A siRNA that targets and intervenes in the expression of the C1ql2 gene in grass carp and its application

Abstract

This invention discloses a siRNA for targeting and intervening in the expression of the grass carp C1ql2 gene and its application. The nucleotide sequence of the upstream primer of the siRNA is shown in SEQ ID NO.3, and the nucleotide sequence of the downstream primer of the siRNA is shown in SEQ ID NO.4. The siRNA for targeting and intervening in the expression of the grass carp C1ql2 gene of this invention, when injected into grass carp, achieves an inhibition efficiency of over 50% on the grass carp C1ql2 gene, demonstrating the advantage of high knockdown efficiency. Simultaneously, the knockdown of the grass carp C1ql2 gene downregulates the expression of immune-related genes such as CR1, C3, C5, C7, TNF-α, IL-1β, and IL-8 in the grass carp intestinal tissue, significantly alleviating intestinal inflammatory responses induced by bacterial LPS and MDP, and providing a basis for the development of intestinal immunomodulators for fish.

Description

Technical Field

[0001] This invention belongs to the field of molecular biology technology, specifically relating to a siRNA that targets and intervenes in the expression of the C1ql2 gene in grass carp and its application. Background Technology

[0002] In recent years, the rapid expansion of intensive aquaculture has exacerbated the pressure on farmed fish, leading to an increase in the incidence and severity of various fish diseases (Pulkkinen et al., 2010). Intensive aquaculture increases the spread of fish diseases, and intestinal inflammation is becoming a major cause of high morbidity and mortality in farmed fish, resulting in significant economic losses annually (Dias et al., 2016; Huo, Wang, Xiao, Yang, & Su, 2022). In freshwater fish, intestinal inflammation caused by bacteria (such as Aeromonas hydrophila) is particularly prevalent. -Safińska, 2018). Grass carp, belonging to the Cyprinidae family of freshwater fish, is one of the most farmed fish in China and even the world (Miao & Wang, 2020). It is popular among consumers due to its simple diet, delicious meat, rich nutrition, and rapid growth. However, under intensive aquaculture conditions, bacterial enteritis induced by bacterial infections leads to a significant decline in grass carp production. Therefore, research on the immune defense mechanisms of grass carp will contribute to the effective prevention and treatment of its diseases.

[0003] Immune defense depends on the host's ability to recognize pathogenic microorganisms. The complement component C1q, with its typical C1q globular (gC1q) domain, has been identified as an important recognition molecule in the classical complement pathway (Bally et al., 2019; Zong et al., 2019). Together with C1r and C1s, it constitutes the pathogen recognition unit (Kishore & Ghebrehiwet, 2020; Vadászi et al., 2022) and can activate the classical complement pathway. The activated classical complement pathway has multiple functions, including the destruction of target microorganisms and the clearance of pathogens (Wang, Zhang, & Wang, 2008).

[0004] Proteins containing a C1q domain (C1qDC) all share the same gC1q domain, and this class of proteins is known as the C1qDC family. C1qDCs have been identified in multiple species and possess immune functions such as bacterial recognition (Lv et al., 2018), antibacterial activity (L.Wang et al., 2017), activation of the complement pathway (Li, Zhang, & Sun, 2023), and retroviral clearance (Liang et al., 2022). C1q-like proteins (C1ql) belong to one class of C1qDCs. In 1999, Berube et al. discovered the human C1ql gene, which was identified as an aging-related gene (Bérubé et al., 1999). In mice, researchers have identified three C1ql proteins (C1ql1, C1ql2, and C1ql3), which are three secreted proteins that function in vivo (Iijima, Miura, Watanabe, & Yuzaki, 2010). In zebrafish, researchers have found that the C1ql gene is associated with apoptosis and hematopoiesis (Mei, Zhang, Li, Lin, & Gui, 2008). However, the immune function of the C1ql gene in bony fishes has not yet been fully investigated.

