Pelteobagrus fulvidraco circRNA and application thereof

Abstract

The invention discloses a pelteobagrus fulvidraco circRNA (Ribonucleic Acid) and application thereof, the circRNA is named as circPIK3C2A, and the nucleotide sequence of the circRNA is shown as SEQ ID NO.1. The pelteobagrus fulvidraco circRNA is named as circPIK3C2A. The circPIK3C2A provided by the invention is highly in positive correlation with the infection duration of aeromonas veronii. After the synthesized circPIK3C2A interferent (si-circPIK3C2A) is transfected to liver cells of the pelteobagrus fulvidraco, the expression of autophagy genes PIK3C2A and DDIT4 can be obviously promoted, the expression of apoptosis genes CASP3 and TRAF2 is inhibited, and the regulation and control effects of the circPIK3C2A on the autophagy, apoptosis and antibacterial processes of the pelteobagrus fulvidraco under bacterial infection are effectively proved.

Description

Technical Field

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

[0002] Yellow catfish (Pelteobagrus fulvidraco) is an important freshwater specialty economic fish in my country with high nutritional value and no intramuscular spines. With the vigorous development of yellow catfish aquaculture, its high-density intensive aquaculture model has caused frequent outbreaks of bacterial diseases. On the one hand, the rapid deterioration of water quality has created an environment that is not conducive to the healthy growth of fish, and on the other hand, the abuse of drugs by farmers has also aggravated the development of the disease. Among them, Aeromonas veronii, as the main pathogen of yellow catfish, is known for its high infectivity and rapid incidence. It can infect humans, mammals and aquatic organisms across species, which not only threatens the survival of yellow catfish, but also poses a potential risk to human health. In recent years, yellow catfish aquaculture has frequently encountered large-scale concentrated outbreaks of bacterial diseases, resulting in severe damage to most fish ponds in the country, farmers have no harvest, and the economic losses are huge and difficult to estimate, which seriously hinders the sustainable development of the yellow catfish industry. Therefore, it is of great significance to study the innate immune response of yellow catfish in response to bacterial infection.

[0003] CircRNA is a new type of non-coding RNA molecule with a closed-loop structure formed by reverse splicing of the source gene, which is not easily degraded and widely exists in eukaryotes. It has the characteristics of organization, timing, disease specificity and high conservation among species. A large number of studies have shown that circRNA plays a key regulatory role in biological processes such as reproductive development, immunity and stress response. It can act as a competitive endogenous RNA (ceRNA) to adsorb specific miRNA, directly participate in the protein translation process, and form a complex interaction network with RNA binding proteins (RBPs) or other non-coding RNA. In recent years, circRNA therapy has attracted much attention due to its excellent stability and has shown great potential in the treatment of various diseases in mammals, especially in the fields of infectious diseases, tumors, and rare diseases, providing new hope for protein replacement therapy and the development of new vaccines. However, the application of circRNA in the prevention and control of diseases in lower vertebrates is still limited, and its involvement in the innate immune response mechanism of fish in response to bacterial infection remains to be studied. Summary of the invention

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

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

[0006] A yellow catfish circRNA, the circRNA is named circPIK3C2A, and its nucleotide sequence is shown as SEQ ID NO.1.

[0007] Use of the reagent for inhibiting the expression of the above-mentioned yellow catfish circRNA in the preparation of a drug for regulating yellow catfish's resistance to Aeromonas veronii infection.

[0008] Preferably, the reagent is si-circPIK3C2A, whose nucleotide sequence is shown in SEQ ID NO.8.

[0009] Preferably, si-circPIK3C2A is transfected into yellow catfish hepatocytes at a transfection concentration of 100 nM.

[0010] Preferably, the regulation is at least one of the following (1)-(3):

[0011] (1) Regulate the expression of autophagy-related source gene PIK3C2A and autophagy gene DDIT4;

[0012] (2) regulating the expression of apoptosis genes CASP3 and TRAF2;

[0013] (3) Regulate the number of Aeromonas veronii in the hepatocytes of yellow catfish.

[0014] Application of the above-mentioned yellow catfish circRNA in the preparation of antibacterial preparations.

