CN106256904B - Genetic marker related to immune traits of grass carp hemorrhagic disease - Google Patents

Abstract

The invention belongs to the technical field of fish genetic marker screening and preparation, and particularly relates to a genetic marker related to immune traits of grass carp hemorrhagic disease. The genetic marker is obtained by screening from a C7N1 gene. The genetic marker is obtained by sequencing transcriptome of a grass carp individual level resistance population and a susceptible population, sequencing transcriptome of a cell level resistance and susceptible cell, correlation analysis and infection verification, and is shown as SEQ ID NO: 1, and the following components: a substitution mutation of one base is carried out at the 2145 th base of the C7N1 gene, so that the 715 th amino acid of the grass carp C7N1 protein is changed. The grass carp reovirus infection test shows that: the mortality rate of the heterozygous genotype grass carp is obviously lower than that of the homozygous grass carp, thereby indicating that the basic group mutation of the grass carp C7N1 gene is related to the hemorrhagic disease character of the grass carp. The invention provides a new genetic marker resource for the breeding of the immune-related traits of the hemorrhagic disease of the grass carp.

Description

Genetic marker related to immune traits of grass carp hemorrhagic disease

Technical Field

The invention belongs to the technical field of fish genetic marker screening and preparation, and particularly relates to a genetic marker related to immune traits of grass carp hemorrhagic disease. The genetic marker is obtained from a C7N1 gene. The invention also relates to application of the genetic marker in association analysis of the genetic marker of the grass carp hemorrhagic disease immune trait.

Background

Grass carp (Ctenophagogon idella, the English name grass carp) is an important freshwater fish in China, and the yield of the grass carp accounts for about 18% of the total yield of freshwater fish (Rao YL et al 2015. insects into the aquaculture grass carp. J Immunol Res, 670437). Grass carp hemorrhagic disease is the most serious disease in the fingerling stage of Grass carps, has long onset season, wide epidemic range and high mortality rate, often causes great economic loss to culturists, and has the pathogen of Grass Carp Reovirus (GCRV), wherein the virus consists of 11 double-stranded ribonucleic acid (dsRNA) segments and two layers of capsid proteins. With the rapid development of sequencing technologies, new genes and their functions are continuously being explored. At present, the human, murine, zebrafish C7N1 gene sequence has been reported (Wan D et al 2004 Large-scale cDNA transformation for genes related to cancer. Proc Natl Acad Sci USA 101(44):15724 and 15729), but there is no study on its molecular structure and function.

The C7N1 gene is located on chromosome 7 of grass carp, and the coded protein is a novel protein and consists of 993 amino acids. Even in higher mammals, no relevant reports on their biological functions have been found. Bioinformatics analysis of C7N1 revealed that the gene contains 3 exons and 2 introns, and that the amino acid sequence of the gene has 72% similarity to the homologous sequence of zebrafish, which was found to play an important role in the metabolic pathways of cysteine and methionine.

At present, the research on the cloning and the function of grass carp immunity-related genes is greatly improved. On the basis of such abundant theoretical research, it is necessary to find an efficient and feasible method for breeding grass carp hemorrhagic disease resistant varieties. Traditional breeding methods are time-consuming and labor-intensive, and genetic marker-based breeding methods are increasingly being adopted by researchers. The development of molecular breeding technology for aquatic animals has been in the genome era after decades, and has been advancing toward the ambitious goal of molecular breeding. With the completion of whole genome sequencing of many economic organisms, it is urgent to deeply mine useful biological information from massive genetic sequence information, and further to convert the knowledge into technology and apply the technology to production practice. Among them, genetic polymorphism identification is an important part of the research in the post-genome era. Through correlation analysis and linkage analysis, the variation of gene structure and the variation of phenotype are related, and genes and markers related to economic traits are searched, so that the genes and markers are used for molecular genetic breeding. On the Sinilabeo avellana, there have been reports of breeding new species using microsatellite marker breeding methods, and this report demonstrates that molecular genetic breeding is an effective breeding method (Sahu DK et al 2013.Identification of reproduction-related genes and SSR-markers through expressed sequence tags analysis of amonsoon breeding Labcarp roha, Haemohita (Haimmiton) Gene 524 (524) (1-14)). In 1 month 2015, Qiuguang reviewed the application of important economic traits in molecular Genetic breeding of fish in journal of Science China Life Sciences, analyzed the characteristics of molecular breeding, and provided important basis for breeding of disease-resistant and stress-resistant strains, vaccine development and disease management (Tong JG et al 2015.Genetic and Genetic analysis for molecular antigens and vaccine in molecular breeding of cultured fish Science China Life Sciences (58) (178-. Meanwhile, comparative genomics is also widely adopted in research. Grass carp, zebra fish and gobiocypris rarus belong to the same family of carpidae, but show different disease resistance to GCRV infection, researches show that the mortality rate of grass carp infected with GCRV is 50-90%, the mortality rate of gobiocypris rarus can reach 100%, and the zebra fish is not lethal. Provides a direction for exploring the anti-virus immune mechanism of the three, wherein the difference of the three on the gene level can be more widely excavated by comparing genomics, and lays a solid theoretical foundation for further research.