[0005] RNA interference (RNAi) is a widely used gene silencing technology in recent years. It uses a small interfering RNA (siRNA), a double-stranded RNA of 20-25 nucleotides, to easily and efficiently knock down the expression of specific genes in vivo, thereby affecting their corresponding biological functions (Y. Wang et al., 2023). Due to its specific gene knockdown effect, siRNA has become an important tool for studying gene function and drug targets. However, the application of RNAi technology to the C1ql2 gene in higher mammals and fish has not yet been reported. Therefore, obtaining an siRNA that targets and intervenes in the expression of the C1ql2 gene in grass carp is of great significance for achieving high-quality fish genetic breeding and can provide reference data for human disease prevention research. Summary of the Invention

[0006] The technical problem to be solved by the present invention is to overcome the shortcomings of the prior art and provide a siRNA with high knockdown efficiency for targeted intervention of grass carp C1ql2 gene expression and its application.

[0007] To solve the above-mentioned technical problems, the present invention adopts the following technical solution:

[0008] A siRNA that targets and intervenes in the expression of the C1ql2 gene in grass carp, wherein the nucleotide sequence of the upstream primer of the siRNA is shown in SEQ ID NO.3, and the nucleotide sequence of the downstream primer of the siRNA is shown in SEQ ID NO.4.

[0009] The above-mentioned siRNA is further improved, and the amino acid sequence of the grass carp C1ql2 gene is shown in SEQ ID NO.1, and the nucleotide sequence of the grass carp C1ql2 gene is shown in SEQ ID NO.2.

[0010] As a general technical concept, the present invention also provides the application of the above-mentioned siRNA that targets and intervenes in the expression of the grass carp C1ql2 gene in the specific knockdown of the grass carp C1ql2 gene expression.

[0011] Further improvements to the above application include the following steps: injecting siRNA that targets and intervenes in the expression of the grass carp C1ql2 gene into grass carp to achieve specific knockdown of the grass carp C1ql2 gene expression.

[0012] As a general technical concept, the present invention also provides an application of the above-mentioned siRNA that targets and intervenes in the expression of the grass carp C1ql2 gene in identifying the function of the grass carp C1ql2 gene.

[0013] Further improvements to the above application include the following steps: injecting siRNA that targets and intervenes in the expression of the grass carp C1ql2 gene into grass carp to specifically knock down the expression of the grass carp C1ql2 gene, thereby downregulating the expression of immune genes CR1, C3, C5, C7, TNF-α, IL-1β, and IL-8 in the intestinal tissue of grass carp.

[0014] As a general technical concept, the present invention also provides the application of the above-mentioned siRNA that targets and intervenes in the expression of the grass carp C1ql2 gene in the preparation of grass carp antibacterial drugs.

[0015] As a general technical concept, the present invention also provides the application of the above-mentioned siRNA that targets and intervenes in the expression of the grass carp C1ql2 gene in the preparation of grass carp immunomodulators.

[0016] Compared with the prior art, the advantages of the present invention are as follows:

[0017] This invention provides a siRNA that targets and intervenes in the expression of the grass carp C1ql2 gene. When injected into grass carp, the siRNA exhibits an inhibition efficiency of over 50% on the grass carp C1ql2 gene, demonstrating high knockdown efficiency. Simultaneously, the knockdown of the grass carp C1ql2 gene downregulates the expression of immune-related genes such as CR1, C3, C5, C7, TNF-α, IL-1β, and IL-8 in the grass carp intestinal tissue. It effectively inhibits the expression of key genes in the classical complement pathway and genes related to cellular inflammatory factors, thereby participating in the immune process induced by bacterial pathogens. This provides a theoretical basis for the development of immunomodulators for fish. The siRNA of this invention, which targets and intervenes in the expression of the grass carp C1ql2 gene, identifies gene function by specifically knocking down the expression of the grass carp C1ql2 gene. It can be applied to the genetic breeding of high-quality fish and provides reference data for research on human disease prevention, possessing significant economic and scientific value. Attached Figure Description

[0018] Figure 1 This is a diagram showing the knockdown effects of different types of siRNAs on the grass carp C1ql2 gene in Example 1 of the present invention.

[0019] Figure 2 This is a diagram showing the effect of targeted intervention on the expression of the C1ql2 gene in grass carp under LPS stimulation conditions in Example 2 of the present invention.

[0020] Figure 3 This is a diagram illustrating the effect of targeted intervention on the expression of the C1ql2 gene in grass carp under LPS stimulation on the CR1 immune gene in Example 2 of the present invention.

