Analysis Of DEGs In Myzus Persicae Under Trans-anethole Stress And Study On Antioxidant Function Of Superoxide Dismutase

Myzus persicae is one of the most important agricultural and forestry pests in the world.The long-term use of chemical pesticides has caused serious resistance to M.persicae.Botanical insecticides are toxic to pests,and it is difficult to produce resistance to pesticides,which provides an effective alternative to chemical synthetic pesticides.Trans-anethole is the main component of essential oil such as Illicium verum and Foeniculum vulgare.Previous studies have shown that trans-anethole has high toxicity to M.persicae.However,there are few studies on how M.persicae responds to trans-anisole stress at the molecular level.In this study,transcriptome sequencing was performed on the third instar larvae of M.persicae and untreated control group after 48 h treatment with Trans-anethole sublethal dose by high-throughput sequencing technology.Bioinformatics tools analyze Differentially Expressed Genes in M.persicae under trans-anethole stress.Screening important stress response genes and studying their functions.This study lays a foundation for further development of Botanical insecticide using trans-anethole.The main results are as follows:1.Toxicity of trans-anethole to 3rd instar nymphs of peach aphid showed that trans-anethole had high toxicity to M.persicae,and LC₅₀ was 3.2 mg/L at 48 h.2.Transcriptome sequencing results three treatment groups were 4.14×10⁷,4.87×10⁷,4.69×10⁷ raw reads.Three control groups obtained 4.93×10⁷,4.75×10⁷ and 5.16×10⁷ raw reads.By filtering the data and removing the low-quality reads,3.86×10⁷,4.57×10⁷,4.4×10⁷ clean reads,and 4.59×10⁷,4.53×10⁷,4.85×10⁷ clean reads were obtained from each treatment and control group.By comparing Clean Reads with the reference genome sequence,it was found that about 93%of the treatment group and the control group could be localized to the reference genome on average.By analyzing differential gene expression between the treatment and control groups,559 DEGs were obtained,including318 up-regulated genes and 241 down-regulated genes.3.Through enrichment analysis of DEGs,the DEGs in GO database 559 were divided into 46 functions.67 DEGs enriched in 18 metabolic pathways in the KOG database.Due to the lack of research on antioxidase genes,SOD genes were selected as the main research object for further study.4.The SODs information of M.persicae was obtained by NCBI online query.The SOD1 protein sequence of M.persicae had two Cu/Zn SOD specific sequences 43-53 bp（GFHIHEFGDNT）,137-148 bp（GNAGARIACGVI）,there are 7 metal binding sites for Cu²⁺and Zn²⁺,and two cysteines（SS）binding sites.The SOD2 protein sequence of M.persicae has a Mn SOD specific sequence183-189 bp（DVWEHAYY）and four Mn²⁺metal-binding sites.There were two Cu/Zn SOD specific sequences 71-81 bp（GFHVHEKGDVT）and 165-176 bp（GHAGSRVACGVI）in the SOD3 protein of M.persicae.Seven Cu²⁺and Zn²⁺metal-binding sites indicated that the SODs of three M.persicae was highly conserved during evolution.The phylogenetic tree analysis showed that SOD genes of three aphids were closely related to three SOD genes of the cotton aphid and three SOD genes of the pea aphid.5.RT-q PCR was used to determine the expression patterns of SOD genes in three aphids at different ages and under four adverse environmental stresses including trans-anethole,temperature,ultraviolet and permethrin.The results showed that the expression levels of SOD genes in three aphids increased with age.Under 3.2 mg/L trans-anethole stress,the SOD gene expression levels of three aphid species were significantly up-regulated compared with the control group,which was consistent with the differentially expressed genes in transcriptional sequencing.Under different temperature stresses,the SOD expression levels of the three peach aphids were significantly up-regulated at 0°C compared with those of the control group（25°C）.Under UV stress,the expression levels of SOD genes in the three peach aphids were up-regulated most significantly at 1.5 h compared with those in the control group（0 h）.Under the stress of 20 mg/L permethrin,the SOD gene expression levels of the three aphids increased most significantly at 36 h compared with the control group（0h）.6.SOD1 of M.persicae was selected as the main research object.The p Cold II-Mp SOD1plasmid was successfully constructed,and Trans B（DE3）was used as the prokaryotic expression strain to obtain the recombinant protein.The recombinant protein was purified by combining the His-tag with the Ni column,dialysis and enzyme activity detection.Finally,a specific band at about 15.7 k Da was obtained,which was consistent with the target band,and the enzyme activity was determined to be 1.03 U/mg.7.The activity of Cu Zn/Mn SOD was detected in M.persicae treated with 3.2 mg/m L trans-anethole.The results showed that trans-anethole induced the increase of Cu Zn/Mn SOD activity and the most obvious increase occurred at 72 h.8.On the basis of overexpression of SOD1 in E.coli,the antioxidant function of the transformed recombinant protein p Cold II-Mp SOD1 was analyzed under paraquat and H2O2stress.The results showed that the survival rate of E.coli cells containing p Cold II-Mp SOD1recombinant protein was significantly higher than that of p Cold II E.coli cells,indicating that SOD1 plays an important role in antioxidant damage.