region of-40 bp to-35 bp.4.Analysis of transcriptional regulation of ABCA3 gene by Tgc-Myc and its regulatory effect on heavy metal Cd stress/detoxification-related genes in blood clam.RNA interference experiments on Tgc-Myc showed that the expression of Tgc-Myc at 48 h was significantly down-regulated to 40.27 % of that of the blank control group,and the expression of its regulated downstream gene ABCA3 and seven other heavy metal Cd stress-related genes(including antioxidant enzyme genes,transporter genes,genes regulating energy metabolism and proliferation growth genes)were all down-regulated to about 50 % of the blank control.In summary,our results indicate that ABCA3 gene acts as a target gene of transcription factor Tgc-Mycand its transcriptional expression is positively regulated by Tgc-Myc;Tgc-Myc is involved in the stress response and detoxification response of blood clam under cadmium stress induction.The results of the study not only enriched the knowledge of the molecular mechanism of heavy metal enrichment in shellfish,but also laid the theoretical foundation for the healthy culture of mud clam and the selection and breeding of low cadmium-enriched strains.