[0015] Preferably, the pathogenic bacteria of the antibacterial preparation is Aeromonas veronii.

[0016] An antibacterial preparation for yellow catfish to resist Aeromonas veronii comprises the yellow catfish circRNA.

[0017] The above-mentioned yellow catfish circRNA, or the use of the reagent for inhibiting the expression of the above-mentioned yellow catfish circRNA in the cultivation of yellow catfish varieties resistant to Aeromonas veronis, wherein the reagent is si-circPIK3C2A, and its nucleotide sequence is shown in SEQ ID NO.8.

[0018] Preferably, the ability of yellow catfish to resist Aeromonas veroni infection is improved by increasing the expression level of the above-mentioned yellow catfish circRNA.

[0019] The beneficial effects of the present invention are as follows:

[0020] The circPIK3C2A provided by the present invention is highly positively correlated with the infection duration of Aeromonas veronii. After the synthetic circPIK3C2A interferor (si-circPIK3C2A) is transfected into yellow catfish hepatocytes, the expression of autophagy genes PIK3C2A and DDIT4 can be significantly promoted, and the expression of apoptosis genes CASP3 and TRAF2 can be inhibited, effectively proving the regulatory effect of circPIK3C2A on autophagy, apoptosis and antibacterial processes of yellow catfish under bacterial infection. The present invention proves by experiments that si-circPIK3C2A can promote yellow catfish to be infected with Aeromonas veronii, so it is reasonable to speculate that circPIK3C2A has corresponding antibacterial effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 The expression level of circPIK3C2A in yellow catfish after being infected with Aeromonas vermiformis for different time periods in Example 1;

[0022] Figure 2 The identification results of circPIK3C2A in Example 2: A is the PCR amplification of circPIK3C2A using convergent and divergent primers; B is the Sanger sequencing peak diagram of the RT-PCR product; C is the RT-PCR detection of circPIK3C2A and PIK3C2A before and after RNAse R digestion;

[0023] Figure 3 The effects of circPIK3C2A interferors (si-circPIK3C2A) in Example 3 on the antibacterial ability of yellow catfish: A is a schematic diagram of the synthesis of three circPIK3C2A interferors; B is the knockdown effect of si-circPIK3C2A on circPIK3C2A under Aeromonas veronii infection conditions; C is the plate culture of each treatment group; D is the colony statistics of each treatment group;

[0024] Figure 4 The expression changes of the source gene PIK3C2A (A) and the autophagy gene DDIT4 (B) after the expression of circPIK3C2A was reduced under the infection condition of Aeromonas veronii in Example 4;

[0025] Figure 5 The expression changes of apoptosis-related genes after reducing circPIK3C2A expression under Aeromonas vernix infection conditions in Example 5: A is the CASP3 gene, and B is the TRAF2 gene. DETAILED DESCRIPTION

[0026] The present invention is further described in detail below with reference to the accompanying drawings and specific embodiments.

[0027] 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.

[0028] The experimental animals used in the following examples were yellow catfish, from Nanjing Fisheries Research Institute, weighing 8.74±1.88g, uniform in size, and healthy. First, they were raised in a circulating water tank with a water temperature of 27±1.0°C and a pH of 7.50±0.20 for two weeks, during which aeration maintained normal oxygen supply, and commercial feed (Jiaji Feed Co., Ltd., Zhenjiang) was fed twice a day. Feeding was stopped 24h before the experiment.

[0029] Example 1 Discovery of circRNA (circPIK3C2A)

[0030] Yellow catfish of uniform size (8.74±1.88g) were randomly sorted into experimental water tanks. The infected group yellow catfish were intraperitoneally injected with 100μL Aeromonas vernix suspension, and the control (Ct) group yellow catfish were injected with the same amount of sterile PBS buffer under the same conditions. The infection of yellow catfish was observed in time after injection, and MS-222 was used for anesthesia at 2h (2hpi), 4h (hpi), 6h (6hpi), 8h (8hpi), 12h (12hpi) and 24h (24hpi) after infection. The liver tissue samples were collected for subsequent sequencing and experiments. According to the standards of the Illumina HiSeq2500 sequencing platform, the liver tissues of the control group, the 6h infection group and the 12h infection group were used to construct sequencing libraries and high-throughput sequencing. After obtaining high-quality sequences (HQT) through Fastp (v0.12.4) and Bowtie 2 (v2.2.8), these HQT sequences were aligned with the reference genome of yellow catfish using Tophat 2 (v2.0.3.12), the sequences that were continuously aligned with the genome were removed, and the unmapped sequences with splicing sites were retained. Finally, circRNAs were identified using the program find_circ.

