SiRNA screening system for intervening chlamys farreri chain metabolism and application of siRNA screening system

Abstract

The invention belongs to the technical field of biology, and particularly relates to a siRNA screening system for intervening chlamys farreri chain metabolism and application of the siRNA screening system. The siRNA screening system is used for inhibiting expression of metabolism genes of chlamys farreri, the metabolism genes are CYP46A1, GSTM1 and ABCF2, the nucleotide sequences of the siRNA are shown as SEQ ID NO.1-11, and the siRNA screening system comprises five kinds of siRNA aiming at the CYP46A1, three kinds of siRNA aiming at the GSTM1 and three kinds of siRNA aiming at the ABCF2. The invention provides siRNA for intervening expression of Chlamys farreri metabolic genes (CYP46A1, GSTM1 and ABCF2), and key technical indexes of application of RNAi outside model organisms are perfected at the same time. The technical content of the invention is of great significance for identifying genes playing a key role in a metabolic process, researching and developing a prevention and control technology for harmful substances in scallops, further cultivating new varieties with stress resistance characteristics, and promoting healthy development of scallop culture industry.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a screening system for siRNA that intervenes in the chain metabolism of scallops and its application. Background Technology

[0002] Metabolic mechanisms play a crucial role in the response of the scallop to exogenous harmful substances such as paralytic shellfish toxins (PSTs). Currently, the widely accepted metabolic pathway for harmful substances in organisms is a three-phase chain metabolic system composed of cytochrome P450, glutathione S-transferase, and ABC transporters. In this pathway, cytochrome P450 is responsible for the initial modification of harmful substances through oxidation, reduction, or hydrolysis; glutathione S-transferase further catalyzes the binding of primary metabolites with glutathione, forming a more water-soluble complex; finally, the ABC transporters actively transport the conjugate to the extracellular space, completing the toxin metabolism process. Although some genes and family members involved in this pathway have been identified in scallops, current research lacks systematicity and has not fully elucidated the molecular metabolic mechanisms of the scallop in response to PST pollution.

[0003] In recent years, RNA interference (RNAi) technology, as a highly efficient reverse genetics tool, has been widely used in gene function verification and research on biological stress resistance. Unlike forward screening methods such as transcriptome sequencing, RNAi achieves targeted gene silencing by introducing small interfering RNA (siRNA) to guide the specific degradation of target gene mRNA. This technology has been successfully developed in model organisms such as Drosophila, Caenorhabditis elegans, and mammalian cells, and has achieved significant results in agricultural pest control and the verification of the function of plant and animal resistance / stress resistance genes. However, the application of RNAi technology in aquatic shellfish is still in its early stages, and related technical details need further refinement. Currently, commonly used RNAi delivery methods include immersion, feeding, and injection. Due to the special environment of aquatic organisms, injection has become the main delivery method in current RNAi research in aquatic animals because of its high interference efficiency and ease of dosage control. However, in practical applications, the effective implementation of RNAi technology still faces a series of technical challenges, among which the selection of interfering fragments is particularly critical. Specific challenges include: 1. The design of siRNA must balance specificity and efficiency to avoid off-target effects; 2. The choice of delivery system directly affects the interference effect, requiring comprehensive consideration of vector type, delivery route, and tissue targeting; 3. Optimization of interference dosage and treatment timing requires precise regulation based on the physiological state and gene expression characteristics of the target species. These technical bottlenecks limit the large-scale application of RNAi in aquatic economic species such as the scallop, and also restrict the breeding process of related stress-resistant varieties. Summary of the Invention

[0004] Based on the above research background, this invention proposes a method for intervening in the metabolic genes of the scallop (…). CYP46A1 , GSTM1 , ABCF2 The expression of siRNA was improved, and key technical indicators for the application of RNAi outside of model organisms were also improved.

[0005] The primary objective of this invention is to provide a screening system for siRNAs that intervene in the chain metabolism of scallops, used to inhibit the expression of metabolic genes in scallops.