[0021] Figure 4 This is a diagram illustrating the effect of targeted intervention on the expression of the C1ql2 gene in grass carp under LPS stimulation on the C3 immune gene in Example 2 of the present invention.

[0022] Figure 5 This is a diagram illustrating the effect of targeted intervention on the expression of the C1ql2 gene in grass carp under LPS stimulation on the C5 immune gene in Example 2 of the present invention.

[0023] Figure 6 This is a diagram illustrating the effect of targeted intervention on the expression of the C1ql2 gene in grass carp under LPS stimulation on the C7 immune gene in Example 2 of the present invention.

[0024] Figure 7 This is a diagram illustrating the effect of targeted intervention on the expression of the C1ql2 gene in grass carp under LPS stimulation on the TNF-α immune gene in Example 2 of the present invention.

[0025] Figure 8 This is a diagram illustrating the effect of targeted intervention on the expression of the C1ql2 gene in grass carp under LPS stimulation on the IL-1β immune gene in Example 2 of the present invention.

[0026] Figure 9 This is a diagram illustrating the effect of targeted intervention on the expression of the C1ql2 gene in grass carp under LPS stimulation on the IL-8 immune gene in Example 2 of the present invention.

[0027] Figure 10 This is a diagram showing the effect of targeted intervention on the expression of the C1ql2 gene in grass carp under MDP stimulation conditions in Example 3 of the present invention.

[0028] Figure 11 This is a diagram illustrating the effect of targeted intervention on the expression of the C1ql2 gene in grass carp under MDP stimulation conditions on the CR1 immune gene in Example 3 of the present invention.

[0029] Figure 12 This is a diagram illustrating the effect of targeted intervention on the expression of the C1ql2 gene in grass carp under MDP stimulation conditions on the C3 immune gene in Example 3 of the present invention.

[0030] Figure 13 This is a diagram illustrating the effect of targeted intervention on the expression of the C1ql2 gene in grass carp under MDP stimulation in Example 3 of the present invention on the C5 immune gene.

[0031] Figure 14 This is a diagram illustrating the effect of targeted intervention on the expression of the C1ql2 gene in grass carp under MDP stimulation in Example 3 of the present invention on the C7 immune gene.

[0032] Figure 15 This is a diagram illustrating the effect of targeted intervention on the expression of the C1ql2 gene in grass carp under MDP stimulation on the TNF-α immune gene in Example 3 of the present invention.

[0033] Figure 16 This is a diagram illustrating the effect of targeted intervention on the expression of the C1ql2 gene in grass carp under MDP stimulation on the IL-1β immune gene in Example 3 of the present invention.

[0034] Figure 17 This is a diagram illustrating the effect of targeted intervention on the expression of the C1ql2 gene in grass carp under MDP stimulation on the IL-8 immune gene in Example 3 of the present invention. Detailed Implementation

[0035] The present invention will be further described below with reference to the accompanying drawings and specific preferred embodiments, but this does not limit the scope of protection of the present invention. All materials and instruments used in the following embodiments are commercially available.

[0036] Example 1:

[0037] A method for obtaining siRNA that targets and intervenes in the expression of the C1ql2 gene in grass carp according to the present invention includes the following steps:

[0038] (1) Template preparation

[0039] RNA was extracted from grass carp using the Trizol method, and then processed according to PrimeScript. TM The II First Strand cDNA Synthesis Kit (Takara) procedure involves reverse transcription to obtain a cDNA template.

[0040] (2) PCR amplification

[0041] Using primers C1ql2-F and C1ql2-R, cDNA was used as a template for PCR amplification to obtain the ORF sequence of C1ql2. The nucleotide sequence of primer C1ql2-F is 5'-TCAGGTAAAGATGTCCT-3'; the nucleotide sequence of primer C1ql2-R is 5'-AGACTCAGTAATGCCAATAA-3'. The PCR amplification conditions were: 98℃ pre-denaturation for 2 min; 98℃ denaturation for 10 s, 40℃ annealing for 30 s, 72℃ extension for 2 min 30 s, for 30 cycles; and a final extension at 72℃ for 5 min.

