Assessment kit and assessment method for disease-resistant characters of oysters
By screening genes BMP7, EP4 and IL17 as disease-resistant trait indicators, real-time fluorescence quantitative PCR evaluation kits and methods were constructed, which solved the problem of difficult to quickly evaluate the disease-resistant traits of oysters in the prior art, and achieved the effect of rapid screening of high disease-resistant oyster varieties.
Patent Information
- Application Number
- CN202510715055.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-05-30
- Publication Date
- 2025-08-12
AI Technical Summary
The existing technology lacks methods to quickly evaluate the disease resistance of oysters, which leads to lag in the prevention and control of oyster diseases and affects the development of the breeding industry.
Based on the transcriptome data of Hong Kong oysters infected with Vibrio algae, the genes BMP7, EP4 and IL17 were screened as key indicators of disease resistance traits, and real-time fluorescence quantitative PCR evaluation kits and methods were constructed to calculate disease resistance through detection of gene expression through toxicity experiments.
It has achieved rapid and effective evaluation of oyster disease resistance traits, screened out highly disease-resistant varieties, and improved the disease resistance and economic value of oyster farming.
Smart Images
Figure CN120464754A_ABST
Abstract
Description
Technical Field
[0001] The present invention belongs to the technical field of aquaculture and disease-resistant variety breeding, and more specifically relates to a kit and method for evaluating the disease resistance of oysters. Background Art
[0002] Oysters are a major marine shellfish cultivated worldwide. Their widespread distribution, high yield, and high nutritional value make them highly sought after by consumers. However, with the development of high-density, intensive, and factory-style oyster farming, the environmental capacity is unable to meet the rapid expansion of aquaculture, leading to a deteriorating aquaculture environment and frequent mass oyster mortality events, resulting in significant economic losses.
[0003] Vibrio alginolyticus ( Vibrio alginolyticus Diseases caused by pathogenic microorganisms such as spores ( spores) are one of the main causes of mass oyster mortality. Compared to the rapid development of the oyster aquaculture industry, oyster disease control technology lags significantly, severely hindering the industry's growth and improving quality and efficiency. In addition to developing disease control drugs, breeding oyster varieties with high disease resistance is also crucial for oyster disease control. Oyster disease resistance directly impacts the yield, quality, and economic value of aquaculture varieties. Assessing oyster disease resistance and selecting highly resistant varieties and strains are crucial to successful aquaculture.
[0004] Although methods for selecting superior Vibrio-resistant oyster strains have been reported, these methods are complex to implement. Parents with different genetic backgrounds must first be selected to construct a base breeding population. Once the progeny are one year old or older, they serve as a reference population. Furthermore, challenge experiments using a highly virulent strain of Vibrio alginolyticus as a model pathogen are required to assess the resistance phenotype of the reference population. Genotypic data is then obtained to construct a model. Consequently, there is still a lack of methods for rapidly assessing oyster disease resistance. Developing an efficient and rapid method for assessing disease resistance in oysters is crucial for addressing current industry bottlenecks. Summary of the Invention
[0005] The present invention addresses the shortcomings of the prior art and provides a kit and method for evaluating oyster disease resistance. The kit and method can rapidly assess oyster disease resistance to Vibrio spp., thereby enabling the screening of highly resistant oyster varieties, strains, or populations.
[0006] The first object of the present invention is to provide a kit for evaluating disease resistance traits of oysters.
[0007] The second object of the present invention is to provide a method for detecting genes BMP7 、 EP4 and IL17 Application of a reagent for the relative expression amount of oysters in the preparation of a product for evaluating the disease resistance traits of oysters.
[0008] The third object of the present invention is to provide a method for evaluating disease resistance traits of oysters.
