CN109576275B - Cynoglossus semilaevis antibacterial disease related gene and application method thereof - Google Patents
Cynoglossus semilaevis antibacterial disease related gene and application method thereof Download PDFInfo
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Abstract
The invention provides an anti-bacterial disease related gene screened from cynoglossus semilaevis, the nucleotide sequence of the gene is SEQ ID NO. 1, and the sequence of the coded protein is SEQ ID NO. 2. The gene provided by the invention is related to the cynoglossus semilaevis antibacterial disease, the expression quantity of the gene in intestines is related to the disease resistance of a fish body, and the higher the expression level is, the stronger the disease resistance of the fish body is; the in vitro recombinant expressed protein can obviously inhibit the proliferation of several gram-negative bacteria, so the method has application values in aspects of cynoglossus semilaevis disease-resistant family breeding, disease control, feed additive and bactericide development and the like.
Description
Technical Field
The invention belongs to the technical field of aquatic product genetic breeding, and particularly relates to a cynoglossus semilaevis antibacterial disease related gene and an application method thereof.
Background
The Cynoglossus semilaevis (Cynoglossus semilaevis) is mainly distributed in coastal areas of China, is an important seawater economic cultured fish in China, has delicious meat and rich nutrition, is deeply loved by consumers, and has good market demand and culture and development potential. However, due to the intensive culture mode with high density and environmental pollution, bacterial diseases such as ascites, fin rot and tail rot of the cynoglossus semilaevis frequently occur, especially skin and other ulcers caused by vibrio harveyi are the most serious, which causes huge economic loss and seriously hinders the development of the cynoglossus semilaevis culture industry. At present, the prevention and treatment of fish vibrio harveyi disease mainly depends on antibiotics, but the abuse of drugs can not only cause the problems of drug residue, drug resistance, environmental pollution and the like, but also have potential threat to human health. In recent years, people are keenly looking for drugs capable of replacing traditional antibiotics, and with the emergence of more and more antibiotic-resistant strains, the antibacterial proteins are widely regarded in the fields of aquaculture industry, pharmaceutical industry, food additives and the like.
The antibacterial protein is a biological macromolecule with antibacterial activity and coded by genes, and is an important component of a life defense system. Its main functions are to resist the invasion of pathogenic microorganism, improve the disease resistance of organism itself, and maintain the continuation and normal operation of life activities. The antibacterial protein has a resisting effect on gram-positive bacteria and gram-negative bacteria, and can inhibit or kill fungi, mold, viruses and the like. A plurality of antibacterial proteins are separated and purified from microorganisms, plants, animals and human bodies, and antibacterial peptides are synthesized by utilizing a genetic engineering recombination technology, so that the antibacterial proteins from different sources become more and more important in the field of aquaculture. Research shows that the antiviral infection capacity of the penaeus monodon can be remarkably improved by adding the antibacterial peptide Mytilin into the culture feed of the penaeus monodon; the antibacterial peptide is added into the feed, so that the disease resistance of the litopenaeus vannamei and the litopenaeus vannamei can be obviously improved; the recombinant antibacterial peptide Chelonianin can effectively control the inflammation of tilapia infected with Vibrio harveyi and improve the survival rate of the tilapia. The research of antibacterial protein is rapidly advanced in recent years, and with the increasing maturity of protein research technology, the antibacterial protein becomes an effective way for developing new disease-resistant genes.
At present, some research reports about cynoglossus semilaevis immune related genes exist, and mainly homologous genes of immune related genes reported in mammals are obtained through a homologous cloning method. Such as interferons, lectins, histocompatibility complex families, cytokine families, complement factors, tumor necrosis factors, chemokines, lysozymes, transcription factors, actin-activating protein (dctn5), signal transduction and transcriptional activation activators (stat5), R-spondins secretion blushing, and the like. However, due to the existence of species specificity, the proteins encoded by these homologous genes do not necessarily have the function of disease-resistant immunity in fish species such as cynoglossus semilaevis, and particularly do not necessarily have the function of resisting specific pathogenic bacteria of cynoglossus semilaevis. In addition, the expression level and difference of the immune related genes in the disease-resistant and susceptible families of the cynoglossus semilaevis are not reported, and the genes are difficult to develop into marker genes of the bacterial disease resistance of the cynoglossus semilaevis. Therefore, the relevant gene of the cynoglossus semilaevis itself against a specific pathogen is preferably selected from the cynoglossus semilaevis itself. In view of this, it is very important to screen a new gene with tissue-specific expression, bacterial disease resistance-related function and disease resistance family dominant expression by combining the whole genome re-sequencing data and the immune tissue transcriptome data of the disease-resistant and non-disease-resistant families of cynoglossus semilaevis and using bioinformatics technologies such as whole genome association analysis (GWAS). Further researching the function of the related gene of the cynoglossus semilaevis for resisting bacterial diseases and the action mechanism of the related gene in disease-resistant immunity, exploring the technical approaches and the like applied to the cynoglossus semilaevis in disease-resistant breeding and disease prevention and control, and providing theoretical basis and gene markers or gene products for the cynoglossus semilaevis in disease-resistant breeding and disease prevention and control. The method has important significance and application value for breeding and disease prevention and control of fish such as cynoglossus semilaevis.
