| Rhynchocypris lagowskii(Dybowski,1869)is a unique omnivorous small fish species,with an extremely high economic value and market potential.Currently,the culturing of Rhynchocypris lagowskii(Dybowski,1869)is relatively extensive and is being promoted on a large scale.Even those culturing of the species is expanding,the artificial breeding techniques used such as nutritional needs,supporting bait,and seed breeding are still immature.In the course of breeding practice,they are often susceptible to oxidative stress and their growth becomes hindered or even get sick.Subsequently,this causes huge stock losses and economic losses to farmers.The nuclear transcription factor NF-E2 related factor 2(nuclear factorery throid 2-related factor 2,Nrf2)is the most important key protein in regulating the body's antioxidant stress.Nuclear entry initiates the Nrf2-Keap1-ARE antioxidant signaling pathway and promotes the expression of downstream effector genes,thus enhancing the antioxidant capacity,thereby protecting cells from oxidative stress.However,there is no relevant report on the research of this fish at present,which is detrimental to the culturing and breeding of this fish.Therefore,analyzing the internal mechanism of Rhynchocypris lagowskii(Dybowski,1869)'s resistance to oxidative stress,strengthening the research on non-specific immunity of Rhynchocypris lagowskii(Dybowski,1869),strengthen the research of Rhynchocypris lagowskii(Dybowski,1869)non-specific immunity,and enhance the immunity of the fish is the key to solving the above bottlenecks in the industry.In this paper,the juveniles(13.25±0.21)g of Rhynchocypris lagowskii(Dybowski,1869)were used in the experiment of this study,and the related research on its Pld-Nrf2 gene was conducted.First,the RACE-PCR technology was used to clone the full-length c DNA of the Pld-Nrf2 gene of the species.Secondly,RT-q PCR technology was used to detect the tissue distribution of Rhynchocypris lagowskii(Dybowski,1869)Pld-Nrf2 gene,and the cloned Rhynchocypris lagowskii(Dybowski,1869)Pld-Nrf2 gene phylogenetic tree,isoelectric point of protein,hydrophobic site encoding protein,protein two Bioinformatics analysis of secondary structure,protein tertiary structure,and transmembrane region.Finally,intraperitoneal injection of Nrf2 agonist tert-butyl hydroquinone(t BHQ)and Nrf2 inhibitor dichlorobenzimidazole furan-type riboside(DRB),through the detection of related antioxidant enzyme activities and antioxidant substances,and use Relative to q PCR technology to detect the response of the antioxidant system of the Rhynchocypris lagowskii(Dybowski,1869).The study aimed to verify the antioxidant regulation function of the Rhynchocypris lagowskii(Dybowski,1869)Pld-Nrf2 gene;to explore it physiological metabolic mechanism against oxidative stress.This study consists of four parts of experiments.The results and conclusions are as follows:1.Rhynchocypris lagowskii(Dybowski,1869)nuclear transcription factor Pld-Nrf2 gene cloneIn this study,the full-length sequence of Rhynchocypris lagowskii(Dybowski,1869)Pld-Nrf2 gene was cloned for the first time using homology cloning and RACE PCR technology,with a length of 2613 bp.The coding region of the gene is from 134-1900,and the CDS region is 1767 bp.2.Rhynchocypris lagowskii(Dybowski,1869)nuclear expression factor Pld-Nrf2 tissue expression distribution and bioinformatics analysisBioinformatics analysis was performed on the full length of Rhynchocypris lagowskii(Dybowski,1869)nuclear transcription factor Pld-Nrf2.The Pld-Nrf2 protein has 588 amino acids,ATG is the start codon,AAC is the last codon of the sequence,and TAG is the stop codon.The open reading frame(ORF)is 1323 bp,tailed signal(AATAAA),and Poly-A tail.The theoretical molecular weight and isoelectric point of the protein are 66157.09 Da and 4.60.No signal peptide,SMART and CDD predictions show that the protein contains a BZIP domain;the protein encoded by the Pld-Nrf2 gene has no transmembrane region,so amino acids are located on the cell membrane surface.The protein encoded by the Pld-Nrf2 gene has a certain hydrophobicity,with a maximum hydrophobicity index of 2.86 and a minimum The hydrophobic index is-4.1.The phylogenetic tree was constructed by the NJ method for 1000-subset analysis,and the closest relationship with the same genus Pimephales.Real-time q PCR results showed that Pld-Nrf2 gene was expressed in various tissues of Rhynchocypris lagowskii(Dybowski,1869),especially in liver tissues(P<0.05).3.Effect of t BHQ on Rhynchocypris lagowskii(Dybowski,1869)liver and pancreas antioxidant enzymes and relative expression of antioxidant gene m RNAIntraperitoneal injection of 4 different concentrations of t BHQ solutions of 0mg/kg,25 mg/kg,50 mg/kg and 75 mg/kg were used to measure antioxidant enzyme activity and genes at 12 h,24 h,36 h and 48 h.The results show that t BHQ can increase the activity of SOD in serum and liver,?-GCS,CAT and other antioxidant enzymes,and T-GSH and T-AOC levels(P<0.05),but reduced MDA content(P<0.05).through RT-q PCR detection technology we learned that after intraperitoneal injection of t BHQ,increased liver Pld-Nrf2,CAT,and Cuzn-SOD m RNA expression(P<0.05),decreased expression of inhibitor protein Keap1 m RNA(P<0.05).4.Effect of DRB on Rhynchocypris lagowskii(Dybowski,1869)liver and pancreas antioxidant enzymes and relative expression of antioxidant gene m RNABy intraperitoneal injection of 4 different concentrations of DRB solutions of 0mg/kg,10 mg/kg,25 mg/kg,and 50 mg/kg,antioxidant enzyme activities and genes were measured at 12 h,24 h,48 h and 72 h.The results showed that with the injection concentration Gradually increasing and the accumulation of response time,the levels of MDA in serum and liver significantly increased(P<0.05),the activity of antioxidant enzymes(CAT,T-SOD,and ?-GCS)and the levels of antioxidant T-GSH also decreased(P<0.05),the total resistance The oxidative capacity was significantly reduced(P<0.05).by RT-q PCR detection technology,we learned that the intraperitoneal injection of DRB reduced the expression of Pld-Nrf2,Cuzn-SOD,and CAT m RNA in the liver(P<0.05),and increased the expression of the inhibitory protein Keap1 m RNA(P<0.05).Based on the results of the experiment,it can concluded that the unknown gene cloned in this study is initially identified as Pld-Nrf2 by bioinformatics analysis such as evolutionary tree.The injection of Nrf2 promoter t BHQ and the inhibitor DRB significantly interfered with the antioxidant capacity of Rhynchocypris lagowskii(Dybowski,1869).The oxidizing ability indicates that the Pld-Nrf2 gene of Rhynchocypris lagowskii(Dybowski,1869)can regulate the body's antioxidant function,thereby protecting fish from oxidative damage. |
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