[0009] The above-mentioned purpose of the present invention is achieved through the following technical solutions: Based on the transcriptome data of Hong Kong oysters infected with Vibrio alginolyticus, the present invention combines experiments such as real-time fluorescence quantitative PCR and statistical analysis to obtain key indicator molecules that can be used to reflect the resistance of oysters to diseases caused by Vibrio alginolyticus, namely, genes BMP7 、 EP4 and IL17 Based on this, the present invention has constructed an evaluation kit and an evaluation method that can effectively evaluate the disease resistance traits of oysters.
[0010] The present invention provides an oyster disease resistance trait evaluation kit, which contains a gene for detecting BMP7 、 EP4 and IL17 Real-time fluorescence quantitative PCR primers for the relative expression levels of
[0011] Specifically, the gene BMP7 The nucleotide sequence of the gene is shown in SEQ ID NO.1; EP4 The nucleotide sequence of the gene is shown in SEQ ID NO.2; IL17 The nucleotide sequence is shown in SEQ ID NO.3.
[0012] Specifically, the disease resistance trait is a trait of resistance to diseases caused by Vibrio alginolyticus.
[0013] As an optional embodiment, the kit is used to detect the gene BMP7 The nucleotide sequences of the PCR primers are shown in SEQ ID NO. 10 to 11; used to detect the gene EP4 The nucleotide sequences of the PCR primers are shown in SEQ ID NO. 12 to 13; used to detect the gene IL17 The nucleotide sequences of the PCR primers are shown in SEQ ID NOs. 14 to 15.
[0014] Specifically, the oyster is Hong Kong oyster.
[0015] Specifically, the gene is detected BMP7 、 EP4 and IL17 When the relative expression level of oyster β -actin gene was used as the internal reference gene.
[0016] Specifically, the β The accession number of the -actin gene in the NCBI database is AF026063.
[0017] Specifically, the nucleotide sequences of the primers used to detect the internal reference gene are shown in SEQ ID NOs. 16-17.
[0018] The present invention claims protection for detecting genes BMP7 、 EP4 and IL17 Application of a reagent for the relative expression amount of oysters in the preparation of a product for evaluating the disease resistance traits of oysters.
[0019] Specifically, the disease resistance trait is a trait of resistance to diseases caused by Vibrio alginolyticus.
[0020] Specifically, the oyster is Hong Kong oyster.
[0021] The present invention also provides a method for evaluating the disease resistance of oysters, or a method for evaluating the disease resistance (disease resistance) of oysters against diseases caused by Vibrio alginolyticus using the kit of the present invention, that is, a method for using the kit, comprising the following steps: S1. Oysters to be evaluated for disease resistance were challenged with Vibrio alginolyticus. Hemocytes were collected 0, 3, and 6 hours after challenge, and total RNA was extracted and reverse-transcribed into cDNA. S2. Using the cDNA obtained in S1 as a template, β -actin gene was used as the internal reference gene, and the gene was detected by real-time fluorescence quantitative PCR. BMP7 、 EP4 and IL17 The relative expression levels of the cytokines at 0 h, 3 h, and 6 h after the challenge were obtained. S3. Calculate genes separately BMP7 、 EP4 and IL17 The ratio of the relative expression level at 3 h and 6 h to the relative expression level at 0 h was converted to obtain the gene BMP7 、 EP4 and IL17 The 3 h and 6 h scores of the gene are averaged, and the gene score of the gene is taken as the average score of the gene scores of the three genes. If the average score of the gene scores of the three genes is greater than 90, the disease resistance is strong, otherwise it is weak; Among them, genes BMP7 、 EP4 and IL17 The conversion method of 3 h and 6 h scores is: if the gene BMP7 or EP4 If the ratio of the relative expression level at 3 h or 6 h to that at 0 h is ≥3, the score is 100 points; if the ratio is 2≤<3, the score is 90 points; if the ratio is 1≤<2, the score is 80 points; if the ratio is <1, the score is 70 points; IL17If the ratio of the relative expression level at 3 h or 6 h to that at 0 h was ≥20, the score was 100 points; if the ratio was 10≤<20, the score was 90 points; if the ratio was <10, the score was 80 points.