Disclosure of Invention
The invention provides a cynoglossus semilaevis antibacterial disease related gene and application thereof as a disease resistance evaluation marker for the first time, and the cynoglossus semilaevis antibacterial disease related gene is used for preparing recombinant protein and identifying the antibacterial activity of a recombinant expression product. Provides gene sequences and gene markers for the breeding of disease-resistant improved cynoglossus semilaevis seeds and provides a technical method for the research and development of green feed additives and antibacterial agents.
The invention provides an anti-bacterial disease gene screened from cynoglossus semilaevis, the sequence of the gene coding protein is SEQ ID NO. 2;
one specific nucleotide sequence of the gene is SEQ ID NO. 1;
the gene provided by the invention is suitable for being used as a marker gene for screening the cynoglossus semilaevis family against bacterial diseases.
The invention also provides application of the antibacterial disease related gene in detecting the disease resistance of cynoglossus semilaevis;
the invention provides a method for detecting the related gene of the bacterial disease, which is to detect the expression amount of the gene in the intestine of the cynoglossus semilaevis; the higher the expression level, the stronger the ability to resist bacterial diseases.
The detection primer used in the method has the following sequence information:
an upstream primer: 5'-GACCAACTGCCACCATACTCC-3' (SEQ ID NO:3)
A downstream primer: 5'-TCGTCCTCCAAGCCAAACC-3' (SEQ ID NO: 4);
the invention provides a recombinant expression strain for recombinant expression of the protein;
the protein is used for preparing a product for preventing and treating the vibriosis half-smooth tongue sole;
the protein of the invention is also used for preparing feed additives and antibacterial agents.
The gene provided by the invention is related to immune disease resistance, and the in vitro recombinant expressed protein can obviously inhibit the proliferation of gram-negative bacteria, so that the gene has application values in the aspects of cynoglossus semilaevis disease control, feed additives, bactericides and the like.
Drawings
FIG. 1: the full-length cDNA sequence of the related gene of the cynoglossus semilaevis for resisting bacterial diseases and the amino acid sequence coded by an open reading frame:
SEQ ID NO. 1 is the full-length sequence of cynoglossus semilaevis antibacterial related cDNA.
SEQ ID NO. 2 is the amino acid sequence of the open reading frame code of the Cynoglossus semilaevis Gunther related to the resistance to bacterial diseases.
FIG. 2: expression levels of the antibacterial disease related genes in various tissues of healthy adult cynoglossus semilaevis:
wherein, RT-PCR (reverse transcription-polymerase chain reaction) is carried out at a ratio of 2-1 to semi-quantitatively detect the expression level of the related gene of the antibacterial disease in each tissue of the healthy adult cynoglossus semilaevis. English abbreviations of the organization therein: l is liver; sp is spleen; k is kidney; i, intestines; gi is gill; h, heart; br: a brain; st is stomach; bl is blood; hk is head kidney.
2-2: and (3) quantitatively detecting the expression level of the gene in each tissue of the healthy adult cynoglossus semilaevis by qRT-PCR. Wherein the abscissa represents different tissues of the cynoglossus semilaevis, and the ordinate represents the expression amount of genes in different tissues, wherein English abbreviations of the tissues are as shown in figure 2-1.
FIG. 3: and (3) analyzing the expression of the cynoglossus semilaevis antibacterial disease related gene in immune related tissues after vibrio harveyi infection. The abscissa represents the time points after infection, and the ordinate represents the expression amounts of the genes associated with bacterial diseases in different tissues. A total of 6 tissues were examined, including: intestine (Intestine), Gill (Gill), Skin (Skin), Kidney (Kidney), Liver (Liver), Spleen (Spleen).
FIG. 4: induced expression, separation, purification and identification of recombinant protein of cynoglossus semilaevis antibacterial disease related gene:
(A) induction expression of recombinant protein, M: protein marker, 1: control group did not induce expression, 2: control induced expression, 3: expression was not induced in the recombinant proteome, 4: inducing expression of the recombinant proteome;
(B) performing solubility analysis and purification on the recombinant protein, wherein M is a large molecular weight protein marker, 1 is supernatant obtained after ultrasonic crushing, and 2 is precipitation obtained after ultrasonic crushing;
(C) after purification and dialysis of the recombinant protein, M is a high molecular weight marker, and 1 is the purified and dialyzed recombinant protein.