[0006] Intervention in metabolic genes CYP46A1 The siRNA nucleotide sequences are shown in SEQ ID NO. 1~5, respectively, and the genes involved in the intervention are... GSTM1 The siRNA nucleotide sequences are shown in SEQ ID NO. 6-8, and the genes involved in the intervention are... ABCF2 The siRNA nucleotide sequences are shown in SEQ ID NO. 9-11, and the siRNA screening system includes [specific siRNA selection methods]. CYP46A1 There are 5 types of siRNA, targeting GSTM1 There are three types of siRNAs, and one that targets... ABCF2 There are three types of siRNAs, and their sense and antisense strand nucleonucleotide sequences are shown in the table below.

[0007] Preferably, the siRNA screening system fragment is selected from... CYP46A1 -3、 GSTM1 -1 and ABCF2 -1.

[0008] against CYP46A1 There are five types of siRNA, whose nucleotide sequences are shown in SEQ ID NO. 1~5, respectively, targeting... GSTM1 There are three types of siRNA, whose nucleotide sequences are shown in SEQ ID NO. 6~8, respectively, and [the following is a list of specific siRNAs]. ABCF2 There are three types of siRNA, and their nucleotide sequences are shown in SEQ ID NO.9~11 respectively.

[0009] A second objective of this invention is to provide the application of the above-mentioned siRNA screening system in silencing the chain metabolic pathway of *Ctenopharynx scallop*.

[0010] Furthermore, 10 μg of siRNA selection system fragment / 100 μL PBS was injected into the adductor muscle of the scallop.

[0011] Furthermore, this secondary intervention, administered on day 1 and day 4 of exposure, can prolong the silencing effect, with a silencing efficiency of 22.0%–98.5%.

[0012] A third objective of this invention is to provide the above-mentioned siRNA screening system as a research tool for elucidating the metabolic mechanism of the scallop.

[0013] The fourth objective of this invention is to provide the application of the above-mentioned siRNA screening system in the development of shellfish toxin control technology and the cultivation of new stress-resistant and high-yielding germplasm resources.

[0014] In this study investigating the metabolic mechanism of PSTs in the scallop, the present invention screened genes related to PST metabolism by combining reference transcriptome analysis. CYP46A1 , GSTM1 and ABCF2 This invention is aimed at... CYP46A1 , GSTM1 and ABCF2 The designed siRNA can significantly inhibit CYP46A1 , GSTM1 and ABCF2 The expression of RNAi demonstrates its effective application in bivalve scallops.

[0015] The beneficial effects of this invention are: The siRNA provided by this invention significantly reduced the levels of siRNA in the scallop *Ctenopharynx*. CYP46A1 , GSTM1 and ABCF2 The expression of PSTs was increased, and the accumulation of PSTs in scallops was also increased. P <0.05). Furthermore, changes in PSTs components after siRNA treatment provide a new direction for understanding PST metabolism in *Ctenopharynx scallop*. This invention proposes a method for intervening in metabolic genes in *Ctenopharynx scallop* (…). CYP46A1 , GSTM1 , ABCF2 This invention expresses siRNA and improves key technical indicators for the application of RNAi outside model organisms. The technical content of this invention is of great significance for identifying genes that play a crucial role in metabolic processes, developing control technologies for harmful substances in scallops, and ultimately cultivating new varieties with stress resistance, thus promoting the healthy development of the scallop aquaculture industry. Attached Figure Description

[0016] Figure 1 Different siRNAs were used to treat different tissues of scallops. CYP46A1 , GSTM1 and ABCF2 Expression levels of the gene in the relative negative control group (NC); Figure 2 For silence CYP46A1 , GSTM1 and ABCF2 Graph showing the relative expression levels of NC in the posterior hepatopancreas; Figure 3 This is a graph showing the change in PSTs accumulation after siRNA treatment; In the diagram, EXP represents the exposure group, and CYP represents the injection group. CYP46A1 -3 interference exposure group, GST is injected GSTM1 The interference exposure group with a value of -1, ABC represents the injection group. ABCF2 -1 interference exposure group; Figure 4 Figure showing the percentage content of PSTs components in gonads and gills after siRNA treatment; In the diagram, EXP represents the exposure group, and CYP represents the injection group. CYP46A1 -3 interference exposure group, GST is injected GSTM1 The interference exposure group with a value of -1, ABC represents the injection group. ABCF2 Interference exposure group of -1. Detailed Implementation