[0022] Specifically, in S1, 80–100 μL of a 1.0×10 9 The alginolytic Vibrio spp. liquid with a CFU / mL was placed at 27±0.5℃ for the challenge experiment.
[0023] Specifically, the oysters to be evaluated for disease resistance in S1 are oysters randomly collected from the same oyster species, strain or population.
[0024] Specifically, the oyster is Hong Kong oyster.
[0025] Specifically, in S2, for detecting the gene BMP7 The nucleotide sequences of the PCR primers are shown in SEQ ID NO. 10 to 11; used to detect the gene EP4 The nucleotide sequences of the PCR primers are shown in SEQ ID NO. 12 to 13; used to detect the gene IL17 The nucleotide sequences of the PCR primers are shown in SEQ ID NOs. 14 to 15; the nucleotide sequences of the primers used to detect the internal reference gene are shown in SEQ ID NOs. 16 to 17.
[0026] In a specific embodiment of the present invention, PerfectStart @ Real-time fluorescence quantitative PCR was performed using the Green qPCR SuperMix kit. The reaction system was as follows: 5 μL qPCR enzyme, 0.4 μL cDNA template, 0.4 μL each of primer F and primer R, and double-distilled water to 10 μL.
[0027] The reaction procedure adopted a three-step method, which was as follows: pre-denaturation at 95°C for 5 min; 45 cycles of 95°C for 10 s, 60°C for 15 s, and 72°C for 15 s; then 95°C for 10 s, 65°C for 60 s, and 95°C for 1 s to establish the melting curve, and finally cooling at 37°C for 30 s.
[0028] The present invention has the following beneficial effects: Based on the transcriptome data of Hong Kong oysters infected with Vibrio alginolyticus, the present invention screened out 9 differentially expressed immune-related genes. Based on the qRT-PCR detection results of the differentially expressed immune-related genes at different times after Vibrio alginolyticus infection, key indicator molecules (genes) that can be used to reflect the resistance of oysters to diseases caused by Vibrio alginolyticus were obtained by digitalization. BMP7 、 EP4 and IL17) and constructed a corresponding evaluation kit and evaluation method. The evaluation method was to test the gene by conducting a Vibrio alginolyticus challenge experiment on oysters. BMP7 、 EP4 and IL17 The relative expression levels of the three genes are then scored based on their expression. Finally, the average score of the three genes is calculated. Oysters with a score above 90 are considered to have good disease resistance traits. The evaluation kit and evaluation method of the present invention can be used to screen for highly disease-resistant oyster varieties, strains, or populations, facilitating the cultivation of disease-resistant oysters. BRIEF DESCRIPTION OF THE DRAWINGS
[0029] Figure 1 Differentially expressed immune-related genes BMP7 、 EP4 、 IL17 、 NFYA 、 HSP90 、 TRAF6 、 BIRC2 、 EGFR and RIG-I The expression of different time after the attack of Vibrio alginolyticus; * in the figure indicates significant difference. P <0.05; ** indicates extremely significant difference. P <0.01; ns indicates not significant; the results are expressed as mean ± standard error (N = 3).
[0030] Figure 2 Figure 1 is the result of non-specific immune enzyme activity test of Hong Kong oysters during the Vibrio alginolyticus challenge period; Figure A is the result of superoxide dismutase (SOD) activity test; Figure B is the result of malondialdehyde (MDA) content test; * indicates significant difference in the figure. P <0.05; ** indicates extremely significant difference. P <0.01; ns indicates no significant difference.
[0031] Figure 3 is the survival rate of Hong Kong oysters during the Vibrio alginolyticus challenge period (N=10); ** in the figure indicates extremely significant differences. P <0.01. DETAILED DESCRIPTION
[0032] The present invention will be further described below with reference to the accompanying drawings and specific examples, but the examples do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.
[0033] Unless otherwise specified, the reagents and materials used in the following examples were commercially available.