FIG. 5: analysis chart of in vitro bacteriostatic activity of recombinant protein:
(1) the effect of the recombinant protein of the related gene of the bacterial disease resistance on inhibiting Vibrio harveyi (Vibrio harveyi) is shown schematically, and 0 is PBS as a negative control group; ampicillin 1 at 10mg/L was used as a positive control; 2. 3, 4 are 3 different concentrations of recombinant protein as experimental groups: wherein 2 is 0.6mg/ml, 3 is 0.12mg/ml, and 4 is 0.054 mg/ml.
(2) The effect of the recombinant protein of the bacterial disease-resistant related gene for inhibiting Vibrio parahaemolyticus (Vibrio parahaemolyticus) is schematically shown, and 0 is PBS as a negative control group; ampicillin 1 at 10mg/L was used as a positive control; 2. 3, 4 are 3 different concentrations of recombinant protein as experimental groups: wherein 2 is 0.6mg/ml, 3 is 0.12mg/ml, and 4 is 0.054 mg/ml.
(3) The effect of the recombinant protein of the related gene of the bacterial disease for inhibiting Edwardsiella (Edwardsiella tarda) is shown schematically, and 0 is PBS as a negative control group; ampicillin 1 at 10mg/L was used as a positive control; 2. 3, 4 are 3 different concentrations of recombinant protein as experimental groups: wherein 2 is 0.6mg/ml, 3 is 0.12mg/ml, and 4 is 0.054 mg/ml.
FIG. 6: expression levels of cynoglossus semilaevis antibacterial disease related genes in various tissues of disease-resistant and susceptible families in 2014:
wherein the 6-1: RT-PCR semi-quantitative detection of the expression quantity of the Cynoglossus semilaevis disease-resistant related gene in each tissue of the 2014 and susceptible families. The abbreviation of English letters of each tissue is added with r in front to represent a disease-resistant family, added with s in front to represent a susceptible family, L is liver, and Sp is spleen; k is kidney; gi is gill; i, intestines; bl is blood.
6-2: qRT-PCR quantitative determination of the expression level of the relevant gene of Cynoglossus semilaevis Gunther in 2014 in the tissues of disease-resistant and susceptible families. Wherein the abscissa represents different tissues of the cynoglossus semilaevis, and the ordinate represents the expression amount of genes in different tissues. SF (Susceptible family) represents susceptible family, represented by black column, RF (Resistantiferous family) represents disease-resistant family, represented by gray column; gi is gill; i, intestines; bl is blood; k is kidney; sp is spleen; l is liver.
FIG. 7: expression level of relevant gene of Cynoglossus semilaevis Gunther for resisting bacterial diseases in each tissue of the Cynoglossus semilaevis Gunther system in 2017:
7-1, RT-PCR semiquantitative detection of the expression quantity of the Cynoglossus semilaevis antibacterial disease related gene in each tissue of the disease-resistant and susceptible families in 2017. Wherein, the abbreviation of English letters of the organization is added with r in front to represent a disease-resistant family, the addition of s in front to represent a susceptible family, L is liver, and Sp is spleen; k is kidney; gi is gill; i, intestines.
7-2: and (3) quantitatively detecting the expression quantity of the cynoglossus semilaevis gene in each tissue of the disease-resistant and susceptible families in 2017 by using qRT-PCR. Wherein the abscissa represents different tissues of the cynoglossus semilaevis, and the ordinate represents the expression amount of genes in different tissues. SF (Susceptible family) represents susceptible family, represented by black column, RF (resistant family) represents disease-resistant family, represented by gray column; gi is gill; i, intestines; k is kidney; sp is spleen; l is liver.
Detailed Description
In order to make the objects, technical lines and advantages of the present invention more apparent, examples of the present invention are described in detail below with reference to the accompanying drawings.
Example 1: cloning and sequence analysis of full-length cDNA of cynoglossus semilaevis antibacterial disease related gene
The applicant, Chensong forest and the like, firstly, carries out whole genome re-sequencing and whole genome association (GWAS) analysis on a cynoglossus semilaevis anti-vibrio harveyi disease family and a susceptible family which are screened out previously by adopting a conventional method, discovers an SNP marker linked with the cynoglossus semilaevis anti-vibrio harveyi disease shape, further positions the SNP site on a fragment, and then carries out RT-PCR and RACE-PCR amplification verification on the fragment to obtain the full-length cDNA sequence of the gene.
