SiRNA targeting intervention of ATF4a gene expression of gynnis and application thereof

Abstract

This invention discloses a siRNA targeting the expression of the ATF4a gene in *Syngonium spp.* and its applications. The siRNA is a double-stranded RNA composed of a sense strand and an antisense strand. When transfected into *Syngonium spp.* muscle cells, the siRNA targeting the expression of the ATF4a gene exhibits an inhibition efficiency of over 50%, demonstrating high knockdown efficiency. Simultaneously, the knockdown of the ATF4a gene downregulates the expression of key purine metabolism genes such as AMPD1, PPAT, ADSL, and ATIC in *Syngonium spp.* muscle cells, significantly inhibiting the synthesis and deposition of inosine monophosphate in muscle cells. This invention demonstrates that ATF4a is a key transcription factor positively regulating inosine monophosphate synthesis in *Syngonium spp.*, and this siRNA can provide a reliable target model and theoretical basis for identifying fish flavor regulation mechanisms.

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 ATF4a gene in the bream and its application. Background Technology

[0002] Fish, due to their low fat and high protein content, have become an important source of protein for humans. However, with the rapid development of intensive aquaculture, farmed fish generally face the problem of declining muscle quality, such as reduced flavor, nutrient loss, and decreased palatability. *Hefang bream* is a novel hybrid fish bred through distant hybridization and backcrossing, derived from *Clouthead bream* (…). Megalobrama amblycephala ,♀) and Culter alburnus ( Culter alburnus The bream was bred through hybridization of male and female bream and two rounds of selective breeding. The *Hefang* bream exhibits good growth performance and meat quality, with its muscle rich in nutrients and flavor compounds such as protein, unsaturated fatty acids, and umami amino acids. Inosine monophosphate (IMP) is an important flavor compound in fish muscle, and its content is closely related to the umami quality of the fish meat. Therefore, elucidating the synthesis and regulatory mechanism of IMP in the muscle of the *Hefang* bream is of great significance for in-depth research on the formation of its meat flavor.

[0003] ATF4 (activating transcription factor 4) is an important transcription factor involved in amino acid sensing, metabolic regulation, and cellular stress response, playing a crucial role in nutrient response and metabolic process regulation. Studies in mammals have shown that ATF4 participates in purine metabolism and IMP synthesis by regulating downstream related genes. However, its specific role in the synthesis of IMP, a flavor compound in the muscle of *Heterodon spp.* remains unclear. While most vertebrates have only one homologous gene for ATF4, two subtypes (ATF4a and ATF4b) have been found in some fish (such as blunt snout bream and zebrafish). Currently, there are few studies on specific functional interventions targeting the ATF4a gene in *Heterodon spp.*, and there is a lack of effective intervention methods to reduce its expression level. Therefore, establishing a method to specifically inhibit the expression of the ATF4a gene in *Heterodon spp.* is an important technical foundation for further elucidating the mechanism of action of ATF4a in IMP synthesis and flavor formation in the muscle of *Heterodon spp.*

[0004] RNA interference (RNAi) is a gene regulation technique that uses small interfering RNA (siRNA) to specifically recognize and promote the degradation of target mRNA, thereby inhibiting the expression of target genes. siRNAs are typically double-stranded RNAs of about 20-25 nucleotides, possessing high sequence specificity and ease of operation, and have been widely used in gene function research and gene expression regulation. However, because the interference effect of siRNAs is closely related to factors such as the selection of the target sequence, the structural characteristics of the target gene itself, and sequence differences between species, siRNA sequences obtained in existing studies are often difficult to directly apply to other species. Therefore, screening for siRNAs with high interference efficiency and specificity based on the sequence characteristics of the *Heterodon spp.* ATF4a gene and establishing corresponding gene expression inhibition methods are of great significance for conducting functional studies of the *Heterodon spp.* ATF4a gene and further clarifying its regulatory role in muscle IMP synthesis and flavor formation. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to overcome the deficiencies and defects mentioned in the background art above, and to provide a siRNA with high knockdown efficiency for targeted intervention of ATF4a gene expression in Heterodon bream and its application.