Relevant research suggests that we can combine basic theoretical research with production practice. And performing association analysis on the genetic polymorphism of the grass carp C7N1 gene and grass carp hemorrhagic disease to obtain disease-resistant related genetic markers. In the research, the sequencing and PCR-RFLP technology developed in the early stage provides exact technical guarantee for the research of genetic polymorphism.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and screen a genetic marker related to the immune trait of the hemorrhagic disease of the grass carp. The genetic marker is obtained by screening from a C7N1 gene, an SNP of the C7N1 gene is found, and further, the genetic marker can be applied to association analysis of the genetic marker of the immunity character of the hemorrhagic disease of the grass carp.

In the invention, the applicant carries out transcriptome sequencing on resistant/susceptible spleen, head kidney tissues and monoclonal CIK cells of grass carps after GCRV infection, and correlation analysis finds that one SNP of the C7N1 gene is closely related to grass carp resistance. Infection experiments prove that: the base mutation in the C7N1 gene is obviously related to grass carp hemorrhagic disease.

The invention screens mutation sites related to the grass carp hemorrhagic disease immune traits by means of a high-throughput sequencing method, verifies candidate sites by utilizing PCR amplification and Sanger sequencing technologies, carries out genotyping on the verified sites by utilizing PCR or PCR-RFLP technologies, and carries out correlation analysis on the typing result and grass carp hemorrhagic disease immune traits of natural grass carp populations, thereby identifying and obtaining genetic markers related to the grass carp hemorrhagic disease immune traits and providing new genetic markers for auxiliary selection of breeding grass carp varieties resistant to GCRV infection.

The technical scheme of the invention is as follows:

the applicant identifies that the grass carp C7N1 gene has the same mutation site on individual and cell levels by a high-throughput sequencing method, and the complete cDNA sequence of the grass carp C7N1 gene is shown as SEQ ID NO: 1, the preparation method is as follows. Through sequence alignment, a single base substitution mutation exists at 2145 of the sequence coding region, and the mutation does not generate special enzyme cutting polymorphism. However, this mutation results in an amino acid change (corresponding amino acid sequence is shown in SEQ ID NO: 2), which may affect the function of the C7N1 gene of grass carp. After amplifying a fragment containing the mutant base by the designed specific primer, sequencing confirms that the mutant site is: g or A (the sequence is shown in SEQ ID NO: 3, and the sequence is a sequence mutant fragment shown in SEQ ID NO: 1). In addition, the transcriptome sequencing results indicated that: the two types of grass carp C7N1 genes have expression differences in resistant and susceptible grass carp populations and CIK cells (see Table 1). In order to verify the difference, the genetic typing and data statistical analysis of the grass carps with resistance and susceptibility after infection prove that the site mutation has a significant correlation with the anti-hemorrhagic disease character: the number of individuals with a genotype heterozygous in the resistant population is greater than in the susceptible population. Therefore, the locus is feasible as a genetic marker for predicting and breeding the immune trait of the hemorrhagic disease of the grass carp.