(1) The experimental method comprises the following steps: trizol method for extracting RNA, synthesizing cDNA and PCR amplification
Extracting RNA by a Trizol method: taking about 100mg of tissue blocks, putting the tissue blocks into an enzyme-free centrifuge tube added with 500 mu L of Trizol reagent, and grinding the tissue blocks on a grinding ice-cream stick; after the tissue is ground, 500 mul Trizol reagent is added, the mixture is kept stand for 10min and then is centrifuged at 4 ℃/12000rpm for 10min, and the precipitate is discarded; adding 200 μ L chloroform, shaking vigorously for 30s, standing for 5min to mix thoroughly, centrifuging at 12000rpm for 15min at 4 deg.C; sucking supernatant, adding isopropanol with the same volume, fully and uniformly mixing, standing at room temperature for 10min, centrifuging at 4 ℃ and 12000rpm for 10min, and removing supernatant; washing twice with 75% ethanol, centrifuging at 12000rpm at 4 deg.C for 3min, and carefully discarding the supernatant; drying at room temperature for 5min to remove ethanol completely; adding a proper amount of RNase-Free H2O, fully dissolving the RNA precipitate; detecting the total RNA quality by agarose gel electrophoresis, and determining the RNA purity and concentration by an RNA/DNA spectrophotometer; the extracted RNA was stored at-80 ℃.
And (3) cDNA synthesis: the extracted RNA is subjected to reverse transcription according to the instruction of a TaKaRa reverse transcription kit, and the synthesized cDNA is stored at the temperature of-20 ℃.
And (3) PCR amplification: based on the sequence of the fragment defined by the SNP marker, a longer intermediate fragment is amplified by RT-PCR at one time, and then the 5 'terminal sequence and the 3' terminal sequence are amplified by RACE PCR technology.
The RACE PCR reaction was divided into two rounds. The method comprises the following specific steps:
one round of: the Touchdown PCR reaction system and reaction conditions are as follows:
and (2) two rounds: the nested PCR reaction system and the reaction conditions are as follows:
first round of PCR product dilution 50 or 100 times served as amplification template for the second round.
And cutting and recovering the PCR products of the two rounds, purifying the PCR products, connecting the PCR products with a cloning vector pEASY-T1, and sequencing the positive clones. The gene cloning procedure was performed according to the description of the pEASY-T1 vector. (3) Acquisition of full-Length cDNA and homology analysis
Splicing and alignment analysis of sequences: splicing the sequence obtained by sequencing by using DNA Star software to finally obtain a full-length cDNA sequence; open reading frames were sought and translated using ORF finders; the cDNA overall length of the finally obtained gene is 1835bp (SEQ ID NO:1), and comprises 21bp of 5 'noncoding region, 1545bp of open reading frame and 269bp of 3' noncoding region. The open reading frame of the gene encodes a 514 amino acid protein (SEQ ID NO:2) (FIG. 1).
Example 2: analysis of expression levels of cynoglossus semilaevis antibacterial disease related genes in different tissues of normal fish
(1) Design of quantitative primers and verification of primer specificity
Designing a target gene quantitative amplification primer according to the antibacterial disease related gene sequence, carrying out common PCR amplification, electrophoresis detection and clone sequencing, if an electrophoresis strip is single and bright and no strip exists in negative control, carrying out clone sequencing analysis to obtain the target gene sequence, and preliminarily judging the specificity of the primer; and in addition, performing fluorescence quantitative PCR (polymerase chain reaction) on the machine, observing a peak image, a CT (computed tomography) value and amplification efficiency, and if the fluorescence quantitative PCR finds that the peak image is single and the amplification efficiency of the internal reference gene and the target gene is almost completely consistent, proving that the primers are specific and can be used for performing real-time quantitative PCR experiments. The quantitative primers for the Actin gene were derived from published articles in the Chensylin laboratory.
The primer sequences are as follows:
an upstream primer: 5'-GACCAACTGCCACCATACTCC-3'
A downstream primer: 5'-TCGTCCTCCAAGCCAAACC-3'
(2) Method for detecting expression level of antibacterial disease related gene in different tissues by using fluorescent quantitative PCR and semi-quantitative PCR
Firstly, taking 9 tissues of healthy cynoglossus semilaevis, namely liver, spleen, kidney, intestine, gill, head kidney, stomach, brain and blood, extracting RNA by a Trizol method and performing reverse transcription to obtain cDNA.
Fluorescent quantitative PCR: the relative expression quantity of the antibacterial disease related gene in each tissue is detected by carrying out fluorescent quantitative PCR reaction by using the primers. The reaction system is as follows: 10 μ L SYBR, 0.4 μ L primer-F, 0.4 μ L LPrimer-R, 0.4 μ L RoxRD II, 1 μ L cDNA template, 7.8 μ L ddH 20. The reaction conditions are as follows: 30s at 95 ℃; 95 ℃ for 5s, 60 ℃ for 34s, 40 cycles.
Semi-quantitative PCR: taking cDNA obtained by reverse transcription as a template, firstly carrying out common PCR reaction by using a quantitative primer of the reference gene actin, and carrying out electrophoresis detection. And adjusting the concentration and the dosage of the template until the brightness of the electrophoresis bands of the PCR products of all tissues is consistent. Then, the common PCR reaction of the target gene is carried out according to the adjusted concentration and dosage, and the electrophoresis detection is carried out.