[0006] To solve the above-mentioned technical problems, the technical solution proposed by this invention is as follows: In a first aspect, the present invention provides an siRNA that targets and intervenes in the expression of the ATF4a gene of the bream, wherein the siRNA is a double-stranded RNA composed of a sense strand and an antisense strand, wherein the sense strand is 5'-CCGACUCACCUACCAGAUAtt-3' and the antisense strand is 5'-UAUCUGGUAGGUGAGUCGGtt-3'.

[0007] The siRNA that targets and intervenes in the expression of the ATF4a gene of *Heterodon spp.* is preferably the nucleotide sequence of the ATF4a gene as shown in SEQ ID NO:2.

[0008] Secondly, the present invention provides an application of the siRNA that targets and intervenes in the expression of the ATF4a gene of *Hemiberleinii* in the specific knockdown of the ATF4a gene expression of *Hemiberleinii*.

[0009] In the above application, preferably, the method of specifically knocking down the ATF4a gene expression of *Heterodon spp.* using siRNA includes the following steps: transfecting the siRNA that targets and intervenes in the expression of the ATF4a gene of *Heterodon spp.* into *Heterodon spp.* muscle cells to achieve specific knockdown of the ATF4a gene expression of *Heterodon spp.*

[0010] Thirdly, the present invention provides the application of the siRNA that targets and intervenes in the expression of the ATF4a gene of *Syngonium syngastum* in downregulating the expression of key genes for purine synthesis in *Syngonium syngastum* muscle cells, wherein the key genes for purine synthesis include at least one of AMPD1, PPAT, ATIC, ADSL, GART, GARS, and PAICS.

[0011] In the above application, preferably, the method of downregulating the expression of key genes for purine synthesis in *Heterodon spp.* muscle cells using siRNA includes the following steps: transfecting *Heterodon spp.* ATF4a gene expression with siRNA into *Heterodon spp.* muscle cells to downregulate the expression of key genes for purine synthesis pathway in *Heterodon spp.* muscle cells.

[0012] Fourthly, the present invention provides an application of the siRNA that targets and intervenes in the expression of the ATF4a gene of *Hemiberleinii* in inhibiting the synthesis of inosine monophosphate in *Hemiberleinii* muscle cells.

[0013] In the above-described application, preferably, the method of using siRNA to inhibit inosine synthesis in *Heterodon spp.* muscle cells includes the following steps: transfecting the siRNA that targets and intervenes in the expression of the *Heterodon spp.* ATF4a gene into *Heterodon spp.* muscle cells to downregulate inosine synthesis in the *Heterodon spp.* muscle cells.

[0014] Preferably, the inosinic acid is glutamate-induced inosinic acid.

[0015] Compared with the prior art, the beneficial effects of the present invention are as follows: 1. This invention provides a siRNA that targets and intervenes in the expression of the ATF4a gene in *Heterodon spp.* When transfected into *Heterodon spp.* muscle cells, the siRNA exhibits an inhibition efficiency of over 50% on the ATF4a gene, demonstrating high specificity and knockdown efficiency. Furthermore, knocking down the ATF4a gene not only significantly downregulates the expression of key genes in the purine synthesis pathway, such as AMPD1, PPAT, ATIC, ADSL, GART, GARS, and PAICS, in *Heterodon spp.* muscle cells, and inhibits the synthesis of the intracellular flavor precursor inosine monophosphate (IMP), but also effectively blocks the activation of AMPD1 and the promoting effect of exogenous glutamate on IMP synthesis. This fully elucidates the regulatory role of ATF4a in mediating glutamate regulation of the *Heterodon spp.* IMP synthesis metabolic pathway, providing a theoretical basis for developing feed additives to improve the flavor quality of fish muscle.

[0016] 2. The siRNA of the present invention, which targets and intervenes in the expression of the ATF4a gene of the bream, identifies gene function by specifically knocking down the gene. It can not only be used as a key molecular tool for the genetic improvement and molecular breeding of high-quality fish, but also provides an important reference for the targeted improvement of meat quality and flavor in aquaculture, and has great economic and scientific research value. Attached Figure Description

[0017] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0018] Figure 1 This is a diagram showing the knockdown effect of different types of siRNAs on the ATF4a gene of *Heterodon spp.* in Example 1 of the present invention.