To amplify the fragment containing this site, applicants designed specific primer pairs as shown below:

a forward primer: 5'-GACTGAGAATGCTGTGAAAGATG-3' the flow of the air in the air conditioner,

reverse primer: 5'-CAATGAGGTGGGATTTTAGTGA-3' are provided.

Specifically, the technical scheme of the invention is as follows:

a genetic marker related to immune traits of grass carp hemorrhagic disease has a nucleotide sequence shown in a sequence table SEQ ID NO: 1, there is a substitution mutation of one base at 2145 of the sequence, i.e., a base G to a base A.

Sequence listing SEQ ID NO: 1 is shown in a sequence table SEQ ID NO: 2, and a 1 amino acid substitution mutation at 715 of the sequence.

The applicant provides a primer pair for detecting said genetic marker, the sequence of which is as follows:

a forward primer: 5'-AAGGGCTGCGGATCGGAT-3'

Reverse primer: 5'-CCTGCCTGACAGACATTTATTGC-3' are provided.

The applicant provides a method for screening genetic markers related to the immune trait of grass carp hemorrhage, comprising the following steps:

1) grass carp CIK cells are divided into resistant individuals and susceptible individuals through a Grass Carp Reovirus (GCRV) infection experiment, wherein Grass carp CIK cells of a natural population are divided into two types of resistance and susceptibility through single cell sorting and GCRV infection experiments by flow cytometry;

2) extracting RNA of head kidney, spleen and monoclonal CIK cells of the grass carp, and screening genetic markers through transcriptome sequencing;

3) and verifying the splicing result of the C7N1 gene in the transcriptome by using mixed pool sequencing, wherein the cDNA sequence of the C7N1 gene is shown as SEQ ID NO: 1 is shown in the specification;

4) typing the C7N1 gene by using a PCR combined 3.0% agarose gel electrophoresis method;

5) the trait association analysis was performed using online analysis software SHEsis (http:// analysis. bio-x.cn/myanalysis. php).

The genetic marker related to the hemorrhagic disease immune trait of the grass carp can be applied to the detection of the hemorrhagic disease immune trait of the grass carp.

The primer pair disclosed by the invention can be applied to immune trait detection of grass carp hemorrhage.

Compared with the prior art, the invention has the following beneficial effects:

the invention screens mutation sites related to the immunity character of grass carp hemorrhage by combining high-throughput sequencing results on individual and cell levels, selects differentially expressed genes to carry out individual test verification by combining the sites so as to excavate genetic marker sites related to disease resistance characters, and simultaneously compares grass carp genes with homologous genes of zebra fish and gobiocypris rarus to analyze the base types of the mutation sites in different species. Compared with the traditional screening of the single-gene nucleotide polymorphic sites, the screening has the advantages of high flux, accurate detection, high speed and more reliable obtained results. The SNP mutation site obtained by the invention lays a theoretical foundation for the breeding of grass carp hemorrhage resistance in the future.

For a more detailed technical solution, refer to the embodiments in the description of the drawings and the detailed description of the invention.

Drawings

Sequence listing SEQ ID NO: 1 is the complete cDNA sequence (1-2982) of the C7N1 gene of the present invention, and a SNP site is present at base 2145 of the sequence (base A after mutation is shown at base 2145).

Sequence listing SEQ ID NO: 2 is the amino acid sequence encoded by the C7N1 gene. The sequence length is 993aa (stop codon TGA is not translated into amino acids). There is a 1 amino acid substitution at position 715 of the sequence (Met by Ile).

Sequence listing SEQ ID NO: 3 is the target fragment (117bp) amplified by the primer designed for genotyping the C7N1 gene mutation of this SNP, and the mutation exists at the 28 th base of the fragment (namely, G-A mutation). (the base shown at the 28 th base is a mutated base A)

Sequence listing SEQ ID NO: 4 and 5 are sequences of primer pairs for detecting the genetic marker of the present invention. As follows:

a forward primer: 5'-AAGGGCTGCGGATCGGAT-3'

Reverse primer: 5'-CCTGCCTGACAGACATTTATTGC-3' are provided.

FIG. 1: the general technical process schematic diagram of the invention.