The results of both PCR reactions showed: besides high expression in tissues such as intestine and gill, the expression level of the gene in other tissues of cynoglossus semilaevis is extremely low (FIG. 3).
Example 3: analysis of relative expression quantity of cynoglossus semilaevis antibacterial disease related gene in immune related tissue after Vibrio harveyi infection
Adopting Vibrio harveyi preserved in Chenpinlin laboratory to carry out Vibrio harveyi infection experiment on cynoglossus semilaevis. Setting a control group and an experimental group, wherein the control group is injected with 0.01mol/L sterile PBS, and the injection concentration of the experimental group is 1.0 multiplied by 104cfu of Vibrio harveyi in an injection dose of about 0.1mL/10 g. At six time points of 0h, 12h, 24h, 48h, 72h and 96h after injection, 6 tissues of liver, spleen, kidney, intestine, gill and skin were taken. The expression level of the gene involved in the bacterial disease was determined in the 6 tissues by the fluorescent quantitative PCR method. The primers, reagents and experimental methods used were as described above.
The results showed that the expression levels of the anti-bacterial disease associated genes in each tissue were up-regulated to different degrees after the injection of Vibrio harveyi: wherein in the intestine, the expression level of the related genes for resisting bacterial diseases is always increased from 12h to 48h, and is highest in 12 h; in gills, the expression level of genes is always increased from 48h to 96h except 12h and 24 h; in spleen, kidney and skin, the expression level of the gene is almost always in an ascending state from 12h to 96h, and reaches a maximum value at 72 h; in the liver, the expression level of the genes associated with bacterial diseases was higher from 24h to 96h than that in the control group, except for 12 h. The expression levels of all 5 tissues except the intestine were highest at 72 hours. These results indicate that, after the stimulation of vibrio harveyi, the expression level of the bacterial disease-resistant related gene in the immune-related tissue with extremely low expression level of the bacterial disease-resistant related gene is also obviously increased under normal conditions except for intestines and gills, indicating that the cynoglossus semilaevis bacterial disease-resistant related gene has strong immune defense effect on the infection of vibrio harveyi (fig. 3).
Example 4 analysis of in vitro recombinant expression of the screened gene and its product bacteriostatic activity (1) construction method of cynoglossus semilaevis antibacterial disease related gene recombinant expression vector:
the complete protein coding region sequence of the related gene of the antibacterial disease is amplified by PCR reaction by adopting high-fidelity blunt-end Pfu enzyme. And (3) treating the purified PCR product by using T4 ligase, and inserting the PCR product into a Peasy E1 prokaryotic expression vector. After the sequencing is correct, the recombinant expression plasmid in the positive clone bacterial strain is extracted. The recombinant expression plasmid was transformed into competent BL21(DE3) plyS engineering bacteria. After induction, purification, dialysis and renaturation, recombinant expression product antibacterial disease related recombinant protein is obtained, and the result is shown in figure 4. The specific scheme for achieving the aim is as follows:
1. the construction method of the antibacterial disease related gene recombinant expression vector comprises the following steps:
and (3) taking the multi-tissue mixed cDNA of the cynoglossus semilaevis as a template, and cloning the sequence of the ORF region of the related gene of the anti-bacterial disease by adopting high-fidelity enzyme to obtain a protein coding region. The PCR product was purified, inserted into pMD18-T vector and sequenced. Connecting the clone with the correct sequence with a full-type golden peasy E1 expression vector to obtain an E1-antibacterial disease related recombinant vector, sequencing to confirm positive clone, and extracting a target plasmid. E1-antibacterial disease-associated plasmid was transformed into competent BL21(DE3) plyS bacteria and used after sequencing correctly.
2. Induced expression of recombinant proteins and analysis of expression products
A single clone of BL21(DE3) plyS-E1-antibacterial disease-related gene strain was inoculated into 1mL of LB liquid medium containing AMP + at a final concentration of 50. mu.g/mL, cultured at 37 ℃ at 200rpm for 8 hours, and then cultured in a medium of 1: 500 inoculation ratio was inoculated into 200ML LB medium for ampicillin resistance, and expanded to OD600Is 0.4-0.6. Adding IPTG at the final concentration of 1mM, and inducing at 28 deg.C and 200rpm for 6-12 h. SDS-PAGE electrophoresis detection of protein expression is carried out on three kinds of bacterial liquids of 1ml of induced expressed bacterial liquid, a control group empty carrier and non-induced bacterial liquid at the same time, and the result shows that protein bands which are expressed differently between 50KD and 70KD appear (figure 4A). The induced bacterial liquid is centrifuged for 5-10min at 8000r/min at 4 ℃, all thalli are collected, then lysate is added (lysate: 10ml of binding buffer, DNAse I20U, PMSF 400 mul (50mM), lysozyme 400 mul (10mg/ml) is added in each gram of thalli), wherein, the preparation method of the binding buffer is as follows, 3.8g of sodium phosphate and 14 g of sodium chloride are added in each 1L of sterile water.61g, imidazole 1.02g), room temperature for half an hour, followed by ultrasonication in ice bath for 20min under conditions of 0.5s sonication and 2s pause. Adding 2 xSDS sample Buffer into the supernatant of the bacterial liquid and the crushed bacterial precipitation according to the proportion of 1:1, and boiling for 5 min; SDS-PAGE electrophoresis detection expression results show that the protein content in the crushed thallus precipitate is obviously higher than that of the supernatant, and the anti-bacterial disease related recombinant protein mainly exists in the form of inclusion bodies, and the results are shown in FIG. 4B.
