Fish circRNA and application thereof

Abstract

The invention discloses a fish circRNA and application thereof, the fish is pelteobagrus fulvidraco, the circRNA is named circArid4b, and the nucleotide sequence of the circRNA is shown as SEQ ID NO.1. The invention further discloses a preparation method of the circRNA. The fish circRNA provided by the invention can regulate and control Arid4b expression of a source gene of the fish circRNA under hypoxia and bacterial infection conditions, influence apoptosis pathways and inflammatory reactions, and finally regulate and control antibacterial reactions. The invention further provides an interferent of the circRNA, the circRNA interferent can be used as a fish immunomodulator, infection of Aeromonas veronii on pelteobagrus fulvidraco under the low-oxygen condition is remarkably inhibited, and a new mode is provided for efficiently improving the antibacterial immunity of fish under the low-oxygen environment.

Description

Technical Field

[0001] The invention belongs to the technical field of molecular biology, and specifically relates to fish circRNA and applications thereof. Background Art

[0002] Yellow catfish (Pelteobagrus fulvidraco) has high nutritional value and no intramuscular spines. It is an important freshwater economic fish in my country. However, during the breeding process, yellow catfish is extremely susceptible to infection by pathogens. Its main pathogen Aeromonas veronii has the characteristics of strong infectivity and high morbidity. It can infect aquatic organisms such as humans, animals, and fish, which not only threatens the survival of yellow catfish, but also endangers human health. At the same time, due to the poor tolerance of yellow catfish to hypoxia, the hypoxia of water bodies caused by high-density breeding, high temperatures in summer, and global warming has caused the immunity of yellow catfish to decline rapidly, which has aggravated the frequent occurrence of bacterial diseases. The large-scale death of yellow catfish has also further reduced the dissolved oxygen in the water, forming a vicious circle. Therefore, it is of great significance to study the innate immune response of yellow catfish in response to bacterial infection in a hypoxic environment.

[0003] CircRNA is a new type of non-coding RNA molecule characterized by a covalently closed circular structure, which is not easily degraded and widely present in eukaryotes. It has the characteristics of organization, timing, disease specificity and high conservation. A large number of studies have shown that circRNA plays a key regulatory role in biological processes such as growth and development, immunity and stress response. Its mechanism of action includes but is not limited to: adsorbing specific miRNA as a competitive endogenous RNA (ceRNA), directly participating in the protein translation process, and forming a complex interaction network with RNA binding proteins (RBPs) or other non-coding RNAs. However, the application of circRNA in fish disease prevention and control is currently rare, and it is still unclear whether fish circRNA is involved in responding to hypoxic stress. Summary of the invention

[0004] In view of the shortcomings of the prior art, the present invention provides a fish circRNA and its application. The study of the circRNA regulatory mechanism of yellow catfish in response to bacterial infection under hypoxic conditions not only enriches the understanding of the fish circRNA immune mechanism, but also provides a new idea for the development of new prevention and control strategies for yellow catfish aquaculture diseases under hypoxic conditions.

[0005] The present invention is achieved through the following technical solutions:

[0006] A fish circRNA, wherein the fish is yellow catfish, the circRNA is named circArid4b, and its nucleotide sequence is shown in SEQ ID NO.1.

[0007] The application of the reagent for inhibiting the expression of the above-mentioned fish circRNA in yellow catfish's resistance to Aeromonas veronii infection.

[0008] Preferably, the reagent is siRNA, and its nucleotide sequence is shown in SEQ ID NO.6.

[0009] Preferably, the yellow catfish is placed in a hypoxic environment and the siRNA is injected intraperitoneally.

[0010] Preferably, the injection dose is 1.3 μg / g yellow catfish body weight.

[0011] Use of the reagent for inhibiting the expression of the above-mentioned fish circRNA in the preparation of antibacterial preparations.