[0031] Among the identified circRNAs, we found a new circRNA formed by alternative splicing of the autophagy gene PIK3C2A (Phosphatidylinositol-4-Phosphate 3-Kinase Catalytic Subunit Type 2Alpha), whose expression was significantly upregulated 12 hours after Aeromonas vernix infection (P < 0.05), and we named it circPIK3C2A. The expression of circPIK3C2A was verified by RT-qPCR, and the RT-qPCR reaction system is shown in Table 1 below:

[0032] Table 1 RT-qPCR reaction system

[0033]

[0034] 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.

[0035] The results are as follows Figure 1 As shown in the figure, 6 hours after Aeromonas vernix infection, the expression of circPIK3C2A was significantly upregulated (P < 0.05). Moreover, with the extension of infection time, the expression of circPIK3C2A was further upregulated, and was highly positively correlated with the infection duration of Aeromonas vernix (within 24 hours) (r > 0.95). Therefore, we selected circPIK3C2A for the next study.

[0036] Example 2 Identification of circRNA (circPIK3C2A)

[0037] By comparing the yellow catfish genome, it was found that the source gene PIK3C2A of circPIK3C2A was located on chromosome 13, and circPIK3C2A was formed by head-to-tail reverse splicing of exons 2, 3, and 4 of the PIK3C2A gene. The length of the mature sequence after splicing was 1359bp, and its nucleotide sequence was shown in SEQ ID NO.1. The genomic DNA (gDNA) and total RNA (Total RNA) of the liver samples collected in Example 1 were extracted respectively, and the extracted RNA was reverse transcribed into cDNA according to the instructions of the cDNA reversal reagent. Then, using gDNA and cDNA as templates, convergent primers and divergent primers were used to amplify circPIK3C2A respectively. The PCR reaction system is shown in Table 2, and the primer sequences are as follows:

[0038] Convergent primers circPIK3C2A-convergent:

[0039] F (SEQ ID NO.2): 5'-TTCAGCCCGTATCCGTTACA-3';

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

[0041] Divergent primers circPIK3C2A-divergent:

[0042] F (SEQ ID NO.4): 5'-AGACGGTAGTTGCAGTGTGA-3';

[0043] R (SEQ ID NO. 5): 5'-CGCAAAGCCTCATCCTTACC-3'.

[0044] Table 2 PCR reaction system

[0045]

[0046] The results are as follows Figure 2 As shown in Figure 2B, when divergent primers were used, only cDNA could amplify circPIK3C2A (expected fragment length was 243 bp), while no amplification product was observed from gDNA.

[0047] The RT-PCR products of circPIK3C2A were subjected to Sanger sequencing, as Figure 2 As shown in A, the product sequence is single and successfully crosses the splicing site of circPIK3C2A.

[0048] After verifying its circular structure, we further verified the stability of circPIK3C2A. TotalRNA was digested with RNAse R in a 37°C water bath for 40 minutes, and then transferred to a 70°C water bath and heated for 10 minutes to inactivate RNAse R. Subsequently, RT-PCR technology was used to detect mRNA and circRNA using cDNA as a template, using the convergent primers of PIK3C2A and the divergent primers of circPIK3C2A, respectively. The RNAse R digestion system is shown in Table 3, the divergent primer sequences of circPIK3C2A are shown in SEQ ID NO.4 and SEQ ID NO.5, and the convergent primers of PIK3C2A PIK3C2A-convergent are as follows:

[0049] F (SEQ ID NO.6): 5'-CCTCGGAAAGTTTGAAGGCAT-3';

[0050] R (SEQ ID NO. 7): 5'-ACCTCTAGGGAAGTGGCTGT-3'.

