Method for identifying sturgeon pathogen infection resistant gene
By designing specific primers and RT-qPCR technology, the anti-pathogenic gene of sturgeon was identified, the detection problems in the existing technology were solved, the immune regulation mechanism of sturgeon was revealed, and the basis for disease prevention and treatment was provided.
Patent Information
- Application Number
- CN202510391626.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-03-31
- Publication Date
- 2025-07-01
- Estimated Expiration
- Not applicable · inactive patent
AI Technical Summary
The prior art is difficult to effectively detect and analyze sturgeon anti-pathogenic infection genes, which leads to difficulties in preventing and treating sturgeon diseases.
Specific primers were designed to PCR amplify from the sturgeon cDNA template, analyze the gene structure and amino acid sequence, and detect the expression distribution of anti-pathogenic genes in different organs by RT-qPCR, and analyze the expression patterns of genes after pathogen infection.
The effective identification of sturgeon anti-pathogenic genes has been achieved, revealing its immune regulation mechanism in viral and bacterial infections, and providing an important basis for disease prevention and treatment.
Smart Images
Figure BDA0005337582230000031 
Figure HDA0005337582240000011 
Figure HDA0005337582240000021
Abstract
Description
Technical Field
[0001] The present study specifically relates to a method for detecting genes against pathogen infection in sturgeons. Background Art
[0002] Sturgeons belong to the class of chondrostei, and their taxonomic status is between cartilaginous fish and bony fish. They are living fossils for studying the evolution of fish and vertebrates, and also important branch nodes on the evolutionary tree of the biological immune system. Currently, sturgeons are still facing various disease threats, especially bacterial diseases, including Mycobacterium, Pseudomonas alcaligenes, Edwardsiella tarda, Elizabethkingia meningoseptica, and Citrobacter freundii.
[0003] The p53 tumor suppressor gene exists widely in various organisms as a transcription factor and plays a central role in maintaining genomic stability, cell cycle arrest, cellular senescence, cell differentiation, DNA repair, autophagy, apoptosis, and innate immune regulation. When the host is infected with a DNA virus, viral genome replication triggers the p53-mediated apoptotic response, thus initiating the DNA damage response. p53 also plays a role in the innate immune response to RNA viruses. In addition, p53 is also involved in regulating the immune response and has antibacterial activity. Therefore, in-depth study of the immune regulation mechanism of the sturgeon p53 gene is of great significance for the prevention and treatment of sturgeon diseases. Summary of the Invention
[0004] In view of this, the purpose of the present invention is to provide a method for detecting genes against pathogen infection in sturgeons, and using this method, the problem of pathogen infection in sturgeons can be prevented or treated.
[0005] To achieve the above purpose, the present invention has identified fish genes against pathogens in sturgeons.
[0006] The method for identifying the gene against pathogen: Design specific primers, use sturgeon cDNA as a template for PCR amplification, and analyze its gene structure and amino acid sequence.
[0007] Preferably, the specific primers are designed according to the genes against pathogens in the sturgeon transcriptome library.
[0008] The present invention has also detected the expression distribution of the gene against pathogen in different organs by RT-qPCR.
[0009] Preferably, the sampled organs are spleen, blood, brain, gill, skin, heart, head kidney, mid kidney, liver, intestine, and muscle.
[0010] The present invention has also analyzed the expression pattern of the mRNA of the gene against pathogen in sturgeons after pathogen infection.
[0011] Preferably, the sturgeon pathogens include the viral RNA analog poly I:C and Mycobacterium marinum.
[0012] In vivo and in vitro experiments were conducted to detect the changes in anti-pathogen genes.
[0013] According to the detailed description of the present invention, anti-pathogen genes in sturgeon can be effectively identified. Detailed implementation manners
[0014] The present invention provides a research method for effectively identifying anti-pathogen genes in sturgeon when infected with pathogens.
[0015] The present invention identified fish anti-pathogen genes in sturgeon and analyzed the gene structure and amino acid sequence. The specific method includes: obtaining the anti-pathogen gene sequence from the sturgeon transcriptome library, designing specific primers through Primer 6.0 software, and performing PCR amplification using cDNA as a template. After purifying the product, it was ligated to the pMD19-T vector, transformed into E. coli DH5α competent cells, and positive clones were screened and verified by sequencing. Then, multiple sequence alignment, gene similarity and identity analysis, signal peptide prediction, open reading frame (ORF) and amino acid sequence analysis, phylogenetic tree construction, protein physicochemical properties and secondary structure analysis were carried out on the gene.
[0016] The present invention detected the expression distribution of anti-pathogen genes in different organs by RT-qPCR. The specific method includes: collecting samples of spleen, blood, brain, gill, skin, heart, head kidney, mid kidney, liver, intestine and muscle tissues from healthy sturgeon. Total RNA was extracted and its purity was detected. Qualified RNA was synthesized into cDNA through a reverse transcription kit. Using cDNA as a template, according to the operation of the fluorescence quantitative kit, the relative expression level of sturgeon anti-pathogen gene mRNA was calculated. The experimental methods of the present invention can be prepared by conventional methods.