3. Purification and verification of cynoglossus semilaevis antibacterial disease related recombinant protein
Denaturing the thallus precipitate with 7M urea, centrifuging to collect supernatant, filtering with 0.45 micron microporous membrane, purifying with HisTrap FF column, and specifically:
first, the purification column was washed with 3-5mL of sterile deionized water, then incubated with 5mL of a binding buffer (20mM/LNa3PO4, 0.5M NaCl, 20mM/L imidazole), the collected supernatant containing the protein of interest was slowly passed through the purification column at a flow rate of 1-2mL/min, then the incubated purification column was washed with 5mL of a binding solution, then 5mL of an elution buffer (20mM/L Na) was used3PO4, 0.5M NaCl, 500mM/L imidazole) to elute the protein of interest. And collecting the purified target protein, and carrying out protein size detection by adopting SDS-PAGE electrophoresis with the concentration of 12%. The confirmed target protein is dialyzed by urea with reduced concentration gradient to assist protein folding, so as to obtain renaturated protein, and the electrophoresis result shows a single band (see figure 4C). And (4) quantitatively analyzing the dialyzed protein by using a BCA protein quantitative kit.
4. Analysis of antibacterial activity of purified cynoglossus semilaevis antibacterial disease related recombinant protein
And (3) detecting the bacteriostatic activity of the recombinant protein by adopting an Oxford cup bacteriostatic zone method. Selecting gram-negative bacteria: detecting the bacteriostatic effect of three pathogenic bacteria such as Edwardsiella (Edwardsiella tarda), Vibrio parahaemolyticus (Vibrio parahaemolyticus) and Vibrio harveyi (Vibrio harveyi), culturing the pathogenic bacteria to logarithmic phase, diluting the bacteria with PBS solution to a concentration of 10%7～108One/ml is ready for use. Making a common cultureAnd (5) culturing the substrate, wherein the thickness of the substrate is about 0.5cm, and placing the substrate into 5 sterilized oxford cups for later use. Mixing the pathogenic bacteria liquid with the solid culture medium which is not solidified when the temperature is reduced to about 40 ℃, and pouring the mixture onto a solid plate with the depth of 0.5cm to prepare the sandwich culture medium. 10mg/L of ampicillin, 0.6mg/ml, 0.12mg/ml and 0.054mg/ml of renaturated protein are respectively added into an oxford cup. After 12h incubation in incubators at 28 ℃ or 37 ℃ (depending on the bacterial species), the size of the zone of inhibition was observed and measured. The results show that the recombinant protein shows obvious bacteriostatic activity on Edwardsiella (Edwards diella tarda), Vibrio parahaemolyticus (Vibrio parahaemolyticus) and Vibrio harveyi (Vibrio harveyi). The bacteriostatic activity of the recombinant protein on Edwardsiella (Edwardsiella tarda), Vibrio parahaemolyticus (Vibrio parahaemolyticus) and Vibrio harveyi (Vibrio harveyi) is enhanced with increasing protein concentration. At low concentrations, the recombinant protein had the lowest bacteriostatic activity against Vibrio harveyi (Vibrio harveyi) at the same protein concentration, but at higher protein concentrations (0.6mg/mL), the recombinant protein had higher bacteriostatic activity against Edwardsiella (Edwardsiellatarda), Vibrio parahaemolyticus (Vibrio parahaemolyticus) and Vibrio harveyi (Vibrio harveyi). The bacteriostatic effect on Vibrio harveyi (Vibrio harveyi) was weak at a recombinant protein concentration of 0.054mg/mL (FIG. 5).
Example 5 analysis of expression levels of anti-bacterial disease-related genes in different tissues of the disease-resistant and susceptible families of Cynoglossus semilaevis Gunther
1. Making a cynoglossus semilaevis disease-resistant and susceptible family: the disease-resistant family and susceptible family of cynoglossus semilaevis are established by adopting the method invented by the applicant in Chensong forest (Chensong forest, etc., 2010).