FIG. 2: schematic diagram of resistance and susceptibility of grass carp CIK cells. Description of reference numerals: c1 is control group, R2 is resistant cell, S3 is susceptible cell; a, B, C, D, E, F, G and H respectively represent that the infection time of the cells is 0H,6H,12H,24H,48H,72H,96H and 120H.

FIG. 3: schematic representation of transcriptome sequencing strategy. Description of reference numerals: and respectively sequencing RNA of tissues and cells by adopting a double-tail sequencing method, and splicing and assembling.

FIG. 4: agarose electrophoresis picture of DNA extraction. Description of reference numerals: lane 1 in the figure: DNA molecular weight standards (marker 4); lanes 2-6: extracted tissue DNA.

FIG. 5: PCR amplifies the agarose electrophoresis picture containing the mutation site fragment. Description of reference numerals: lane 1 in the figure: DNA molecular weight standard (marker 1); lanes 2-9: amplified target fragment.

FIG. 6: and amplifying different genotype electrophoresis pictures after enzyme cutting of the fragment containing single base mutation by using a specific primer. Description of reference numerals: lane 1 in the figure: m, DNA molecular weight standard (marker 1); lane 2: AB, heterozygous individual; lane 3: AA, homozygous individual; lane 4: GG, homozygous individual.

FIG. 7: histogram of the statistics of the ratio of the three genotypes after infection with Grass carp reovirus (Grass carp reovirus, GCRV). Description of reference numerals: "+" indicates significant difference.

FIG. 8: the cDNA sequence of the C7N1 gene in the invention. R at base 2145 of this sequence is an allelic mutation (A/G) (underlined).

FIG. 9: the amino acid sequence (stop codon TGA untranslated amino acid) encoded by C7N1 gene in the present invention. A1 amino acid substitution is present at position 715 of the sequence, indicated by I and underlined.

FIG. 10: in the present invention, 117bp of the target fragment amplified by the primer designed for genotyping the gene mutation of SNP is underlined and R at 28 bases of the indicated sequence is an allelic mutation (A/G).

Detailed Description

Example 1: sequencing of spleen, head kidney and monoclonal CIK cell transcriptome of grass carp

(1) Infection assay

The aquarium used in this example was cleaned by soaking in 0.05% potassium permanganate solution for 24 hours, and then filled with clear water, the oxygen pump and the heating rod were installed, the temperature was set at 28 ℃ to 30 ℃, and the oxygen pump was turned on to prepare the temporary rearing of test grass carp. Grass carp is randomly divided into five groups (60 fish/group) before the Grass Carp Reovirus (GCRV) infection, temporarily cultured in a prepared aquarium, fed with palatable pellet feed twice at fixed point, fixed time and fixed amount every day, and cleaned of residual bait and metabolic waste in time. About one week, a virus infection test was prepared after the daily physiological activities of the fish school were normal. The GCRV-097 strain stock solution is a gift from the institute of aquatic organisms, academy of sciences of China. The viral suspension used for infection was a suspension collected after laboratory infection testing by the applicant and stored at-80 ℃ after activation. Penicillin and streptomycin were added to the virus suspension at 100IU/mL each before virus injection to prevent bacterial infection during injection. Four groups of grass carps were selected as the infected group, and the other group was the control group. The abdominal cavity and dorsal fin muscle of the infected grass carp were carefully injected with 0.1mL/g body weight of GCRV-097 virus suspension, and the control group was injected with the same dose of physiological saline.

The grass carp CIK cell line is from China center for type culture Collection (CCTCC for short, China, Wuhan university), the culture temperature of the grass carp CIK cell line is 28 ℃, and CO is CO₂At a concentration of 5%, 10% fetal bovine serum, and DMEM (purchased from Gibico, USA). Single Cell sorting culture and GCRV infection were performed by flow cytometry (BD FACSAria TMIII Cell Sorter, USA), so that resistant CIK cells and susceptible CIK cells were selected.