[0017] To more clearly illustrate the overall concept of the present invention, the technical solution of the present invention will be further described in detail below with reference to the embodiments and accompanying drawings.

[0018] Unless otherwise specified, in the following implementation plan, reagents or instruments whose manufacturers are not indicated are all conventional products that can be purchased commercially.

[0019] Unless otherwise specified in the examples, the conditions shall be performed according to the standard conditions or the conditions recommended by the manufacturer.

[0020] Source of experimental organisms: Collected from the aquaculture area of ​​Lingshan Bay, Jiaonan, Qingdao (35°43′-35°56′N, 120.01′E). Two-year-old *Ctenophora* scallops (120°10′E) had an average soft tissue weight of 14.3 ± 1.2 g, a shell length of 64.9 ± 0.7 mm, a shell width of 60.3 ± 0.9 mm, and a shell height of 17.1 ± 1.0 mm, and were found to be free of PSTs. Before the experiment, the *Ctenophora* scallops were temporarily held in continuously aerated seawater at 16 ± 1 ℃ and a salinity of 28–32 for 2 days without being fed any food. *Alexandrium microphyllum* (GY-H46) was purchased from Shanghai Guangyu Biotechnology Co., Ltd. The scallops were gradually expanded using f / 2 medium in 1–, 5–, 10–, and 50–L culture flasks. The culture process was carried out in a constant temperature incubator at 20 ± 1 ℃, a light intensity of 6000 lx, and a photoperiod of 12 h:12 h. The main PSTs products of GY-H46 are GTX1-4, with a single-cell toxicity of 2.69-3.44 fmol.

[0021] Example 1 Based on the results of reference-guided transcription sequencing, a target was designed using siDirect (http: / / sidirect2.rnai.jp) to target... CYP46A1 , GSTM1 and ABCF2 siRNA fragments, including those targeting CYP46A1 Five types were designed, targeting GSTM1 and ABCF2 Three types of siRNA were designed for each application. The fragment sequences are shown in Table 1. All siRNA fragments were synthesized by Shanghai Sangon Biotech Co., Ltd., and dissolved in RNase-free PBS as per the instructions to prepare 10 μg siRNA fragment / 100 μL PBS.

[0022] Table 1. Base sequences of siRNA fragments

[0023] First, we will target CYP46A1 Preliminary experiments were conducted to screen for the most suitable siRNA. The experiment lasted for 3 days and included a blank control group, a negative control group (NC), and an interference group. Scallops in the blank control group received no injection, scallops in the negative control group received 100 μL of negative control siRNA, and scallops in the interference group received 10 μg of siRNA fragment / 100 μL PBS. Injections were performed on day 1 of the experiment at the adductor muscle. During the experiment, scallops were fed only non-toxic bait algae (algal paste made from *Phaeodactylum tricornutum*). Since no significant difference in gene expression levels was observed between the blank control group and the negative control group, a blank control group was not included in subsequent preliminary experiments.

[0024] against GSTM1 and ABCF2 The preliminary experiment lasted 7 days, and the rest were the same as... CYP46A1 Consistent with preliminary experiments, scallop visceral masses, gonads, gills, mantle membranes, and adductor muscles were collected on ice at hours 1 and 6 and on days 1, 3, 5, and 7. Each sample consisted of a mixture of three scallops. Samples were stored at -80 °C until analysis.