[0017] The present invention also analyzed the expression pattern of sturgeon anti-pathogen gene mRNA after pathogen infection. The specific method includes: using pathogens to infect sturgeon fish bodies and / or cell lines, and collecting cell samples and / or tissue samples. Total RNA was extracted and its purity was detected. Qualified RNA was synthesized into cDNA through a reverse transcription kit. Using cDNA as a template, according to the operation of the fluorescence quantitative kit, the relative expression level of sturgeon anti-pathogen gene mRNA at different times was calculated. The experimental methods of the present invention can be prepared by conventional methods.
[0018] The following introduces an identification method of an anti-sturgeon pathogen infection gene provided in the embodiments of the present application, analysis of the p53 gene structure and its mRNA expression pattern in Chinese sturgeon after being attacked by the viral RNA analog poly I:C and Mycobacterium marinum.
[0019] Experimental method:
[0020] 1. The p53 gene sequence was obtained from the Chinese sturgeon transcriptome library. Specific primers were designed using Primer 6.0 software (see Table 1), and PCR amplification was performed with cDNA as the template. After purification of the product, it was ligated to the pMD19-T vector, transformed into competent E. coli DH5α cells, and positive clones were screened and verified by sequencing. Then, its gene structure and evolutionary relationship were analyzed.
[0021] Table 1: Specific primers
[0022]
[0023] 2. Samples of spleen, blood, brain, gill, skin, heart, head kidney, mid kidney, liver, intestine and muscle tissues of healthy Chinese sturgeons were collected. Total RNA of the tissues was extracted and its purity was detected. Qualified RNA was reverse-transcribed into cDNA using a reverse transcription kit. RT-qPCR was used to detect the expression of the p53 gene in Chinese sturgeons (primers are shown in Table 1), with the EF-1α gene of Chinese sturgeons as the internal reference. Using cDNA as the template, and operating according to the fluorescence quantitative kit, the relative expression levels of the p53 gene mRNA in various tissues of sturgeons were calculated.
[0024] 3. The spleen cell line of Chinese sturgeons was stimulated with poly I:C, and the changes in the expression of the p53 gene at 3 h, 6 h, 12 h, 24 h and 48 h after infection were detected. Chinese sturgeons were infected with Mycobacterium marinum, and the changes in the expression of the p53 gene at 4 h, 8 h, 12 h, 24 h and 48 h after infection were detected. Total RNA of the cells was extracted and its purity was detected. Qualified RNA was reverse-transcribed into cDNA using a reverse transcription kit. RT-qPCR was used to detect the expression of the p53 gene in Chinese sturgeons (primers are shown in Table 1), with the EF-1α gene of Chinese sturgeons as the internal reference. Using cDNA as the template, and operating according to the fluorescence quantitative kit, the relative expression levels of the p53 gene mRNA in various tissues of sturgeons were calculated.
[0025] 4. The fluorescence quantitative data were analyzed by one-way ANOVA using SPSS 24.0 software. The data were expressed as mean ± standard deviation (mean ± SD), and the significance level was set at p < 0.05.
[0026] Experimental results:
[0027] 1. Molecular characteristics and evolutionary relationship of the p53 gene in Chinese sturgeons
[0028] The full length of the cDNA of the p53 gene in Chinese sturgeons is 1916 bp, containing an open reading frame of 1173 bp, encoding 390 amino acids. This protein has five highly conserved regions (SEQ ID NO.1 - 2). The DNA-binding domain of p53 in Chinese sturgeons consists of 3 α-helices and 11 β-sheets, and its secondary structure topology is highly similar to that of other species (Figure 1 )。 Its gene contains 11 exons and 10 introns ( Figure 2 )。 Phylogenetic analysis shows that the p53 of Chinese sturgeon is most closely related to that of Amur sturgeon and spotted gar, and has a relatively low consistency with mammals ( Figure 3 )。
[0029] 2. Tissue distribution characteristics of p53 mRNA in healthy Chinese sturgeon
[0030] Detected by RT-qPCR technology, the p53 mRNA of Chinese sturgeon was differentially expressed in all tissues examined. Among them, the expression level was the highest in muscle tissue, followed by skin, blood and head kidney, while the expression level was the lowest in spleen and brain tissues ( Figure 4 )。
[0031] 3. Expression dynamics of p53 in Chinese sturgeon under Poly I:C stimulation
[0032] After stimulating the spleen cell line of Chinese sturgeon with the viral RNA analog poly I:C, the expression level of AsP53 in the experimental group increased significantly after 3 h of stimulation (p<0.05), reached the peak at 12 h, and dropped back to the basal level at 48 h ( Figure 5 )。
[0033] 4. Expression characteristics of p53 in Chinese sturgeon after Mycobacterium marinum infection
[0034] Analysis of peripheral blood lymphocytes of Chinese sturgeon infected with Mycobacterium marinum by qRT-PCR found that the expression level of p53 in Chinese sturgeon was significantly up-regulated 4 h after infection (p<0.01), decreased transiently at 8 h, increased again at 12 h, and showed a gradually decreasing trend from 24 h to 48 h ( Figure 6 )。
[0035] The above content can identify the pathogen-resistant genes of sturgeon and participate in the host's anti-pathogen immune defense: restricting virus diffusion by regulating apoptosis during virus infection and playing a protective role by inhibiting the intracellular survival of pathogens during bacterial infection.