[0034] Example 1 Screening of differentially expressed immune-related genes in response to Vibrio alginolyticus infection in oysters The present invention uses Hong Kong oyster ( Crassostrea hongkongensis ) were used as experimental materials, and Vibrio alginolyticus ( Vibrio alginolyticus ) were used to analyze the transcriptome of oysters after infection with Vibrio alginolyticus and screen for differentially expressed immune-related genes.
[0035] Eighty healthy Hong Kong oysters were selected and randomly divided into two groups: a control group and a challenge group. The control group was injected with 100 μL of phosphate buffered saline (PBS) solution, and the challenge group was injected with 100 μL of 1.0×10 9 CFU / mL of Vibrio alginolyticus liquid was used for Vibrio challenge test at room temperature (27±0.5℃); blood cells of oysters challenged for 0 h, 3 h, 6 h and 12 h were collected for transcriptome library construction.
[0036] To obtain high-quality sequences, adapter sequences, reads containing more than 10% unknown nucleotides, and more than 50% low-quality (q ≤ 20) bases were first removed from the original sequences. The processed sequences were mapped to the Hong Kong oyster genome (reference genome GenBank accession number: GCA_016163765.1), and the highly matched sequences were further annotated. The DESeq2 software was used to perform RNA differential expression analysis between the control group and the challenge group. The differentially expressed genes (DEGs) screened at 3 h and 6 h were sorted according to the log2 (FC) size, and the DEGs were selected based on the sorting results. BMP7 、 EP4 、 IL17 、 NFYA 、 HSP90 、 TRAF6 、 BIRC2 、 EGFR and RIG-I , a total of 9 differentially expressed immune-related genes were used as indicator genes for disease resistance trait evaluation in subsequent experiments.
[0037] Nine differentially expressed immune-related genes screened from the transcriptome BMP7 、 EP4 、 IL17 、 NFYA 、 HSP90 、 TRAF6 、 BIRC2 、 EGFR and RIG-I The nucleotide sequences are shown in SEQ ID NOs. 1 to 9.
[0038] Example 2 Analysis of expression patterns of differentially expressed immune-related genes The same method as in Example 1 was used to conduct a challenge experiment with Vibrio alginolyticus on Hong Kong oysters. Oyster blood cells were collected at 0 h, 3 h, 6 h, and 12 h after the challenge, and the total RNA was extracted. The extracted total RNA was reverse transcribed into cDNA, and the obtained cDNA was used as a template to generate the oyster RNA. β -actin gene (accession number AF026063) was used as an internal reference gene, and the expression patterns of the differentially expressed immune genes screened in Example 1 were analyzed by real-time fluorescence quantitative PCR (qRT-PCR).
[0039] The present invention targets differentially expressed immune-related genes BMP7 、 EP4 、 IL17 、 NFYA 、 HSP90 、 TRAF6 、 BIRC2 、 EGFR 、 RIG-I and internal reference genes β The PCR primers designed for -actin are shown in Table 1. The designed primers were synthesized by Sangon Co., Ltd.
[0040] Table 1 PCR primers
[0041] Using PerfectStart @ Green qPCR SuperMix kit (Beijing Quanshijin Biotechnology Co., Ltd.) was used for real-time fluorescence quantitative PCR experiments. -∆∆Ct The relative expression level of each target gene was calculated by the PCR method; three technical replicates were set for each sample. The reaction system and reaction procedure are as follows: Reaction system (10 μL): 5 μL qPCR enzyme, 0.4 μL cDNA template (total RNA was extracted from oyster hemocytes at 0 h, 3 h, 6 h, and 12 h after infection and reverse transcribed into cDNA as template), 0.4 μL each of primer F and primer R at a concentration of 10 μM, and double-distilled water to make up to 10 μL.
[0042] The reaction procedure was as follows: a three-step method was used; pre-denaturation at 95°C for 5 min; 45 cycles of 95°C for 10 s, 60°C for 15 s, and 72°C for 15 s; then, a melting curve was established by heating at 95°C for 10 s, 65°C for 60 s, and 95°C for 1 s, and finally cooling at 37°C for 30 s.