[0051] Table 3 RNAse R digestion system

[0052]

[0053] The results are as follows Figure 2As shown in C, PIK3C2A mRNA has a band in RNase R (-) and no band in RNase R (+), while circRNA has bands in both RNase R (-) and RNase R (+) samples, indicating that linear RNA is completely digested, while circPIK3C2A is resistant to RNAse R digestion.

[0054] The experimental results of this example demonstrate that the identified circPIK3C2A is a stable circular RNA.

[0055] Example 3 Effects of circPIK3C2A interferors on the antibacterial ability of yellow catfish

[0056] In order to explore the role of circPIK3C2A in the antibacterial immune response of yellow catfish, we designed interferors (siRNA) that inhibited the expression of circPIK3C2A and used them in the yellow catfish anti-Aeromonas veronii infection experiment as follows:

[0057] 1. GENEray was commissioned to synthesize three circPIK3C2A interferors, namely si-circPIK3C2A-1, si-circPIK3C2A-2 and si-circPIK3C2A-3 ( Figure 3 A), and a negative control (si-NC) was synthesized at the same time. After the above three interferors (transfection concentration was 100nM) were transfected into yellow catfish hepatocytes for 24h, the cells were infected with Aeromonas vermiformis for 1h at an MOI (multiplicity of infection) of 3. Subsequently, the total RNA of the cells was extracted using the Trizol method and reversely transcribed into cDNA, and the expression of circPIK3C2A in hepatocytes was detected by RT-qPCR.

[0058] The results are as follows Figure 3 As shown in B, both si-circPIK3C2A-1 and si-PIK3C2A-3 can significantly inhibit the expression of circPIK3C2A (P < 0.05). Since si-circPIK3C2A-3 has a higher knockdown efficiency, we used si-circPIK3C2A-3 in subsequent experiments and named it si-circPIK3C2A. The specific sequence is as follows:

[0059] si-circPIK3C2A (SEQ ID NO. 8): 5'-AUUUGACAGUUCACCUGGUUCTT-3'.

[0060] 2. Set up three treatment groups as follows:

[0061] (1) Ct group: no transfection, serving as blank control;

[0062] (2) si-NC group: transfected with si-NC, used as negative control;

[0063] (3) si-circ group: transfected with si-circPIK3C2A, used as the experimental group.

[0064] The transfection concentration of the si-NC group and the si-circ group was 100 nM. 24 hours after transfection, the cells were infected with Aeromonas veronii for 1 hour at an MOI of 3. The cells were then collected, lysed using Triton x-100, and plated for culture. The plate colony counting method was used to compare the Aeromonas veronii load in each group of cells.

[0065] The results are as follows Figure 3 As shown in C and D, compared with the Ct group and si-NC group, the amount of bacteria in the si-circ group was significantly increased (P < 0.05), that is, after reducing the expression of circPIK3C2A, the antibacterial immune response of yellow catfish was inhibited, indicating that circPIK3C2A plays an important regulatory role in the antibacterial infection of yellow catfish.

[0066] Example 4 Regulatory effects of circPIK3C2A on its source genes and autophagy

[0067] In this example, three treatment groups were set up, namely, Ct group, si-NC group and si-circ group, and the specific treatment method was the same as step 2 in Example 3.

[0068] After 24 hours of transfection, the cells were infected with Aeromonas vermifuge at an MOI of 3 for 1 hour and then the cells were collected. The total RNA of the cells was extracted using the Trizol method and reversely transcribed into cDNA. Then, RT-qPCR was used to detect the expression of PIK3C2A, the source gene of circPIK3C2A, in hepatocytes. The RT-qPCR primer sequences for detecting the expression of the PIK3C2A gene are shown in SEQ ID NO.6 and SEQ ID NO.7.

[0069] The results are as follows Figure 4 As shown in A, there was no significant difference in PIK3C2A between the Ct group and the si-NC group, while PIK3C2A was significantly upregulated in the si-circ group (P < 0.05), indicating that under the condition of Aeromonas vernix infection, circPIK3C2A can negatively regulate the expression of its source gene PIK3C2A.