2. Determination of expression levels of antibacterial disease related genes in different tissues of disease-resistant and susceptible families
(1) 6 tissues of liver, spleen, kidney, intestine, gill and blood of 3 fish of disease-resistant family and susceptible family of cynoglossus semilaevis in 2014 are adopted. The primers, reagents and experimental methods for RNA extraction and quantitative PCR are as described above. The results of both the fluorescent quantitative PCR and the semi-quantitative PCR show that: the expression levels of the antibacterial disease related genes in the liver, kidney and spleen were not significantly different between the disease-resistant family and the susceptible family, while the expression levels of the antibacterial disease related genes in the intestines and gills of the disease-resistant family fish were higher than those of the susceptible family (FIG. 6).
(2)2017 detection of expression levels of antibacterial disease-related genes in different tissues of disease-resistant family and susceptible family
The method adopts 5 tissues of liver, spleen, kidney, intestine and gill of 5 fish of a disease-resistant cynoglossus semilaevis family and an susceptible family in 2017. The RNA extraction method, the primers, the reagents and the experimental method used for quantitative PCR are the same as the previous step. The results of both the fluorescent quantitative PCR and the semi-quantitative PCR show that: the expression levels of the genes in the liver, kidney and spleen were not significantly different between the disease-resistant and susceptible families, and the expression levels of the genes in the intestines and gills of the disease-resistant family fish were higher than those of the susceptible family fish, especially in the intestines (FIG. 7). The results of the disease-resistant family and susceptible family in 2014 are repeated.
Through the detection of the expression levels of the bacterial disease resistance related genes of each tissue of the disease-resistant family and the susceptible family of the cynoglossus semilaevis in 2014 and 2017, the positive correlation between the expression levels of the bacterial disease resistance related genes in intestines and branchia and the disease resistance is shown. The expression level of the antibacterial disease related genes in intestines or branchia can be used as an index of disease resistance for breeding disease-resistant families.
(3) Application method of expression level of antibacterial disease related gene in cynoglossus semilaevis intestine as disease resistance evaluation index
Collecting the intestine of cynoglossus semilaevis family fish, extracting RNA, and performing reverse transcription to obtain cDNA.
Analyzing gene expression level according to a conventional quantitative PCR method, drawing an expression level graph by using actin as an internal reference, and comparing the expression levels of the related genes of the cynoglossus semilaevis in the adult fish intestines of different families, wherein the higher the expression level is, the stronger the disease resistance is. Adult fish in a family line with high expression level of related genes resisting bacterial diseases is selected to breed parent fish for breeding offspring, and the cynoglossus semilaevis fry with improved bacterial disease resistance can be produced. The method of the invention firstly uses the expression level of the related gene of the antibacterial disease in the intestines as the index for evaluating the antibacterial disease capability of the fish body, thereby establishing a molecular method for detecting the disease resistance of the cynoglossus semilaevis, and the method has great application value in the disease resistance test and disease resistance family breeding of the cynoglossus semilaevis and can also be popularized and applied in the disease resistance breeding of other fishes.
Sequence listing
<110> research institute for aquatic products in yellow sea of China institute for aquatic science
<120> Cynoglossus semilaevis related gene for resisting bacterial diseases and application thereof
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<170>SIPOSequenceListing 1.0
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agtgtcttct tgcccgcagc catgctgggc ctttctaaac aagcctcgct ctacctgagt 60