[0043] The differentially expressed immune-related genes BMP7 、 EP4 、 IL17 、 NFYA 、 HSP90 、 TRAF6 、 BIRC2 、 EGFR and RIG-I The expression of different time periods of Vibrio alginolyticus infection is as follows Figure 1 As shown. Figure 1 It can be seen that among the DEGs detected, TRAF6 、 EGFR and BIRC2 The expression level reached the highest at 3 h and was significantly higher than that at 0 h and 6 h. IL17 、 EP4 and RIG-I The expression levels were at the highest level at 3 h and 6 h, which were significantly higher than those at 0 h, and there was no significant difference between 3 h and 6 h. BMP7 、 NFYA and HSP90 The expression level was the highest at 6 h, and was significantly higher than that at 0 h and 3 h.
[0044] Example 3 Analysis of non-specific immune enzyme activity in oysters The same method as in Example 1 was used to challenge Hong Kong oysters with Vibrio alginolyticus. Oyster hemolymph was collected 3 h, 6 h, and 12 h after challenge, and the SOD activity and malondialdehyde (MDA) content were detected using a superoxide dismutase (SOD) assay kit and a malondialdehyde (MDA) test kit (Nanjing Jiancheng Bioengineering Institute).
[0045] The specific operations are as follows: SOD activity assay: Mix 50 μL of collected hemolymph with 1000 μL of Reagent I application solution, 100 μL of Reagent II, 100 μL of Reagent III, and 100 μL of Reagent IV application solution in the kit. Incubate at 37°C for 40 min. Then add 2000 μL of the display reagent and let it stand at room temperature for 5 min. Calculate the SOD activity based on the absorbance value at 550 nm.
[0046] MDA content determination: 100 μL of collected hemolymph was mixed with 100 μL of reagent I in the kit, and 3000 μL of reagent II and 1000 μL of reagent III were added in sequence. The mixture was mixed, and the mixture was incubated in a 95°C water bath for 40 min. After cooling with cold water, the mixture was centrifuged at 4000 rpm for 10 min. The absorbance of the supernatant at 523 nm was measured and the MDA content was calculated.
[0047] During the Vibrio alginolyticus challenge period, the results of non-specific immune enzyme activity detection in the hemolymph of Hong Kong oysters were as follows: Figure 2 As shown; Figure 2 A in the figure is the result of superoxide dismutase (SOD) test; Figure 2B in the table is the result of malondialdehyde (MDA) content determination. Figure 2 As shown in A, compared with the control group, the SOD activity of the challenge group increased by 1.09 times at 3 h ( P <0.01), and increased to 1.14 times after 6 h ( P <0.01). Figure 2 As shown in Figure B, the MDA content in the challenge group increased by 2.25 times at 3 h compared with the control group ( P <0.05), and increased to 3.75 times in 6 h ( P <0.05). The above results indicate that the immune stress response of Hong Kong oysters is activated, and the activation time is consistent with the differentially expressed immune-related genes.
[0048] Example 4 Obtaining key evaluation indicators and constructing an evaluation method for oyster disease resistance traits Based on the real-time fluorescence quantitative PCR experimental results in Example 2, the present invention conducted statistical and significant analysis on the expression changes (sensitivity) of the differentially expressed immune-related genes at different times of challenge. Compared with 0 h, at 3 h of challenge, IL17 The expression level increased to 24.15 times ( P <0.01), EP4 Increased to 20.73 times ( P <0.01), BMP7 Increased to 15.59 times ( P <0.01), TRAF6 increased to 3.67 times ( P <0.01), BIRC2 increased to 2.83 times ( P <0.01), EGFR increased to 2.53 times ( P <0.01), HSP90 Increased to 2.08 times ( P <0.01), RIG-I Increased to 1.65 times ( P <0.01). At 6 h after challenge, compared with 0 h, BMP7 The expression level increased to 30.26 times ( P <0.01), EP4 Increased to 26.06 times ( P <0.01), IL17 Increased to 25.20 times ( P <0.01), NFYA Increased to 15.78 times ( P <0.01), HSP90 increased to 4.03 times ( P <0.01), TRAF6Increased to 2.67 times ( P <0.01), RIG-I Increased to 1.66 times ( P <0.01).