[0042] The PCR products obtained above were detected by electrophoresis, and after gel extraction using an agarose gel extraction kit (Aikerui Biotechnology), they were ligated into the pMD18-T vector (Takara) and transformed into E. coli DH5α (Takara). After PCR detection of positive clones, the samples were sent to Platinum Biotechnology Co., Ltd. for sequencing. Finally, the complete ORF sequence of C1ql2 was obtained. The nucleotide sequence of the grass carp C1ql2 gene is shown in SEQ ID NO.2, and the corresponding amino acid sequence is SEQ ID NO.1.

[0043] The amino acid sequence of the grass carp C1ql2 gene is shown in SEQ ID NO.1, specifically:

[0044]

[0045]

[0046] The nucleotide sequence of the grass carp C1ql2 gene is shown in SEQ ID NO.2, specifically as follows:

[0047]

[0048] (3) siRNA screening

[0049] Grass carp (5g) were randomly divided into 4 groups of 3 fish per group and placed in an indoor recirculating aquaculture system. Different types of siRNA (1μg / g) were injected intraperitoneally into the grass carp: specific siRNA-1, specific siRNA-2, specific siRNA-3, and negative control siRNA-NC. Intestinal tissue samples were collected 24 and 48 hours after injection. The pair of siRNAs with the best interference effect was screened by qRT-PCR. The results are as follows: Figure 1 As shown, siRNA-1 exhibited the best interference effect, specifically targeting the expression of the grass carp C1ql2 gene. The tt nucleotide added to the 3' end of the siRNA targeting the grass carp C1ql2 gene expression serves two purposes: stabilization and suspension for easy unwinding. Table 1 shows information on different types of siRNA, synthesized by Shanghai Jiying Biochemical.

[0050] Table 1 Information on different types of siRNA

[0051]

[0052]

[0053] Figure 1 This is a diagram showing the knockdown effects of different types of siRNAs on the grass carp C1ql2 gene in Example 1 of the present invention. From... Figure 1 It can be seen that siRNA-1 knocked down the grass carp C1ql2 gene by more than 50% after 48 hours. Therefore, siRNA-1 had the best interference effect, and siRNA-1 was selected as the siRNA to target the expression of grass carp C1ql2 gene to complete subsequent experiments.

[0054] Example 2:

[0055] The validation experiment of lipopolysaccharide (LPS) in grass carp C1ql2 gene knockdown included the following steps:

[0056] (1) Grass carp were divided into three groups: NC group, NC+LPS group and siRNA-1+LPS group. NC was siRNA-NC. Three grass carp were in each group. The drug was administered according to body weight. The best time point for siRNA-1 knockdown was 48 hours before LPS was injected to complete the subsequent experiment. The concentration of LPS in the experiment was 10 μg / mL.

[0057] (2) Then, grass carp in each group were anesthetized with 20 mg / L MS-222 (Sigma-Aldrich). Intestinal tissue was collected 24 h later for qRT-PCR analysis. The results are as follows: Figures 2-9 As shown in Table 2, the fluorescent quantitative primers are as follows.

[0058] Table 2 Information on quantitative fluorescence primers

[0059]