[0019] Figure 2 This is a diagram showing the effect of siRNA targeting intervention on ATF4a gene expression in Example 2 of the present invention.

[0020] Figure 3 This is a diagram showing the effect of siRNA targeted intervention on the ATF4a gene of *Bryophyllum hexandrum* on key genes AMPD1, PPAT, ATIC, ADSL, GART, GARS, and PAICS in Example 2 of the present invention.

[0021] Figure 4 This is a diagram illustrating the effect of siRNA-targeted intervention on the ATF4a gene of *Heterodon spp.* on the IMP content in *Heterodon spp.* muscle cells in Example 2 of this invention.

[0022] Figure 5 This is a diagram showing the effect of targeted intervention on the expression of the ATF4a gene in He Fang Bian under glutamate treatment conditions in Example 2 of the present invention.

[0023] Figure 6 This is a diagram showing the effect of targeted intervention on the expression level of the ATF4a gene of *Heterodon spp.* on the expression level of the AMPD1 gene under glutamate treatment conditions in Example 2 of this invention.

[0024] Figure 7 This is a diagram illustrating the effect of targeted intervention on the expression of the ATF4a gene of *Heterodon spp.* on the IMP content in *Heterodon spp.* muscle cells under glutamate treatment conditions in Example 2 of this invention.

[0025] The above is attached Figure 1 , 5 In equations 6 and 7, the letters a, b, c, and d represent the 5% significance level, meaning the difference between groups is statistically significant (95%). If two groups share at least one identical letter, the difference between the two groups is not significant at the corresponding significance level; if the letters in the two groups are completely different, the difference between the two groups is significant at the corresponding significance level.

[0026] The above is attached Figure 2 ,3 In 4, This indicates that p < 0.05, and the result is significant at the 0.05 level. This indicates that p < 0.01, and the result is significant at the 0.01 level. p < 0.001 indicates a highly significant result; ns indicates no change. Detailed Implementation

[0027] To facilitate understanding of the present invention, the present invention will be described more fully and in detail below with reference to the accompanying drawings and preferred embodiments, but the scope of protection of the present invention is not limited to the following specific embodiments.

[0028] Unless otherwise defined, all technical terms used herein have the same meaning as commonly understood by those skilled in the art. The technical terms used herein are for the purpose of describing particular embodiments only and are not intended to limit the scope of the invention.

[0029] Unless otherwise specified, all raw materials, reagents, instruments and equipment used in this invention can be purchased from the market or prepared by existing methods.

[0030] Example 1 A siRNA for targeting and intervening in the expression of the ATF4a gene in *Syngonium spp.* according to the present invention is obtained by the following steps: (1) Template preparation RNA was extracted from the muscle tissue of *He Fang Bian* using the Trizol method, and cDNA templates were obtained by reverse transcription following the procedures of the PrimeScript™ II First Strand cDNA Synthesis Kit (Takara).

[0031] (2) PCR amplification Using primers ATF4a-F and ATF4a-R, cDNA was used as a template for PCR amplification to obtain the ORF sequence of ATF4a. The nucleotide sequence of primer ATF4a-F is 5'-CTATTTGGCAAAATGGCGTCCC-3' (as shown in SEQ ID NO:3); the nucleotide sequence of primer ATF4a-R is 5'-CAACTCAAAACAACGAGGCCC-3' (as shown in SEQ ID NO:4). The PCR amplification reaction conditions were: 95 ℃ pre-denaturation for 5 min; 95 ℃ denaturation for 15 s, 58 ℃ annealing for 15 s, 72 ℃ extension for 1 min 30 s, for 32 cycles; and a final extension at 72 ℃ for 5 min.

[0032] 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 pMD19-T vector (Takara) and transformed into E. coli DH5α (Takara). After PCR detection of positive clones, the samples were sent to Hunan Qingke Biotechnology Co., Ltd. for sequencing. Finally, the complete ORF sequence of ATF4a was obtained. The nucleotide sequence of the ATF4a gene of He Fang Bian is shown in SEQ ID NO:2, and the corresponding amino acid sequence is SEQ ID NO:1.