[0049] Subsequently, the disease resistance evaluation ability of the differentially expressed immune-related genes was digitized based on the sensitivity analysis results, key evaluation indicators were obtained, and an oyster disease resistance evaluation method was constructed.
[0050] 1. Digitalization of the ability to evaluate disease resistance traits based on differentially expressed immune-related genes (immune indicators) The real-time fluorescence quantitative PCR results obtained in Example 2 were analyzed using the statistical interval double ranking method, and no statistical difference was found between the samples ( P >0.05) are in the same interval, otherwise they are in different intervals, and the same interval has the same integral. EP4 and IL17 There was no statistical difference in the expression levels of EP4 The expression peaks of are sorted by high to low, and it ranks second with a score of 90. IL17 The same score is also accumulated. Regarding the score, the index expression score is calculated by first ranking the differentially expressed genes from highest to lowest peak expression. The highest expression peak in the first interval is 100 points, the second interval is 90 points, the third interval is 80 points, and so on. Furthermore, the reaction rate score is calculated by ranking the time at which the highest expression peak of each differentially expressed gene occurs. The first interval is 100 points if the highest expression level occurs within both 3 and 6 hours, the second interval is 90 points, and the third interval is 80 points if the highest expression level occurs within 3 hours. The expression score and reaction rate score are weighted 50%:50% to convert them into a comprehensive score.
[0051] The present invention is based on the real-time fluorescence quantitative PCR results obtained in Example 2, and the scores of the differentially expressed genes calculated according to the above method are shown in Table 2.
[0052] Table 2 Scores of differentially expressed genes
[0053] Combined with the results shown in Table 2, the students with a comprehensive score of 90 or above BMP7 、 EP4 and IL17 Genes are used as key evaluation indicators; BMP7 、 EP4 、 IL17 、 NFYA 、 HSP90 、 TRAF6 、 BIRC2 、 EGFR 、 RIG-I As a general immune indicator molecule.
[0054] A method for evaluating oyster disease resistance traits was constructed using key evaluation indicators.
[0055] 2. Construction of evaluation method for oyster disease resistance based on BMP7 、 EP4 and IL17 The present invention constructs a method for evaluating the disease resistance of oysters to diseases caused by Vibrio alginolyticus using these three key evaluation indicators, and the method comprises the following steps: S1. Take the oysters whose disease resistance traits are to be evaluated and inject 100 μL of 1.0×10 9 CFU / mL of Vibrio alginolyticus were placed in a 27±0.5℃ environment for challenge experiments. Oyster blood cells were collected at 0 h, 3 h, and 6 h after challenge, and their total RNA was extracted and reverse transcribed into cDNA. S2. Using the cDNA obtained in S1 as a template, β -actin gene (accession number AF026063) was used as an internal reference gene, and the gene was detected by real-time fluorescence quantitative PCR (qRT-PCR). BMP7 、 EP4 and IL17 The relative expression levels of the cytokines at 0 h, 3 h, and 6 h after the challenge were obtained. S3. Calculate the score based on the qRT-PCR test results; the calculation method is: BMP7 and EP4 The ratio of the relative expression of the gene at 3 h and 6 h to 0 h was ≥3, which was scored as 100 points; 2≤ratio<3 was scored as 90 points; 1≤ratio<2 was scored as 80 points; ratio<1 was scored as 70 points. The average of the scores of 3 h / 0 h and 6 h / 0 h was used to obtain the gene score. IL17 The ratio of the relative expression levels of the gene at 3 h and 6 h to 0 h was ≥20, scored as 100 points, 10≤<20, scored as 90 points; and ratio <10, scored as 80 points. The scores of 3 h / 0 h and 6 h / 0 h were averaged to obtain the gene score. Finally, the average of the gene scores of the three genes represented the disease resistance of the tested oyster population to diseases caused by Vibrio alginolyticus. Oysters with a score of 90 or above had stronger disease resistance.