[0060] Figure 2 This is a diagram showing the effect of targeted intervention on the expression of the C1ql2 gene in grass carp under LPS stimulation conditions in Example 2 of the present invention. Figure 3 This is a diagram illustrating the effect of targeted intervention on the expression of the C1ql2 gene in grass carp under LPS stimulation on the CR1 immune gene in Example 2 of the present invention. Figure 4 This is a diagram illustrating the effect of targeted intervention on the expression of the C1ql2 gene in grass carp under LPS stimulation on the C3 immune gene in Example 2 of the present invention. Figure 5 This is a diagram illustrating the effect of targeted intervention on the expression of the C1ql2 gene in grass carp under LPS stimulation on the C5 immune gene in Example 2 of the present invention. Figure 6 This is a diagram illustrating the effect of targeted intervention on the expression of the C1ql2 gene in grass carp under LPS stimulation on the C7 immune gene in Example 2 of the present invention. Figure 7 This is a diagram illustrating the effect of targeted intervention on the expression of the C1ql2 gene in grass carp under LPS stimulation on the TNF-α immune gene in Example 2 of the present invention. Figure 8 This is a diagram illustrating the effect of targeted intervention on the expression of the C1ql2 gene in grass carp under LPS stimulation on the IL-1β immune gene in Example 2 of the present invention. Figure 9 This is a graph illustrating the effect of LPS stimulation on the expression of the C1ql2 gene in grass carp on the IL-8 immune gene, as shown in Example 2 of this invention. Figure 2 It can be seen that after LPS treatment, compared with the NC group, the expression of C1ql2 gene was upregulated in the NC+LPS group, and the expression of C1ql2 gene was the lowest in the siRNA-1+LPS group, indicating that siRNA-1 can effectively reduce the expression of C1ql2 gene. Figure 3 It can be seen that the CR1 gene expression level in the siRNA-1+LPS group is basically the same as that in the NC group, and both groups are lower than the NC+LPS group. Figure 4 It can be seen that the C3 gene expression level in the siRNA-1+LPS group and the NC group is lower than that in the NC+LPS group, indicating that knocking down the C1ql2 gene can affect the C3 gene expression. Figure 5 It can be seen that the C5 gene expression level in the siRNA-1+LPS group and the NC group is lower than that in the NC+LPS group, indicating that knocking down the C1ql2 gene can affect the C5 gene expression. Figure 6 It can be seen that the C7 gene expression level in the siRNA-1+LPS group and the NC group is lower than that in the NC+LPS group, indicating that knocking down the C1ql2 gene can affect the C7 gene expression. Figure 7It can be seen that the TNF-α gene expression level in the siRNA-1+LPS group and the NC group is lower than that in the NC+LPS group, indicating that the knockdown of the C1ql2 gene can affect the expression of the TNF-α gene. Figure 8 It can be seen that the IL-1β gene expression level in the siRNA-1+LPS group and the NC group is lower than that in the NC+LPS group, indicating that the knockdown of the C1ql2 gene can affect the expression of the IL-1β gene. Figure 9 It can be seen that the IL-8 gene expression level in the siRNA-1+LPS group and the NC group is lower than that in the NC+LPS group, indicating that the knockdown of the C1ql2 gene can affect the expression of the IL-8 gene.

[0061] Combination Figures 2-9 It can be seen that siRNA targeting the expression of the grass carp C1ql2 gene can specifically knock down the expression of the grass carp C1ql2 gene. At the same time, the knockdown of the grass carp C1ql2 gene can downregulate the expression of immune-related genes such as CR1, C3, C5, C7, TNF-α, IL-1β, and IL-8 in the grass carp intestinal tissue. That is, the knockdown of the C1ql2 gene will inhibit the expression of key genes and inflammatory cytokines in the classical complement pathway. This suggests that the grass carp C1ql2 gene may be involved in the signal transduction of the classical complement pathway and participate in bacterial intestinal immune response.

[0062] Example 3:

[0063] The validation experiment for in vivo muramyl dipeptide (MDP) knockdown of the grass carp C1ql2 gene includes the following steps:

[0064] (1) Grass carp were divided into three groups: NC group, NC+MDP group and siRNA-1+MDP group. NC was siRNA-NC. Three grass carp were in each group. The drug was administered according to body weight. The time point with the best siRNA-1 knockdown effect was selected at 48h before MDP was injected to complete the subsequent experiment. The concentration of MDP in the experiment was 10μg / mL.

[0065] (2) Then, grass carp in each group were anesthetized with 20 mg / L MS-222 (Sigma-Aldrich). Intestinal tissue was collected 24 h later for qRT-PCR analysis. The results are as follows: Figures 10-17 As shown.