[0033] The amino acid sequence encoded by the ATF4a gene of *Heterodon spp.* is shown in SEQ ID NO:1, and is as follows: MSVVSQMSLEDVGALLLGPSSLMADPFGPLLDEDEESALSEGSSSPLSSSYAESSSSLSLLSPSPPASVGCKSELLLLSWLPASELLGAQTEPEQSKGDAFSGMDWMSEKLDLNDFDLDSLIGSYDSDEPPSSPEELLACLDTDMDLDLDSLPFSSSELGLTLPPLPLDLPLPEPPLEPVEIKSEPPSP ARSPDSANTLDLGSEVSVLQAPGIMLTPSHIVVVLAPKDEASSTDDSSDSDSGISISGSPTHLPDTAPSPKPAGSSRTKPYSKPDPDAAAVVTGRVKAGPGAPKVVEKKLKMEQNKTAATRYRQKKRAEQDTLNSECADLEKRNRELAEKAESLSREIKYLKDLMEEVRSAENRRSKAKVQDAVGSS.

[0034] The nucleotide sequence of the ATF4a gene of *Heterodon spp.* is shown in SEQ ID NO:2, and is as follows:

[0035] (3) siRNA screening Laboratory-established *Heterodon spp.* muscle cells were cultured in a 28 °C, 5% CO2 incubator, with the culture medium changed every 48 h. Cells were passaged when confluence reached 80%-90%. 24 h before transfection, cells in logarithmic growth phase were seeded at an appropriate density into 6-well plates. Transfection was performed when cell confluence reached 60%-80%. The transfection procedure followed Lipofectamine. TM The transfection was performed according to the instructions of the 3000 transfection reagent (Thermo Fisher Scientific, USA): each specific siRNA and the transfection reagent were diluted with Opti-MEM serum-depleted medium (Gibco, USA) and gently mixed. The mixture was then incubated at room temperature for 10 min to form a transfection complex. The complex was then evenly added to the culture wells containing cells.

[0036] Cells were collected 24 h after transfection and incubation, and the knockdown effect of different siRNAs on the target gene was detected by qRT-PCR. The qRT-PCR screening results are shown below. Figure 1 As shown, siRNA-2 exhibited the highest interference efficiency, thus obtaining a highly efficient siRNA for targeting and intervening in the ATF4a gene expression of *Heterodon spp.*. To optimize the interference effect, the siRNA for targeting and intervening in the ATF4a gene of *Heterodon spp.* described in this invention incorporates two deoxythymidine nucleotides (tt) as dangling ends at its 3' end. This modification effectively resists nuclease degradation and improves the structural stability of the siRNA; furthermore, the 3' dangling structure facilitates the unwinding of the double strand within the cell, promoting its entry and activation of the RNA-induced silencing complex (RISC). The specific sequence information of the different types of siRNA used in this embodiment is shown in Table 1, all of which were chemically synthesized by Hunan Qingke Biotechnology Co., Ltd.

[0037] Table 1 Information on different types of siRNA (nucleotide sequences are shown in SEQ ID NO:5-12)

[0038] Figure 1 This image shows the knockdown effects of different types of siRNAs targeting the ATF4a gene of *Heterodon spp.* in Example 1 of this invention. From... Figure 1 It can be seen that siRNA-2 knocked down the ATF4a gene of He Fang Bian at 24h by more than 50%. Therefore, siRNA-2 had the best interference effect, and siRNA-2 was selected as the siRNA to target the expression of the ATF4a gene of He Fang Bian to complete the subsequent experiments.

[0039] Example 2 The application of siRNA targeting ATF4a gene expression in *Syngonium syngonium* in downregulating the expression of key genes for purine synthesis and inhibiting inosine monophosphate synthesis in *Syngonium syngonium* muscle cells, as described in Example 1, includes the following experimental steps: (1) Primary muscle cells of *He Fang Bian* were seeded into 6-well culture plates and cultured in a constant temperature incubator at 28 ℃ and 5% CO2. Two treatment groups were set up: a negative control group siNC (transfected with non-specific NC-siRNA) and an experimental group siATF4a (transfected with the best interfering sequence siRNA-2 selected in Example 1), with 4 replicates in each group. When the cell confluence reached 60%-80%, the transfection operation was strictly performed according to the Lipofectamine™ 3000 reagent instructions: the siRNA-2 of Example 1 and the transfection reagent were diluted with Opti-MEM serum-depleted medium (Gibco, USA) and gently mixed, and incubated at room temperature for 10 min to form a transfection complex. Then the complex was evenly added to the culture wells containing cells.