[0056] Among them, qRT-PCR detection of genes BMP7 、 EP4 and IL17 Used to detect gene expression at different time points BMP7The nucleotide sequences of the primers are shown in SEQ ID NO. 10 to 11; EP4 The nucleotide sequences of the primers are shown in SEQ ID NO. 12-13; IL17 The nucleotide sequences of the primers are shown in SEQ ID NOs. 14 to 15; the nucleotide sequences of the primers used to detect the internal reference gene are shown in SEQ ID NOs. 16 to 17; the qRT-PCR reaction system and reaction procedure are the same as in Example 2.
[0057] Example 5 Application of the disease resistance trait evaluation method In the present invention, 20 healthy Hong Kong oysters (the collected oysters belong to the same group) were collected from two farms in different regions (farm I and farm II), and the Vibrio alginolyticus challenge experiment was performed on them according to the evaluation method described in Example 4. Oyster blood lymphocytes were collected at 0 h, 3 h, and 6 h after the challenge, and qRT-PCR detection was performed. BMP7 、 EP4 and IL17 The relative expression levels of the genes (detection method is the same as in Example 2). Based on the qRT-PCR detection results, the average score of the three genes was calculated. The average score of the three genes represents the disease resistance of the oyster population. Oysters with a score greater than 90 have strong disease resistance.
[0058] The results are shown in Table 3.
[0059] Table 3 Evaluation scores of disease resistance traits of Hong Kong oysters
[0060] As shown in Table 3, the oyster populations in Farms I and II had strong disease resistance, with Farm II scoring 91.6 points and Farm I scoring 90.0 points, indicating that the oysters in Farm II had stronger disease resistance than those in Farm I.
[0061] Example 6 Verification of the Accuracy of the Hong Kong Oyster Disease Resistance Evaluation System In the present invention, 20 healthy Hong Kong oysters were collected from farms I and II described in Example 5, respectively, and randomly divided into two groups, a control group and a challenge group. The control group was injected with 100 μL PBS, and the challenge group was injected with 100 μL high concentration (1.0×10 11 CFU / mL) of Vibrio alginolyticus were tested at room temperature (27±0.5℃). During the Vibrio challenge, the oysters were observed regularly. If the shells were found to be open and there was no closing reaction when the soft parts were touched with tweezers, they were considered dead. Dead oysters were picked out in time and the number of dead oysters was recorded.
[0062] The oyster survival rate was calculated as follows: RS = (n1-n2) / n1×100%; Where n1 is the initial number of oysters and n2 is the number of dead oysters.
[0063] The survival rate of Hong Kong oysters during the attack period of Vibrio alginolyticus is as follows: Figure 3 As shown. Figure 3 It can be seen that the survival rate of Hong Kong oysters in the control group of the two farms was always 100% during the Vibrio challenge period. Six hours after the challenge, the survival rate of farm I dropped to 60%, while the survival rate of farm II remained 100%, with a significant difference between the two ( P <0.01); 12 hours after challenge, the survival rate of Hong Kong oysters in Farm I dropped to 0, while the survival rate in Farm II was 70%; the Hong Kong oysters in Farm II did not die until 72 hours after challenge. This indicates that the disease resistance of oysters in Farm I was significantly lower than that in Farm II. This data is consistent with the results in Example 5, demonstrating the accuracy and practicality of the oyster disease resistance evaluation method established by this invention.
[0064] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be considered as equivalent replacement methods and are included in the scope of protection of the present invention.
