Nrf2/ARE Pathway Mediated Trx Oxidative Stress Regulation Mechanism Of Cristaria Plicata

Thioredoxin(Trx)is one of the downstream target genes of Nrf2/ARE antioxidant pathway,which is involved in the process of protecting nucleic acids,resisting oxidative stress and controlling cell apoptosis.When Nrf2/ARE pathway responds to oxidative stress,the transcription factor Nrf2 forms a heterodimer with small MAF protein,and then binds to the antioxidant element(ARE)in the promoter region to regulate the transcription of target genes.In this paper,the mechanism of Cp Nrf2 regulation of thioredoxin expression was studied under stimulation of microcyst toxin.Recombinant plasmid p GBKT7-Nrf2 was transfected into Y2 HGold yeast to detect its self-activation and toxicity.The results showed that p GBKT7-Nrf2 could grow blue plaque on SD/-Trp/X-α-gal/ Ab A solid medium,and the morphology,size and quantity of the plaque growing on SD/-Trp medium was similar to that of the plaque containing p GBKT7.The full length of Nrf2 was self-activated and had no toxic effect on yeast.The Nrf2-ORF sequence fragment deletion experiment verified that the self-activated domain existed in the range of 1038-1560 bp of the Nrf2 open reading frame.Real-time fluorescence quantitative PCR was utilized to analyze the changes of thioredoxin Cp Trx gene expression in Cristaria plicata under microcystin-MC stress.The experimental data showed that the expression of Cp Trx m RNA was significantly up-regulated in gill and kidney tissues.Nrf2 and Keap1 gene were knocked down by specific small RNA.Comparison with the control group,the expression of Cp Trx m RNA in Keap1 knockdown group was significantly upregulated in both gill and kidney tissues under microcystins stress.In contrast,Cp Trx m RNA expression in Nrf2 knockdown group was significantly down-regulated.The promoter DNA sequence of Cp Trx gene(2423 bp)was amplified by chromosomal step method using the DNA template of C.plicata.The online database analysis showed that a variety of transcription factor binding sites and cis elements was on promoter region,including TATA box,CAAT box,ARE(anti-oxidative stress element),AP-1(activating protein-1)binding site,nuclear factor NF-k B binding site,etc.Dual-luciferase reports showed that Cp Trx gene promoter had high transcriptional activity,and this basic transcriptional activity persisted after the ARE element mutation.The region of promoter-206 ～ +217bp was a core promoter region,and had forward regulatory elements.Microcystins could not up-regulate the activity of Cptrx gene promoter,and Cp Nrf2 gene could inhibit the activity of Cp Trx gene promoter.Gel shift Assay(EMSA)showed that the Cp Trx promoter could bind to the purified proteins Cp Nrf2 and Cp Maf K in vitro.The binding phenomenon disappeared after the ARE element mutation in promoter region.