[0040] (2) 24 h after transfection, cells from each group were collected, and total RNA was extracted using Trizol reagent and reverse transcribed to synthesize cDNA. Using cDNA as a template, qRT-PCR was used to detect the relative mRNA expression levels of the target gene ATF4a and the key enzyme gene for inosine monophosphate synthesis in the cells. The results are as follows: Figures 2-3 As shown in Table 2, the quantitative fluorescent primers are as follows.

[0041] (3) Cell samples for inosinic acid (IMP) detection were collected, and cells were lysed with 1 mL of distilled water preheated to 95 °C. The lysate was boiled in a 95 °C water bath for 5 min, centrifuged, and the supernatant was filtered through a 0.45 μm filter membrane and then analyzed using an IMP detection kit (Shanghai Enzyme Linked Diagnostics). The results are as follows: Figure 4 As shown.

[0042] Table 2 Information on the primers for quantitative fluorescence (nucleotide sequences are shown in SEQ ID NO:13-28)

[0043] Figure 2 This is a diagram showing the effect of siRNA targeting intervention on ATF4a gene expression in Example 2 of the present invention. Figure 3 This is a diagram showing the effect of siRNA targeted intervention on the ATF4a gene of *Bryophyllum hexandrum* on key genes AMPD1, PPAT, ATIC, ADSL, GART, GARS, and PAICS in Example 2 of the present invention. Figure 4This is a diagram illustrating the effect of siRNA-targeted intervention on the ATF4a gene of *Heterodon spp.* on the IMP content in *Heterodon spp.* muscle cells in Example 2 of this invention.

[0044] Combination Figures 2-4 It can be seen that the siRNA targeting ATF4a gene expression described in this invention can effectively knock down ATF4a gene expression in *Heterodon spp.* Simultaneously, the knockdown of the *Heterodon spp.* ATF4a gene downregulates the expression of key genes in the purine synthesis pathway, including AMPD1, PPAT, ATIC, ADSL, GART, GARS, and PAICS, in *Heterodon spp.* muscle cells, and inhibits IMP synthesis. This indicates that ATF4a may play a core positive regulatory role in the IMP synthesis metabolic pathway of *Heterodon spp.*

[0045] The inhibitory effect of targeted intervention on ATF4a gene expression in *Heterodon spp.* on glutamate-induced inosine monophosphate synthesis was investigated through the following experimental steps: (1) Primary myoblasts of *Heterodon spp.* in logarithmic growth phase were seeded into 6-well plates and cultured in a constant temperature incubator at 28 ℃ with 5% CO2. Four treatment groups were set up: negative control group (siNC + PBS); glutamate treatment group (siNC + Glu); interference group (siATF4a + PBS); and interference + glutamate treatment group (siATF4a + Glu). The interference group was transfected with the optimal interference sequence siRNA-2 selected in Example 1, with four replicates per group. The concentration of Glu in the experiment was 2 mM.

[0046] (2) After 24 hours of interference, Glu or PBS was added and the cells were treated for another 48 hours before collecting the cells from each group. The relative expression levels of relevant genes in each group of cells were detected using qRT-PCR; simultaneously, the intracellular inosine monophosphate (IMP) content in each group of cells was also detected. Results are as follows: Figures 5-7 As shown.