[0012] Preferably, DEPC water is used to dissolve the siRNA having a nucleotide sequence such as that shown in SEQ ID NO. 6, and the concentration is configured to be 20 μM.

[0013] Preferably, the pathogenic bacteria of the antibacterial preparation is Aeromonas veronii.

[0014] An antibacterial preparation for yellow catfish to resist Aeromonas veronii comprises an agent for inhibiting the expression of the above-mentioned fish circRNA.

[0015] The application of the reagents for inhibiting the expression of the above-mentioned fish circRNA in improving the immune ability of yellow catfish.

[0016] The beneficial effects of the present invention are as follows:

[0017] The circArid4b provided by the present invention can regulate its source gene Arid4b, and promote the apoptosis and inflammatory response of yellow catfish cells in response to bacterial infection in a hypoxic environment. The synthesized circArid4b interferor (siRNA) is prepared into an antibacterial preparation, which can significantly inhibit the infection of Aeromonas veronii to yellow catfish in a hypoxic environment after injection into the fish body, that is, improve the antibacterial immunity of yellow catfish in a hypoxic environment. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 The expression level change of circArid4b in yellow catfish after hypoxia and bacterial infection for 6 hours in Example 1;

[0019] Figure 2 The PCR amplification of circArid4b using different primers in Example 2 (A) and the Sanger sequencing peak diagram of the RT-PCR product (B);

[0020] Figure 3 The expression changes of apoptosis-related genes in yellow catfish after hypoxia and bacterial infection for 6 hours in Example 3: A is Traf1, B is Traf2, C is Egfr, D is Gadd45b, E is Nfkbiaa, F is Parp4, G is Arid4b, H is Ctsh, and I is Apoa1;

[0021] Figure 4 The expression changes of inflammation-related genes in yellow catfish after hypoxia and bacterial infection for 6 hours in Example 3: A is TLR5, B is IL-1β, C is C7, D is Itln3, E is Elf2b, F is Ddit4, G is A2m, H is Cyp2j2, and I is CD163;

[0022] Figure 5 This is a result diagram of the knockdown effect of circArid4b interferor (si-circArid4b) on circArid4b under hypoxic infection conditions in Example 4;

[0023] Figure 6 The changes in bacterial load in liver tissue of Pelteobagrus fulvidraco under hypoxia and bacterial infection treatment after reducing circArid4b expression in Example 4: A is a plate image (n=3), and B is a colony statistics image;

[0024] Figure 7 The expression level change of the source gene Arid4b after the expression of circArid4b was reduced under hypoxic infection conditions in Example 5;

[0025] Figure 8 The expression changes of apoptosis-related genes after reducing circArid4b expression under hypoxic infection conditions in Example 6: A is Traf1, B is Traf2, C is Gadd45b, and D is Nfkbiaa;

[0026] Fig. 9 The expression changes of inflammation-related genes after reducing circArid4b expression under hypoxic infection conditions in Example 6: A is TLR5, B is IL-1β, C is Ddit4, and D is A2m. DETAILED DESCRIPTION

[0027] The present invention is further described in detail below with reference to the accompanying drawings and specific embodiments.

[0028] Unless otherwise specified, the technical means used in the following examples are all conventional means well known to those skilled in the art, and the experimental methods without specifying the specific conditions are all conventional methods in the art.

[0029] The experimental animals used in the following examples were yellow catfish, purchased from Chongqing Yongchuan Tangzhongcheng Fishery, weighing 1.5±0.5g, uniform in size, and healthy. They were first raised in a circulating water tank at a water temperature of 27±1.0°C for two weeks, during which aeration maintained normal oxygen supply, and commercial feed (Jiaji Feed Co., Ltd., Zhenjiang) was fed twice a day, and feeding was stopped 24h before the experiment.