[0025] The relative gene expression levels of each tissue are shown in the figure. Figure 1 , Figure 1 Different tissues of scallops under the action of siRNA CYP46A1 Gene Figure 1 (A) GSTM1 Gene Figure 1 (B) and ABCF2 Gene Figure 1 A graph showing the relative expression levels of C in the middle molecule. (From...) Figure 1 Gene silencing was observed in all scallop tissues, indicating that the injected siRNA has tissue-transferability. Real-time quantitative PCR (qRT-PCR) verification (see...) Figure 2): siRNA fragments have a silencing effect on different tissues of scallops, but the degree of effect varies. Figure 2 (A) CYP46A1 -3、 Figure 2 (B) GSTM1 -1 and Figure 2 (C) ABCF2 The silence effect of -1 is better than other fragments. CYP46A1 -3、 GSTM1 -1 and ABCF2 More tissue samples with -1 interference showed lower relative NC expression levels. Furthermore, the relative NC expression levels across different tissues were all less than 0, indicating that siRNA is transmissible between tissues. Regarding silencing time, CYP46A1 -3、 GSTM1 -1 and ABCF2 The stability of -1 lasted for 3 days, achieving a good silencing effect.

[0026] Example 2 720 scallops were randomly divided into 8 groups (see Table 2), with 3 replicates per group, requiring a total of 24 culture frames, with 30 scallops per frame. The experiment lasted 14 days, including a 7-day exposure phase and a 7-day removal phase. During the exposure phase, 1×10⁻⁶ scallops were fed daily. 6 GY-H46 algal cells / shellfish were used as bait during the cleansing phase. Feeding was conducted twice daily, at 8:00 AM and 8:00 PM, with the aquaculture water changed before each feeding. Based on the results of Example 1, [the following was selected]. CYP46A1 -3、 GSTM1 -1 and ABCF2 -1 was used as the siRNA in Example 2. On days 1 and 4 of the experiment, siRNA was injected into the adductor muscle of scallops in the six interference groups at a dose of 10 μg siRNA / 100 μL PBS.

[0027] Table 2 Experimental Grouping Table

[0028] Note: - indicates no siRNA injection, + indicates siRNA injection.

[0029] Nine scallops were collected from each group on days 3, 7, 10, and 14 of the experiment. The adductor muscle was cut off, and the scallops were dried with filter paper before the tissues (hepatopancreas, gonads, gills, mantle, and adductor muscle) were separated on ice. The separated tissues were quickly flash-frozen in liquid nitrogen and stored at -80 °C until analysis.

[0030] Figure 3 siRNA ( CYP46A1 -3, GSTM1 -1, ABCF2-1) PSTs content changes after treatment. The PSTs content in scallops gradually increased over time during the exposure phase. The average PSTs content in the EXP group was 466.58 μg STXeq / kg. CYP46A1 Subsequently, PSTs levels remained higher than in other groups, peaking at 1015.50 μg STXeq / kg on day 7. Except for day 7, PSTs levels in the GST and ABC groups were 461.01 μg STXeq / kg and 376.32 μg STXeq / kg, respectively, both lower than in the EXP group. Overall, the CYP group showed a significant increase in PST accumulation, while the GST and ABC groups showed a decrease. Simultaneously, the silencing of related metabolic genes significantly inhibited the proportion and time of toxin conversion from highly toxic components to less toxic components, reducing the metabolic efficiency of the toxin (see...). Figure 4 ), Figure 4 (A) gonads and Figure 4 Percentage of PSTs components in gills (B)

[0031] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention in any other way. Any person skilled in the art may make changes or modifications to the above-disclosed technical content to create equivalent embodiments. However, any simple modifications, equivalent changes, and modifications made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the protection scope of the present invention.

