miRNA related to oxidative stress damage of duck intestinal mucosa and application thereof

[0033] according to figure 1 As shown, this example proposes to obtain the intestinal oxidative stress-related gene (AKR7A2) and its regulatory miRNA (miRNA-218-5p) through joint analysis of miRNA and mRNA based on NGS technology.

[0034] (1) Experimental animals and sample preparation

[0035] The hatched Jingjiang ducklings were used as the source of experimental materials. They were subjected to high-temperature transport stress treatment for 8 hours, slaughtered immediately after treatment, blood samples and small intestine samples were collected, and the collected small intestine samples were quickly put into liquid cryopreservation in nitrogen;

[0036] After the small intestine samples of the stress group and the control group were sampled, three duckling samples were selected from each group as the small intestine samples of the experimental group and the small intestine samples of the control group, and the selected small intestine samples of the experimental group and

[0044] according to figure 2 , 3 As shown, this example proposes a method for population verification of the correlation between miRNA-218-5p markers and intestinal stress, using real-time fluorescent quantitative PCR (qRT-PCR) to sequence key miRNAs (miRNA-218-5p) and The expression of the gene (AKR7A2) was used for sample identification.

[0045] (1) Experimental sample preparation

[0046] Take 40 newborn Jingjiang ducklings, half of them are treated with transportation stress, and half of them are used as the control group. They are slaughtered after 8 hours of long-distance transportation, and blood samples and small intestine tissue samples are collected. After the small intestine tissue samples are collected, they are quickly frozen in liquid nitrogen. Then, according to the oxidative stress indicators in the blood, 6 individuals with extreme phenotype values ​​were selected for each group.

[0047] (2) Verification of differential expression of miRNA-218-5p

[0048]

[0061] according to Figure 4 , 5 As shown in 6 and 6, this example proposes the regulation of miRNA miRNA-218-5p on AKR7A2 gene expression and oxidative stress response of small intestinal epithelial cells.

[0062] (1) Construction of plasmids and synthesis of miRNA-218-5p mimics

[0063] Construction of AKR7A2 vector: Design primers according to the 3'UTR sequence of duck AKR7A2 on the ensemble, extract the RNA in duck small intestine, and reverse transcribe it into cDNA, and use PCR method to amplify the 3'UTR sequence of AKR7A2, including miRNA-218- 5p binding site, and then after recovery, purification and other steps, the target fragment is cloned into the pmirGLO vector; the specific experimental steps include analysis of the restriction site of the 3'UTR sequence of AKR7A2, and according to the vector sequence information, select PmeI and The AKR7A2 vector was obtained after the two restriction sites of XhoI and PCR products were digested, the target fragments were pur