[0066] Figure 10 This is a diagram showing the effect of targeted intervention on the expression of the C1ql2 gene in grass carp under MDP stimulation conditions in Example 3 of the present invention. Figure 11 This is a diagram showing the effect of targeted intervention on the expression of the C1ql2 gene in grass carp under MDP stimulation in Example 3 of the present invention on the CR1 gene. Figure 12 This is a diagram illustrating the effect of targeted intervention on the expression of the C1ql2 gene in grass carp under MDP stimulation conditions on the C3 immune gene in Example 3 of the present invention. Figure 13 This is a diagram illustrating the effect of targeted intervention on the expression of the C1ql2 gene in grass carp under MDP stimulation in Example 3 of the present invention on the C5 immune gene. Figure 14 This is a diagram illustrating the effect of targeted intervention on the expression of the C1ql2 gene in grass carp under MDP stimulation in Example 3 of the present invention on the C7 immune gene. Figure 15 This is a diagram illustrating the effect of targeted intervention on the expression of the C1ql2 gene in grass carp under MDP stimulation on the TNF-α immune gene in Example 3 of the present invention. Figure 16 This is a diagram illustrating the effect of targeted intervention on the expression of the C1ql2 gene in grass carp under MDP stimulation on the IL-1β immune gene in Example 3 of the present invention. Figure 17 This is a diagram illustrating the effect of targeted intervention on the expression of the C1ql2 gene in grass carp under MDP stimulation on the IL-8 immune gene in Example 3 of the present invention. Figure 10 It can be seen that the C1ql2 gene expression in the siRNA-1+MDP group was lower than that in the NC+MDP group and the NC group, and the difference was significant. This indicates that siRNA-1 can specifically knock down the C1ql2 gene expression level. Figure 11 It can be seen that the expression of the CR1 gene in the siRNA-1+MDP group was lower than that in the NC+MDP group and the NC group. Figure 12 It can be seen that the expression of C3 gene in the siRNA-1+MDP group was lower than that in the NC+MDP group and the NC group, indicating that knocking down the C1ql2 gene can affect the expression of C3 gene. Figure 13 It can be seen that the C5 gene expression in the siRNA-1+MDP group was lower than that in the NC+MDP group and the NC group, indicating that knocking down the C1ql2 gene can affect the C5 gene expression. Figure 14 It can be seen that the C7 gene expression in the siRNA-1+MDP group was lower than that in the NC+MDP group and the NC group, indicating that knocking down the C1ql2 gene can affect the C7 gene expression. Figure 15 It can be seen that the expression of TNF-α gene in the siRNA-1+MDP group is lower than that in the NC+MDP group and the NC group, indicating that the knockdown of C1ql2 gene can affect the expression of TNF-α gene. Figure 16 It can be seen that the IL-1β gene expression in the siRNA-1+MDP group was lower than that in the NC+MDP group and the NC group, indicating that the knockdown of the C1ql2 gene can affect the expression of the IL-1β gene. Figure 17 It can be seen that the IL-8 gene expression levels in the siRNA-1+MDP group and the NC group are basically the same, but both are lower than those in the NC+MDP group, indicating that the knockdown of the C1ql2 gene can affect the expression of the IL-8 gene.

[0067] Combination Figures 15 to 17 The results indicate that specifically knocking down the C1ql2 gene can, to some extent, help reduce intestinal inflammatory damage in grass carp. Figures 10-17It can be seen that siRNA targeting the expression of the C1ql2 gene in grass carp can effectively intervene in the expression levels of the C1ql2 gene, CR1 gene, key genes of the complement pathway (C3, C5, C7), and inflammatory cytokines (TNF-α, IL-1β, IL-8) in grass carp, significantly alleviating the intestinal inflammatory response induced by MDP. This provides a molecular target for the treatment of bacterial enteritis in grass carp and provides a basis for the development of broad-spectrum antibacterial drugs and the screening of immunomodulators.

[0068] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make many possible variations and modifications to the technical solutions of the present invention using the methods and techniques disclosed above, or modify them into equivalent embodiments with equivalent changes, without departing from the spirit and technical essence of the present invention. Therefore, any simple modifications, equivalent substitutions, equivalent changes, and modifications made to the above embodiments based on the technical essence of the present invention without departing from the content of the technical solutions of the present invention shall still fall within the protection scope of the technical solutions of the present invention.

Claims

1. The application of a siRNA that targets and intervenes in the expression of the grass carp C1ql2 gene in the preparation of a drug for treating intestinal inflammation in grass carp, wherein the nucleotide sequence of the upstream primer of the siRNA is shown in SEQ ID NO.3, and the nucleotide sequence of the downstream primer of the siRNA is shown in SEQ ID NO.

4.

2. The application according to claim 1, characterized in that, The amino acid sequence of the grass carp C1ql2 gene is shown in SEQ ID NO.1, and the nucleotide sequence of the grass carp C1ql2 gene is shown in SEQ ID NO.2.

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