[0036] Although the above embodiments have described the present invention in detail, they are only a part of the embodiments of the present invention, rather than all embodiments. People can also obtain other embodiments based on these embodiments without creative efforts, and these embodiments all fall within the protection scope of the present invention. Brief description of the drawings
[0037] Figure 1 : Prediction results of the secondary structure of the DNA binding domain of p53 protein in Chinese sturgeon and other species;
[0038] Figure 2 : Comparison of p53 gene structures between Chinese sturgeon and other vertebrates;
[0039] Figure 3 : Phylogenetic tree of p53 protein;
[0040] Figure 4 : Tissue distribution characteristics of the p53 gene of Chinese sturgeon;
[0041] Figure 5 : Effects of Poly I:C stimulation on the expression of the p53 gene in Chinese sturgeon cells;
[0042] Figure 6 : Effects of Mycobacterium marinum infection on the expression of the 53 gene in peripheral blood lymphocytes of Chinese sturgeon. Legend:
[0043] Figure 1 : Among them, α-helix, β-sheet and random coil are represented by cylinders, arrows and thick lines respectively.
[0044] Figure 2 : Black boxes represent exons, and black horizontal lines represent introns. The exon size (bp) is marked inside the box, the intron size (bp) is marked above the horizontal line, and the intron phase is marked below the horizontal line. The accession numbers of the p53 gene sequences used are as follows: gar (XM_015340733.1), fugu (XM_003966884.3), frog (NM_001088098.1) and human (NM_001126112.3).
[0045] Figure 3: The numerical value of the branch node represents the similarity percentage (%). The GenBank accession numbers of the p53 protein sequences used for comparison are as follows: Acipenser ruthenus XP_034774718, Lepisosteus oculatus XP_015196221, Takifugu rubripes XP_011604971, Salmo salar ACN10490, Gallus gallus NP_990595, Homo sapiens BAC16799, Oncorhynchus mykiss NP_001118164, Danio rerio AAB40617, Mus musculus BAA82344, Ctenopharyngodon idella ASY04226, Carassius auratus XP_026066398, Siniperca chuatsi AWD73450, Paralichthys olivaceus ABQ42582, Xenopus tropicalis NP_001001903, Ovis aries CAA57349, Felis catus BAA05653.
[0046] Figure 4 : The tissues detected include: skin (SK), muscle (M), gill (G), intestine (I), brain (BR), mid-kidney (MK), head-kidney (HK), liver (L), blood (BL), heart (H) and spleen (SP). The data are from 4 independent individuals, and the error bars in the bar graph represent the mean ± standard deviation (n = 4).
[0047] Figure 5 : An asterisk (*) indicates a significant difference from the control group (*p < 0.05, **p < 0.01).
[0048] Figure 6 : An asterisk (*) indicates a significant difference from the control group (*p < 0.05, **p < 0.01).
Claims
1. A method for identifying a gene that resists pathogen infection in sturgeons, characterized in that: The following steps are involved: Identify pathogen resistance genes from the sturgeon transcriptome library and analyze their gene structure and amino acid sequence; Detecting the expression distribution of the anti-pathogen gene in different tissues of healthy sturgeons by RT-qPCR; The expression pattern of the anti-pathogenic gene mRNA after pathogen infection was analyzed.
2. The identification method according to claim 1, characterized in that: The anti-pathogen gene is a p53 gene, the full length of its cDNA is 1916 bp, contains an open reading frame (ORF) of 1173 bp, encodes 390 amino acids, and its GenBank accession number is ON454214.
3. The identification method according to claim 1 or 2, characterized in that: The gene structure analysis includes: Signal peptide prediction, open reading frame (ORF) and amino acid sequence analysis; Secondary structure prediction and phylogenetic tree construction.
4. The identification method according to claim 1, characterized in that: The tissues detected by RT-qPCR include: spleen, blood, brain, gills, skin, heart, head kidney, mesonephros, liver, intestine and muscle.
5. The identification method according to claim 1, characterized in that: The pathogenic infections include viral RNA analog poly I:C stimulation and Mycobacterium marinum infection.
6. The identification method according to claim 5, characterized in that: The poly I:C stimulation experiment uses a sturgeon spleen cell line to detect changes in gene expression at 3h, 6h, 12h, 24h and 48h after infection.
7. The identification method according to claim 5, characterized in that: The Mycobacterium marinum infection experiment uses sturgeon peripheral blood lymphocytes to detect changes in gene expression 4h, 8h, 12h, 24h and 48h after infection.
8. An application of a gene for resisting pathogen infection in sturgeons, characterized in that: The anti-pathogen gene is a p53 gene, which is used to prepare a sturgeon disease prevention and control preparation.
9. The use according to claim 8, characterized in that: The disease prevention and control preparation is used to inhibit virus replication or bacterial intracellular survival, thereby improving the disease resistance of sturgeons.