Claims

1. A screening system for siRNAs intervening in the chain metabolism of *Ctenopharynx scallop*, characterized in that: The siRNA screening system was used to inhibit the expression of metabolic genes in the scallop. Intervention in metabolic genes CYP46A1 The siRNA nucleotide sequences are shown in SEQ ID NO. 1~5, respectively, and the genes involved in the intervention are... GSTM1 The siRNA nucleotide sequences are shown in SEQ ID NO. 6-8, and the genes involved in the intervention are... ABCF2 The siRNA nucleotide sequences are shown in SEQ ID NO. 9-11, and the siRNA screening system includes [specific siRNA selection methods]. CYP46A1 There are 5 types of siRNA, targeting GSTM1 There are three types of siRNAs, and one that targets... ABCF2 There are three types of siRNAs, and their sense and antisense strand nucleonucleotide sequences are shown below. SEQ ID NO.1, fragment name CYP46A1 -1, the justice chain (5'-3') is CCAACACAAUGAGUUUCAUTT, and the antisense chain (5'-3') is AUGAAACUCAUUGUGUUGGTT; SEQ ID NO.2, fragment name CYP46A1 -2, the justice chain (5'-3') is GGUGUUUAAGGAAACACUUTT, and the antisense chain (5'-3') is AAGUGUUUCCUUAAACACCTT; SEQ ID NO.3, fragment name CYP46A1 -3, the justice chain (5'-3') is GGUACAGACAUGGUGGUUATT, and the antisense chain (5'-3') is UAACCACCAUGUCUGUACCTT; SEQ ID NO.4, fragment name CYP46A1 -4, the justice chain (5'-3') is UCGGACCUCAUACCUGUAUTT, and the antisense chain (5'-3') is AUACAGGUAUGAGGUCCGATT; SEQ ID NO.5, fragment name CYP46A1 -5, the justice chain (5'-3') is GCGGUAGAUGGUGUCAAGUTT, and the antisense chain (5'-3') is ACUUGACACCAUCUACCGCTT; SEQ ID NO.6, fragment name GSTM1 -1, the justice chain (5'-3') is GGAACUGUUCGACUUUGUUTT, and the antisense chain (5'-3') is AACAAAGUCGAACAGUUCCTT; SEQ ID NO.7, fragment name GSTM1 -2, the justice chain (5'-3') is CCUACUUUGAACGGUUAUUTT, and the antisense chain (5'-3') is AAUAACCGUUCAAAGUAGGTT; SEQ ID NO.8, fragment name GSTM1 -3, the justice chain (5'-3') is CCUUGGCUGCCUUGAUCAAUTT, and the antisense chain (5'-3') is AUUGAUCAAGGCAGCCAGGTT; SEQ ID NO.9, fragment name ABCF2 -1, the justice chain (5'-3') is GGACAUCGAGACCAUCGAUTT, and the antisense chain (5'-3') is AUCGAUGGUCUCGAUGUCCTT; SEQ ID NO.10, fragment name ABCF2 -2, the justice chain (5'-3') is UCAGAGUUGUCGAGGAAATT, and the antisense chain (5'-3') is UUUCCUCGACAACCUGUGATT; SEQ ID NO.11, fragment name ABCF2 -3, the justice chain (5'-3') is GCAGAAGUUGACACAGAUUTT, and the antisense chain (5'-3') is AAUCUGUGUCAACUUCUGCTT.

2. The siRNA screening system for intervening in the chain metabolism of *Ctenopharynx scallop* as described in claim 1, characterized in that: The siRNA screening system fragments are selected from... CYP46A1 -3、 GSTM1 -1 and ABCF2 -1.

3. The application of the siRNA screening system as described in claim 1 in silencing the chain metabolic pathway of *Ctenopharynx scallop*.

4. The application as described in claim 3, characterized in that: Inject 10 μg of siRNA selection system fragment / 100 μL PBS into the adductor muscle of the scallop.

5. The application as described in claim 4, characterized in that: Injections were administered on day 1 and day 4 of exposure.

6. The siRNA screening system as described in claim 1 is used as a research tool for elucidating the metabolic mechanism of the scallop.

7. The application of the siRNA screening system as described in claim 1 in the development of shellfish toxin control technology and the cultivation of stress-resistant and high-yielding new germplasm resources.

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