[0030] Example 1 Discovery of circRNA (circArid4b)

[0031] Healthy yellow catfish weighing 1.5±0.5g were randomly divided into two groups. In the hypoxia infection (HB) group, yellow catfish were intraperitoneally injected with Aeromonas vermiformis in a hypoxic environment with a dissolved oxygen of 2.0mg / L. The bacterial solution dose was 4×10 5 CFU / g fish. The control (PBS) group was injected with an equal amount of sterile PBS buffer under normoxic environment. After 6h of HB treatment, the two groups of yellow catfish were anesthetized with MS-222, aseptically dissected and their liver tissue samples were collected for subsequent sequencing and experiments. According to the requirements of the Illumina HiSeq2500 sequencing platform, the sequencing library was constructed and high-throughput sequencing was performed. High-quality (HQT) sequences were obtained using Fastp (v0.12.4) and Bowtie 2 (v2.2.8), and the HQT sequences were aligned to the reference genome of yellow catfish using Tophat 2 (v2.0.3.12). Reads that were continuously aligned with the genome were removed, unmapped sequences with splice sites were retained, and circRNAs were identified using the program find_circ.

[0032] Among the identified circRNAs, a new circRNA whose parental gene was related to apoptosis was found and named circArid4b. The results of RT-qPCR and sequencing were consistent, such as Figure 1 As shown, circArid4b was significantly downregulated after 6 h of hypoxia and bacterial infection (P < 0.05).

[0033] Example 2 Identification of circRNA (circArid4b)

[0034] By comparing the genome of yellow catfish, it was found that the source gene Arid4b of circArid4b is located on chromosome 4, and circArid4b is formed by head-to-tail reverse splicing of exons 12-17 of the Arid4b gene. The length of the mature sequence after splicing is 741bp, and its nucleotide sequence is shown in SEQ ID NO.1. Convergent primers and divergent primers were used to amplify circArid4b, respectively. The PCR reaction system is shown in Table 1, and the specific primer sequences are as follows:

[0035] Convergent primer circArid4b-convergent:

[0036] F (SEQ ID NO.2): 5'-CCAAAGAGCTGGTTGTGGAC-3';

[0037] R (SEQ ID NO. 3): 5'-GCAAATAGACCTGAGGCTGC-3'.

[0038] Divergent primer circArid4b-divergent:

[0039] F (SEQ ID NO.4): 5'-GAGGAAGAGTTGGAGCCCTT-3';

[0040] R (SEQ ID NO. 5): 5'-TGTCTTTCCGTAGAACCAGGT-3'.

[0041] Table 1 PCR reaction system

[0042]

[0043] The results are as follows Figure 2 As shown in Figure A, circArid4b (expected fragment length of 229 bp) can be amplified from cDNA only by using divergent primers, while no amplification product was observed from gDNA. The RT-PCR product of circArid4b was subjected to Sanger sequencing, as shown in Figure 2 Figure 2 As shown in B, circArid4b was further verified to be a stable circular RNA.

[0044] Example 3 Changes in the expression of genes related to apoptosis and inflammatory pathways in yellow catfish after HB treatment

[0045] In this embodiment, two treatment groups are set up, namely, the PBS group and the HB group, and the specific treatment method is the same as that in Example 1.

[0046] The total RNA of liver tissue was extracted by Trizol method, and the extracted RNA was reverse transcribed into cDNA according to the instructions of cDNA reverse reagent (Yisheng). RT-qPCR was used to quantitatively analyze the key functional genes in apoptosis and inflammatory response of yellow catfish, among which Traf1, Traf2, Egfr, Gadd45b, Nfkbiaa, Parp4 can promote cell apoptosis, Arid4b, Ctsh, Apoa1 can inhibit cell apoptosis; TLR5, IL-1β, C7, Itln3, Elf2b, Ddit4 can promote inflammatory response, A2m, Cyp2j2, CD163 can inhibit inflammatory response. The RT-qPCR reaction system is shown in Table 2 below:

[0047] Table 2 RT-qPCR reaction system

[0048]

[0049] The above system was placed in the ABI Step One Plus system RT-qPCR instrument and the reaction was carried out according to the 2-step standard procedure. -△△Ct The relative expression was calculated by the method.