[0070] Since PIK3C2A, the source gene of circPIK3C2A, is related to autophagy, we used the same method to detect the expression of the autophagy gene DDIT4 in hepatocytes after knocking down circPIK3C2A. The RT-qPCR primer sequences for detecting DDIT4 gene expression are as follows:

[0071] F (SEQ ID NO.9): 5'-CGGTTTACTGCCACACGATT-3';

[0072] R (SEQ ID NO. 10): 5'-CAGGAAGAGATACGGAACCAGT-3'.

[0073] The results are as follows Figure 4 As shown in B, there was no significant difference in DDIT4 between the Ct group and the si-NC group, while DDIT4 was significantly upregulated in the si-circ group (P < 0.05), indicating that circPIK3C2A is involved in the regulation of autophagy in yellow catfish cells under Aeromonas veronii infection.

[0074] Example 5 Regulatory effect of circPIK3C2A on cell apoptosis

[0075] In this example, three treatment groups were set up, namely, Ct group, si-NC group and si-circ group, and the specific treatment method was the same as step 2 in Example 3.

[0076] 24 h after transfection, cells were infected with Aeromonas vermifuge at an MOI of 3 and collected after 1 h. Total cellular RNA was extracted using the Trizol method and reverse transcribed into cDNA. RT-qPCR was then used to detect the expression levels of apoptosis genes CASP3 and TRAF2 in hepatocytes.

[0077] The RT-qPCR primer sequences for detecting CASP3 gene expression are as follows:

[0078] F (SEQ ID NO. 11): 5'-TGGAGTCCAAAACGGTGACT-3';

[0079] R (SEQ ID NO. 12): 5'-CCCAATGTTGGGGAAGTCCA-3'.

[0080] The RT-qPCR primer sequences for detecting TRAF2 gene expression are as follows:

[0081] F (SEQ ID NO.13): 5'-CCAAGAAGAGGCAAGACGCT-3';

[0082] R (SEQ ID NO. 14): 5'-GCATCAGGGTGACCTTCTGG-3'.

[0083] The results are as follows Figure 5 As shown in A and B, there was no significant difference in the expression levels of CASP3 and TRAF2 in the Ct group and the si-NC group, but they were significantly downregulated in the si-circ group (P < 0.05), indicating that circPIK3C2A can regulate the apoptosis pathway of yellow catfish under Aeromonas veronii infection.

[0084] 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 yellow catfish circRNA, characterized in that The circRNA is named circPIK3C2A, and its nucleotide sequence is shown in SEQ ID NO.

1.

2. Use of an agent for inhibiting the expression of the yellow catfish circRNA as claimed in claim 1 in the preparation of a drug for regulating the yellow catfish's resistance to Aeromonas veronii infection.

3. The use according to claim 2, characterized in that: The reagent is si-circPIK3C2A, and its nucleotide sequence is shown in SEQ ID NO.

8.

4. The use according to claim 3, characterized in that: The si-circPIK3C2A was transfected into yellow catfish hepatocytes at a transfection concentration of 100 nM.

5. The use according to claim 2, characterized in that: The regulation is at least one of the following (1)-(3): (1) Regulate the expression of autophagy-related source gene PIK3C2A and autophagy gene DDIT4; (2) regulating the expression of apoptosis genes CASP3 and TRAF2; (3) Regulate the number of Aeromonas veronii in the hepatocytes of yellow catfish.

6. Use of the Pelteobagrus fulvidraco circRNA as claimed in claim 1 in the preparation of antibacterial preparations.

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

8. An antibacterial preparation for yellow catfish against Aeromonas veronii, characterized in that: Including the yellow catfish circRNA as described in claim 1.

9. Use of the yellow catfish circRNA according to claim 1, or an agent for inhibiting the expression of the yellow catfish circRNA according to claim 1, in the cultivation of yellow catfish varieties resistant to Aeromonas veronis, characterized in that: The reagent is si-circPIK3C2A, and its nucleotide sequence is shown in SEQ ID NO.

8.

10. The use according to claim 9, characterized in that: By increasing the expression level of the yellow catfish circRNA as claimed in claim 1, the ability of yellow catfish to resist Aeromonas veroni infection is improved.

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