(2) Sampling

After GCRV-097 virus injection is finished, the activity condition of the fish body in the aquarium is observed every 3 hours, dead grass carps (namely susceptible grass carps) with grass carp bleeding disease symptoms occurring between 24 hours and 48 hours are collected in time, and sample numbers and records are made. Taking down spleen and head kidney of diseased fish on ice, placing into 1.5mL sterile centrifuge tube containing 800 μ L ice Trizol reagent (purchased from Boehringer Mannheim, Ltd.), carefully grinding, and storing in-80 deg.C refrigerator after no obvious block tissue; the other portion was placed directly into an empty sterile 1.5mL centrifuge tube and stored in a-80 ℃ freezer. The grass carp surviving 7 days after infection test is regarded as resistant individual, and spleen and head kidney samples are taken and stored according to the method.

Resistant and susceptible cells are screened according to the reaction of the monoclonal CIK cells on GCRV, and corresponding uninfected cells are collected respectively and stored according to the method.

(3) Extraction of spleen, head kidney and monoclonal CIK cell total RNA

Total RNA was extracted using the Trizol method (performed according to the instructions of the Trizol reagent). The specific operation method is as follows (the gun heads of all the micropipettes are treated by DEPC water):

1) respectively placing grass carp spleen, head kidney sample and monoclonal CIK cell sample in a 1.5mL centrifuge tube (DEPC water treated) filled with 800 μ L Trizol reagent;

2) after fully grinding, storing on ice for later use (or storing at minus 80 ℃ for a long time for later use);

3) adding 160 μ L chloroform into each tube, covering tightly, shaking vigorously for 15s, and standing at room temperature for 3 min;

4) centrifuging at 12000rpm for 15min at 4 deg.C;

5) preparing a 1.5mL centrifuge tube (DEPC water treated), transferring the colorless upper layer water phase obtained in the step 4) into the centrifuge tube, adding 400 mu L isopropanol, gently mixing uniformly, and standing at room temperature for 10 min;

6) centrifuging at 12000rpm for 10min at 4 deg.C;

7) the supernatant was aspirated off, 800. mu.L of 75% ethanol (DEPC water treated) was added, and the tube wall was rinsed for 5 s;

8) centrifuging at 7500rpm for 5min at 4 deg.C;

9) sucking and removing the supernatant, and air-drying for 10 min;

10) add 100. mu.L ddH₂O (DEPC treated), water bath at 58 deg.C for 10min, and immediately storing at-80 deg.C.

(4) Transcriptome sequencing

And respectively sending the extracted spleen, head kidney and monoclonal CIK cell RNA to Shanghai Mergiz biological medicine science and technology Limited company and Guangzhou Yoto Biotechnology Limited company by using Miseq and HiSeq2500 platforms of Illumeta to sequence, and performing basic analysis such as sequence splicing, differential expression, clustering and the like. The sequencing strategy is shown in FIG. 3. The expression level of C7N1 gene in spleen, head kidney and monoclonal CIK cells is shown in Table 1.

TABLE 1 expression level of C7N1 in transcriptome (FPKM value)

(5) Bioinformatics analysis

And analyzing the SNP sites of the individuals and the cells obtained by sequencing, wherein the resistance and the susceptibility are related, and finding that only one mutation site exists at the levels of the individuals and the cells and is related to the disease resistance. Then, the base types of the sites on the homologous genes of the zebra fish and the gobiocyprisrarus are analyzed through comparison, and the results are shown in table 2.

TABLE 2 base types of mutation sites in grass carp, gobiocypris rarus and zebrafish

Example 2: C7N1 gene verification and correlation analysis

(1) DNA extraction of spleen tissue of grass carp

The genome DNA was extracted by the conventional chloroform method. The specific operation method comprises the following steps:

1) 0.05g of tissue (mung bean size) was put into a 1.5mL EP tube, 200. mu.L of the tissue extract was added, and the mixture was sufficiently ground. Adding 400 μ L tissue extract and 60 μ L1 mg/mL proteinase K, placing in 55 deg.C water bath (about 3h) or 37 deg.C overnight, and digesting until the liquid is clear;

2) adding 600 μ L Tris-saturated phenol, mixing thoroughly for 10min, and centrifuging at 12,000rpm for 10 min;

3) transferring the supernatant to a new EP tube, adding 500 μ L chloroform for extraction, mixing thoroughly for 10min, and centrifuging at 12,000rpm for 10 min;

4) transferring supernatant into new EP tube, adding 2 times volume of glacial ethanol (-20 deg.C), precipitating DNA at-20 deg.C for at least 30min, and centrifuging at 12,000rpm for 5 min;

5) discarding the supernatant, washing with 70% ethanol for 1-2 times, and air drying at room temperature;

6) DNA was dissolved in 50. mu.L of triple distilled water and stored at-20 ℃ for further use. After the extraction of DNA was completed, 2. mu.L of the extracted DNA was electrophoretically detected on a 1% agarose gel (containing 0.01% of the nucleic acid dye Gelview). The results are shown in FIG. 4.