[0050] The results are as follows Figure 3 , 4 As shown in Figure 2, the RT-qPCR results were consistent with the sequencing results. After 6 h of hypoxia infection, the genes promoting apoptosis in yellow catfish were significantly upregulated ( Figure 3 AF, P<0.05), genes inhibiting apoptosis were significantly down-regulated ( Figure 3 medium GI, P<0.05); genes promoting inflammation were significantly upregulated ( Figure 4 AF, P<0.05), genes inhibiting inflammation were significantly downregulated ( Figure 4 GI, P<0.05). This indicates that the apoptosis pathway and inflammatory response pathway of yellow catfish were activated after 6 hours of hypoxia and bacterial infection.

[0051] Example 4 Effects of circArid4b interferors on the antibacterial ability of Pelteobagrus fulvidraco in a hypoxic environment

[0052] In this example, an interferor (siRNA) for inhibiting the expression of circArid4b is provided, named si-circArid4b, and its nucleotide sequence is as follows:

[0053] si-circArid4b (SEQ ID NO. 6): 5'-GACAGUUCACCUGGUUCUATT-3'.

[0054] like Figure 5As shown, si-circArid4b could significantly reduce the expression of circArid4b (P<0.05).

[0055] The above si-circArid4b was prepared into an antibacterial preparation and used in the experiment of yellow catfish against Aeromonas veroni infection, as follows:

[0056] (1) GENEray was commissioned to synthesize si-circArid4b and negative control (si-NC). si-circArid4b was dissolved in deionized water (DEPC-treated water) to prepare an antibacterial preparation with a concentration of 20 μM; at the same time, a si-NC control solution (20 μM) was prepared.

[0057] (2) Healthy yellow catfish (about 0.8 g) were randomly divided into three groups, namely, Ct group, si-NC group and si-circArid4b group, and intraperitoneal injection was performed as follows:

[0058] Ct group: each fish was injected with 6.4 μL sterile PBS as a blank control;

[0059] si-NC group: each fish was injected with 6.4 μL of the above si-NC control solution as a negative control;

[0060] si-circArid4b group: Each fish was injected with 6.4 μL of the above antibacterial preparation solution as the experimental group.

[0061] (3) Hypoxia and Aeromonas vernix infection treatment: After grouping, each fish in each group was intraperitoneally injected with 8 μL of Aeromonas vernix suspension (dose 4×10 5 CFU / g fish), and nitrogen was added to the water until the dissolved oxygen value in the water was stabilized at 2.0 mg / L.

[0062] (4) After 6 h of treatment, the fish in each group were dissected and their liver tissues were aseptically isolated. The load of Aeromonas veroni in the liver tissues of each group was detected and compared by plate count method. The experimental data were statistically analyzed using GraphPad Prism (v9.5.1).

[0063] The results are as follows Figure 6 As shown in A and B, compared with the si-NC group, the amount of bacteria in the si-circArid4b group was significantly reduced (P < 0.05), indicating that si-circArid4b can significantly inhibit the infection of Aeromonas veronii to yellow catfish under hypoxic conditions.

[0064] Example 5

[0065] In this example, three treatment groups were set up, namely, Ct group, si-NC group and si-circArid4b group. The specific treatment method was the same as steps (2) and (3) in Example 4.

[0066] After 6 hours of treatment, the fish in each group were dissected and their liver tissues were aseptically separated. The total RNA of the liver tissues was extracted by Trizol method, and the extracted RNA was reverse transcribed into cDNA according to the instructions of cDNA reverse transcriptase reagent (Yisheng). Then, RT-qPCR was used to detect the expression of Arid4b in liver tissues. The RT-qPCR primer sequences for detecting the expression of Arid4b gene are as follows:

[0067] F (SEQ ID NO.7): 5'-CGACGGATTTGCTGAACGAC-3';

[0068] R (SEQ ID NO. 8): 5'-CAGGCTCCTCAAGAGTCTTCATT-3'.

