Identification And Antioxidative Properties Of The Antioxidant Enzymes CAT And TPX From Pieris Rapae

The small white butterfly,Pieris rapae(Insecta:Lepidoptera:Pieridae),is an insect pest which larvae are one of the main pests harming cruciferous vegetables.Up to now,the control of this insect mainly depends on chemical pesticides,such as lambda-cyhalothrin,chlorantraniliprole and avermectin.However,the excessive use of insecticides will not only aggravate environmental pollution,but also reduce the sensitivity of pests to insecticides,leading to the gradual reduction of control effect.Previous studies have found that insecticides can induce the insect body to produce a large number of reactive oxygen species,which can cause oxidative damage to insects.The antioxidant mechanism in which a variety of enzymes are involved can resist oxidative damage,thus enhancing the tolerance of pests to pesticides indirectly.At present,reports on the antioxidant mechanism of P.rapae are very limited.In view of this,our study focused on catalase(CAT)and thioredoxin peroxidase(TPX)which are key enzymes in the antioxidant mechanism of P.rapae,concentrating on the role of these two enzymes in the resistance of P.rapae larvae which are treated with lambda-cyhalothrin.The research results can reveal the role of antioxidant enzymes in the defense of P.rapae against external discomfort environment at the molecular level,thus providing a theoretical basis for the control of agricultural pests.The main research results are summarized as follows:1.Identification of CAT and TPX genes in P.rapae and bioinformatic analysesA CAT gene(named PrCAT)and five TPX genes(named PrTPXl,PrTPX3,PrTPX4,PrTPX5 and PrTPX6)were identified by retrieving the published genomic and transcriptional databases of Pieris rapae.PrCAT encodes a protein consisting of 509 amino acids,while the number of amino acids of five proteins encoded by PrTPX1,3,4,5 and 6 ranges from 189 to 247.The N-terminal of PrTPX3 protein was predicted to have a mitochondrial transport peptide,suggesting that the protein might be localized in mitochondria,while the N-terminal of PrTPX4 protein was predicted to have a secretory signal peptide,suggesting that the protein might be secreted out of the cell.BLAST results showed that these genes were highly consistent with the homologous genes of Lepidoptera.The phylogenetic results also showed that PrCAT and PrTPX genes were closely related to the direct homologous insects genes of Lepidoptera,but far from the direct homologous genes of other insects.2.Analysis of the expression patterns of CAT and TPX genes in P.rapaeReverse transcription polymerase chain reaction(RT-PCR)results showed that PrCAT and PrTPX were expressed in different tissues and developmental stages of Pieris rapae.Real-time quantitative PCR results showed that the relative expression of PrCAT mRNA varied greatly in different developmental stages of P.rapae.The highest expression level was in fourth-and fifth-instar stages and the second expression level was in adult stages,but the expression level in larvae of 2nd-and 3rd-instar larvae stages as well as in pupae stages was lowest.The expression level of PrCAT gene was almost the same in integument,Malpighian tubes and fat body,but was significantly lower than that in midgut.The expression levels of five PrTPX genes were different.Overall,the expression levels of these five genes were relatively high in the midgut and relatively low in the fifth-instar larvae stages and pupae.After the larvae of P.rapae were treated with sublethal dosage of lambda-cyhalothrin we found that the contents of hydrogen peroxide and malondialdehyde and the expressions of PrCAT and PrTPX genes in larvae's bodies were all significantly increased at the same time.It was speculated that PrCAT and PrTPX genes might be involved in the defense of P.rapae larvae against oxidative stress.3.Antioxidative properties of CAT and TPX proteins in P.rapaeThrough designing gene-specific primers,the prokaryotic expression vectors of PrCAT,PrTPX 1,PrTPX4 and PrTPX6 were constructed and transformed into E.coli,and the recombinant proteins were expressed and purified.The activity of these four recombinant proteins was tested by the catalytic degradation of hydrogen peroxide.The results showed that PrCAT,PrTPXl,PrTPX4 and PrTPX6 proteins all had the activity of scavenging hydrogen peroxide.The antioxidant activity of PrTPX1 protein was further analyzed by the disc diffusion assay.The results showed that the strains transformed with pET30a-PrTPX1 expression plasmid were more tolerant to the ROS inducer Cumene hydroperoxide(CHP)than the control group(E.coli strain transformed with pET30a empty vector),which proved that PrTPX1 protein had the antioxidant activity.