(2) Primer design

Aiming at the replacement of a single base, a pair of primers is designed by utilizing Primer Premier 5 software for genotype detection, and the primers are synthesized by Wuhan engine science and technology limited. The DNA sequences of the primer pairs are shown as follows:

a forward primer: 5'-GACTGAGAATGCTGTGAAAGATG-3'

Reverse primer: 5'-CAATGAGGTGGGATTTTAGTGA-3' are provided.

(2) PCR amplification

The synthesized primer was treated with ddH₂O was diluted to a concentration of 10. mu.M. The total volume of the PCR reaction system is 20 mu L: mix10 μ L, forward primer 0.6 μ L, reverse primer 0.6 μ L, cDNA 1 μ L, double distilled water 7.8 μ L. Amplification conditions: pre-denaturation at 94 ℃ for 1min, denaturation at 94 ℃ for 30s, annealing at 57 ℃ for 30s, extension at 72 ℃ for 30s, 35 cycles, and re-extension at 72 ℃ for 5 min. The PCR product was collected in 5. mu.L and detected by 1% agarose gel electrophoresis, and the results are shown in FIG. 5.

(3) PCR-electrophoretic typing

26 susceptible grass carps and 30 resistant grass carps obtained by infection test are subjected to DNA extraction and PCR amplification, and then 3.0% agarose gel electrophoresis is used for detecting and parting. An exemplary electropherogram for typing is shown in FIG. 6. After typing, the analysis was carried out using the online analysis software SHEsis (http:// analysis. bio-x.cn/myanalysis. php) (Shi YY and He L.2005.SHEsis, a power fundamental software platform for analysis of linkage disequilibrium, halophenylstructure, and genetic association at polymeric location. cell Res,15(2):97-8), the results of which are shown in Table 3.

TABLE 3 grass carp C7N1 genotyping statistics

Table 3 illustrates: the AA genotype and the BB genotype in Table 3 represent homozygotes, and the AB genotype represents heterozygotes; "+" indicates significant difference.

As is clear from Table 1, the C7N1 gene transcript was expressed in less resistant individuals and cells than in susceptible individuals and cells. As can be seen from Table 3 and FIG. 7, the number of resistant individuals was greater than the number of susceptible individuals in the AB genotype individuals; the number of resistant individuals in individuals with the BB genotype was less than the number of susceptible individuals (P < 0.05). Similarly, the number of resistant individuals in the A allele is greater than the number of susceptible individuals, and the number of resistant individuals in the B allele is less than the number of susceptible individuals. Therefore, it can be known that the corresponding encoded amino acid, i.e. "M/I", of this SNP site plays an important role in the function of C7N 1. Therefore, the selection of the heterozygote is favorable for the immune trait selection of the hemorrhagic disease of the grass carps.

Claims (1)

1. As shown in a sequence table SEQ ID NO: 1, the SNP in the C7N1 gene as a molecular marker is applied to the correlation analysis of the resistance of the C7N1 gene coding region to grass carp reovirus under the condition of vitro non-diagnosis, the SNP is characterized in that the base at 2145 has polymorphism, the base type is A or G, and the DNA sequence of a primer pair for amplifying the molecular marker is as follows:

a forward primer: 5'-GACTGAGAATGCTGTGAAAGATG-3' the flow of the air in the air conditioner,

reverse primer: 5'-CAATGAGGTGGGATTTTAGTGA-3', respectively;

extracting grass carp genome DNA to be detected, amplifying the 2145 th SNP site in the C7N1 gene by using the primer pair, and typing, wherein the genotype is associated with resistance of grass carp reovirus by AG.

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