[0069] The results are as follows Figure 7 As shown in the figure, there was no significant difference in Arid4b in the liver between the Ct group and the si-NC group, while Arid4b was significantly downregulated in the si-circArid4b group (P < 0.05), indicating that circArid4b can regulate its source gene Arid4b under hypoxic infection conditions.

[0070] Example 6

[0071] In this example, three treatment groups were set up, namely, Ct group, si-NC group and si-circArid4b group. The specific treatment method was the same as steps (2) and (3) in Example 4.

[0072] After 6 hours of treatment, the fish in each group were dissected and their liver tissues were aseptically separated. The total RNA of the liver tissue was extracted using the Trizol method, and the extracted RNA was reverse transcribed into cDNA according to the instructions of the cDNA reverse transcriptase reagent (Yishen). Then, RT-qPCR was used to detect the expression levels of genes related to the apoptosis pathway and inflammatory response in the liver tissue of the yellow catfish, including important functional genes that promote cell apoptosis, namely Traf1, Traf2, Gadd45b, and Nfkbiaa, as well as important functional genes related to the inflammatory pathway, among which TLR5, IL-1β, and Ddit4 can promote inflammatory response, while A2m can inhibit inflammatory response.

[0073] RT-qPCR results Figure 8 , 9As shown in the figure, there was no significant difference in the expression of the above eight genes in the liver between the Ct group and the si-NC group, while Traf1, Traf2, Gadd45b, Nfkbiaa, TLR5, IL-1β, and Ddit4 were significantly upregulated in the si-circArid4b group ( Figure 8 , Fig. 9 AC, P<0.05), A2m was significantly down-regulated ( Fig. 9 D, P < 0.05), indicating that reducing the expression of circArid4b under hypoxic infection conditions can promote cell apoptosis and inflammatory response in yellow catfish and improve the immune ability of yellow catfish.

[0074] The embodiments described above are only some embodiments of the present invention, rather than all embodiments. The detailed description of the embodiments of the present invention is not intended to limit the scope of the invention claimed for protection, but merely represents selected embodiments of the present invention. The scope of protection of the present invention shall be subject to the scope required by the claims. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work shall fall within the scope of protection of the present invention.

Claims

1. A fish circRNA, characterized in that: The fish is yellow catfish, the circRNA is named circArid4b, and its nucleotide sequence is shown in SEQ ID NO.

1.

2. Use of an agent for inhibiting the expression of fish circRNA as claimed in claim 1 in yellow catfish against Aeromonas veronii infection.

3. The use according to claim 2, characterized in that: The reagent is siRNA, and its nucleotide sequence is shown in SEQ ID NO.

6.

4. The use according to claim 3, characterized in that: The yellow catfish is placed in a hypoxic environment and the siRNA is injected into the abdominal cavity.

5. The use according to claim 4, characterized in that: The injection dose is 1.3 μg / g yellow catfish body weight.

6. Use of an agent for inhibiting the expression of fish circRNA as claimed in claim 1 in the preparation of an antibacterial preparation.

7. The use according to claim 6, characterized in that: The siRNA having a nucleotide sequence as shown in SEQ ID NO. 6 was dissolved in DEPC water and prepared at a concentration of 20 μM.

8. The use according to claim 6, characterized in that: The pathogenic bacteria of the antibacterial preparation is Aeromonas veronii.

9. An antibacterial preparation for yellow catfish against Aeromonas veronii, characterized in that: Comprising an agent for inhibiting the expression of the fish circRNA as claimed in claim 1.

10. Use of the reagent for inhibiting the expression of fish circRNA as claimed in claim 1 in improving the immune ability of yellow catfish.

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