Claims
1. A kit for evaluating disease resistance of oysters, characterized in that: The kit contains a BMP7 、 EP4 and IL17 Real-time fluorescence quantitative PCR primers for the relative expression of The gene BMP7 The nucleotide sequence of the gene is shown in SEQ ID NO.1; EP4 The nucleotide sequence of the gene is shown in SEQ ID NO.2; IL17 The nucleotide sequence is shown in SEQ ID NO.3; the disease resistance trait is a trait of resistance to diseases caused by Vibrio alginolyticus.
2. The kit according to claim 1, wherein For detecting the gene BMP7 The nucleotide sequences of the PCR primers are shown in SEQ ID NO. 10 to 11; used to detect the gene EP4 The nucleotide sequences of the PCR primers are shown in SEQ ID NO. 12 to 13; used to detect the gene IL17 The nucleotide sequences of the PCR primers are shown in SEQ ID NOs. 14 to 15.
3. The kit according to any one of claims 1 or 2, characterized in that The oyster is Hong Kong oyster.
4. The kit according to any one of claims 1 or 2, characterized in that Detection of the gene BMP7 、 EP4 and IL17 When the relative expression level of oyster β -actin gene was used as the internal reference gene.
5. The kit according to claim 4, wherein The nucleotide sequences of the primers used to detect the internal reference gene are shown in SEQ ID NOs. 16-17.
6. Used for gene detection BMP7 、 EP4 and IL17 The use of a reagent for the relative expression amount of oyster in the preparation of a product for evaluating the disease resistance of oysters is characterized in that: The gene BMP7 The nucleotide sequence of the gene is shown in SEQ ID NO.1; EP4 The nucleotide sequence of the gene is shown in SEQ ID NO.2; IL17 The nucleotide sequence is shown in SEQ ID NO.3; the disease resistance trait is a trait of resistance to diseases caused by Vibrio alginolyticus.
7. The application according to claim 6, characterized in that The oyster is Hong Kong oyster.
8. A method for evaluating disease resistance of oysters, characterized in that: The disease resistance trait is a trait of resistance to diseases caused by Vibrio alginolyticus, and the method comprises the following steps: S1. Oysters to be evaluated for disease resistance were challenged with Vibrio alginolyticus. Hemocytes were collected 0, 3, and 6 hours after challenge, and total RNA was extracted and reverse-transcribed into cDNA. S2. Using the cDNA obtained in S1 as a template, β -actin gene was used as the internal reference gene, and the gene was detected by real-time fluorescence quantitative PCR. BMP7 、 EP4 and IL17 The relative expression levels of the cytokines at 0, 3, and 6 h after the challenge were obtained. S3. Calculate genes separately BMP7 、 EP4 and IL17 The ratio of the relative expression level at 3 h and 6 h to the relative expression level at 0 h was converted to obtain the gene BMP7 、 EP4 and IL17 The 3 h and 6 h scores of the gene are averaged, and the gene score of the gene is taken as the average score of the gene scores of the three genes. If the average score of the gene scores of the three genes is greater than 90, the disease resistance is strong, otherwise it is weak; Among them, genes BMP7 、 EP4 and IL17 The conversion method of 3 h and 6 h scores is: if the gene BMP7 or EP4 If the ratio of the relative expression level at 3 h or 6 h to that at 0 h is ≥3, the score is 100 points; if the ratio is 2≤<3, the score is 90 points; if the ratio is 1≤<2, the score is 80 points; if the ratio is <1, the score is 70 points; IL17 If the ratio of the relative expression level at 3 h or 6 h to that at 0 h was ≥20, the score was 100 points; if the ratio was 10≤<20, the score was 90 points; if the ratio was <10, the score was 80 points.
9. The method according to claim 8, wherein In S1, 80-100 μL of a 1.0×10 9 The alginolytic Vibrio spp. liquid with a CFU / mL was placed at 27±0.5℃ for the challenge experiment.
10. The method according to claim 8 or 9, characterized in that: The oyster is Hong Kong oyster.