[0047] Figure 5 This is a diagram illustrating the effect of targeted intervention on ATF4a gene expression in *Heterodon spp.* under glutamate treatment conditions in Example 2 of this invention. Figure 6 This is a diagram showing the effect of targeted intervention on the expression level of the ATF4a gene in *Heterodon spp.* on the expression level of the AMPD1 gene under glutamate treatment conditions in Example 2 of this invention. Figure 7 This is a graph illustrating the effect of targeted intervention on the expression of the ATF4a gene in *Hemiberlesia lataniae* on the IMP content in *Hemiberlesia lataniae* muscle cells under glutamate treatment conditions, as shown in Example 2 of this invention. Figures 5-7As shown, in cells transfected with the negative control sequence (NC-siRNA), the addition of glutamate significantly upregulated the expression of ATF4a and AMPD1 genes and significantly increased intracellular IMP levels. However, after specifically transfecting ATF4a gene knockdown with siRNA-2 of this invention, the expression level of AMPD1 gene increased slightly after the addition of glutamate, but the increase was not significant. Figure 6 Furthermore, no significant increase was observed in intracellular IMP levels. Figure 7 ).

[0048] The above results demonstrate that the siRNA of this invention effectively interferes with ATF4a gene expression, partially blocking the activation of AMPD1 by glutamate and the promoting effect of IMP synthesis. This, to some extent, proves that ATF4a may be a key transcription factor mediating the glutamate-regulated inosine monophosphate synthesis pathway in the muscle cells of *Brachys spp.*, providing a theoretical basis for improving the flavor quality of fish muscle.

Claims

1. A siRNA that targets and intervenes in the expression of the ATF4a gene in the bream, characterized in that, The siRNA is a double-stranded RNA composed of a sense strand and an antisense strand, wherein the sense strand is 5'-CCGACUCACCUACCAGAUAtt-3' and the antisense strand is 5'-UAUCUGGUAGGUGAGUCGGtt-3'.

2. The siRNA targeting the expression of the ATF4a gene in *Brachys syngastricensis* according to claim 1, characterized in that, The nucleotide sequence of the ATF4a gene of the synbromeliad is shown in SEQ ID NO:

2.

3. The application of a siRNA that targets and intervenes in the expression of the ATF4a gene of *Hemiberleinii* as described in claim 1 or 2 in the specific knockdown of the ATF4a gene expression of *Hemiberleinii*.

4. The application of the siRNA according to claim 3 in specifically knocking down the expression of the ATF4a gene in *Syngonium spp.*, characterized in that, The method for specifically knocking down the ATF4a gene expression of *Heterodon spp.* using siRNA includes the following steps: transfecting the siRNA that targets and intervenes in the expression of the ATF4a gene of *Heterodon spp.* into *Heterodon spp.* muscle cells to achieve specific knockdown of the ATF4a gene expression of *Heterodon spp.* 5. The application of a siRNA targeting the expression of the ATF4a gene in *Syngonium syngastum* as described in claim 1 or 2 in downregulating the expression of a key gene for purine synthesis in *Syngonium syngastum* muscle cells, characterized in that... The key genes for purine synthesis include at least one of AMPD1, PPAT, ATIC, ADSL, GART, GARS, and PAICS.

6. The application of the siRNA according to claim 5 in downregulating the expression of key genes for purine synthesis in the muscle cells of *Sinocyclocheilus bream*, characterized in that, The method for downregulating the expression of key genes in purine synthesis in *Heterodon spp.* muscle cells using siRNA includes the following steps: transfecting *Heterodon spp.* ATF4a gene expression with siRNA into *Heterodon spp.* muscle cells to downregulate the expression of key genes in the purine synthesis pathway in *Heterodon spp.* muscle cells.

7. The application of a siRNA that targets the expression of the ATF4a gene of *Hemiberleinii* as described in claim 1 or 2 in inhibiting inosine monophosphate synthesis in *Hemiberleinii* muscle cells.

8. The application of the siRNA according to claim 7 in inhibiting inosine monophosphate synthesis in the muscle cells of *Hemiberlesia lataniae*, characterized in that, The method for inhibiting inosine synthesis in *Heterodon spp.* muscle cells using siRNA includes the following steps: transfecting *Heterodon spp.* ATF4a gene expression with siRNA into *Heterodon spp.* muscle cells to downregulate inosine synthesis in the muscle cells.

9. The application of the siRNA according to claim 7 in inhibiting inosine monophosphate synthesis in the muscle cells of *Hemiberlesia lataniae*, characterized in that, The inosine monophosphate is glutamate-induced inosine monophosphate.

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