cttcttctat ttctgtccct ccaatcctca gtcctcaccc ttactgggac cgcgagccag 120
tgccagtctg ctccctttgt accaggttac aatctggcag gagagggctt taacatagtc 180
actctgcgcc gacaaggagc ctacgtgatt gatatgagga cccacctcaa cccgaatggc 240
acctgtacac tttaccacaa ccctcttcaa ggcaacgcgt tgcaaaaagt gccactctct 300
gtgcatgact ggagatcctt ccgtcagtgc actgcatctc tccacggaag ctaccacagc 360
tctaacagca cactaattga aacatacacc agtctagaca gccatgactg gaagtccgga 420
ctgaatataa gtaaacttgg tggcttaaat gttggtggat caagttccag tgcctacagc 480
tttgcttcaa ccaggagcag agaagaccag cacattttca gccttcacta catgtactgc 540
aaccactact cataccgagt gtcaaacaga ccaaccctga gctctgagtt caggggggat 600
ttggaccaac tgccaccata ctccgcttca acaaaggctg attacaggag aattatagag 660
acatacggca cacattacat caacaaggtt tggcttggag gacgatacag aaggctgtca 720
gctatccgta catgtttgtc cagattgaat ggcttgtcaa cttatcaggc acatgactgc 780
ttgtctctgg gaatcaaatt aagcctgaag ataattcaag gttcatcaga aacacatacc 840
tgctccaaga tcttggagaa catggacact gctgcatcat acagcgctgg gctccaccag 900
cacgtcacag aggtaacagg aggaaatggt tggctcggcg aattttcact ctctggtatt 960
gacgcctctg gctatgaaac atggctgcgc agcctcaaag atcaccctga tgttattcac 1020
tattctctga gaccactgta cgagctggca ctatcgtggt caactatgat tggactgtat 1080
ttggccacac atgattacct caaagaacat ggcgtcagtt ctgggaccag taatccaaga 1140
tgtagcggtc gtcctaacct tgattacaac tgctgtccca tcgagacccg cagaggaaat 1200
ctaagggtga ccatcgtccg tgcttggggt ctgaaaggag atcctgtcgg gaagacagag 1260
gcgtatgtga agatgtggta cggtcaacat taccgtagga cccgtatgat ccgatcaaac 1320
tccccccgtt ggaattcaga ttatgacctt ggaaatgttt atgcccattt gagtcttaag 1380
attgaagtct gggatgaaga tgtgttcaga gacgaccgtc tggggtcatg tgtgaggaac 1440
ctgagacagg ggtcacacac ttttatctgt tcagtccaaa gagggggaat agagatcaga 1500
tactcactca cctgtgaccg ccatctgact ggaaaccagt gccaggacta cagaccagtt 1560
ccatagaaac tcagtctgaa tgaaagaaac aatgaaacat tatattagca tttccactgt 1620
atgagcatta gctttcccga tttttgatca atatcttttc taataaatga catgataggt 1680
gttggtgaaa gttaaaaagg tctgatctat gtcactgtga ttttagatga tttagtaact 1740
gtctacttca tcactttact ttgctaaaaa cagctaataa atacacagaa atgattttaa 1800
gcatcaaaaa aaaaaaaaaa aaaaaaaaaa aaaaa 1835
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<213> Artificial Sequence (Artificial Sequence)
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Phe Leu Ser Leu Gln Ser Ser Val Leu Thr Leu Thr Gly Thr Ala Ser
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Gln Cys Gln Ser Ala Pro Phe Val Pro Gly Tyr Asn Leu Ala Gly Glu
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Gly Phe Asn Ile Val Thr Leu Arg Arg Gln Gly Ala Tyr Val Ile Asp
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Met Arg Thr His Leu Asn Pro Asn Gly Thr Cys Thr Leu Tyr His Asn
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Pro Leu Gln Gly Asn Ala Leu Gln Lys Val Pro Leu Ser Val His Asp
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Trp Arg Ser Phe Arg Gln Cys Thr Ala Ser Leu His Gly Ser Tyr His
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Ser Ser Asn Ser Thr Leu Ile Glu Thr Tyr Thr Ser Leu Asp Ser His
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Asp Trp Lys Ser Gly Leu Asn Ile Ser Lys Leu Gly Gly Leu Asn Val
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Gly Gly Ser Ser Ser Ser Ala Tyr Ser Phe Ala Ser Thr Arg Ser Arg
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Glu Asp Gln His Ile Phe Ser Leu His Tyr Met Tyr Cys Asn His Tyr
165 170 175
Ser Tyr Arg Val Ser Asn Arg Pro Thr Leu Ser Ser Glu Phe Arg Gly
180 185 190
Asp Leu Asp Gln Leu Pro Pro Tyr Ser Ala Ser Thr Lys Ala Asp Tyr
195 200 205
Arg Arg Ile Ile Glu Thr Tyr Gly Thr His Tyr Ile Asn Lys Val Trp
210 215 220
Leu Gly Gly Arg Tyr Arg Arg Leu Ser Ala Ile Arg Thr Cys Leu Ser
225 230 235 240
Arg Leu Asn Gly Leu Ser Thr Tyr Gln Ala His Asp Cys Leu Ser Leu
245 250 255
Gly Ile Lys Leu Ser Leu Lys Ile Ile Gln Gly Ser Ser Glu Thr His
260 265 270
Thr Cys Ser Lys Ile Leu Glu Asn Met Asp Thr Ala Ala Ser Tyr Ser
275 280 285
Ala Gly Leu His Gln His Val Thr Glu Val Thr Gly Gly Asn Gly Trp
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Leu Gly Glu Phe Ser Leu Ser Gly Ile Asp Ala Ser Gly Tyr Glu Thr
305 310 315 320
Trp Leu Arg Ser Leu Lys Asp His Pro Asp Val Ile His Tyr Ser Leu
325 330 335
Arg Pro Leu Tyr Glu Leu Ala Leu Ser Trp Ser Thr Met Ile Gly Leu
340 345 350
Tyr Leu Ala Thr His Asp Tyr Leu Lys Glu His Gly Val Ser Ser Gly
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Thr Ser Asn Pro Arg Cys Ser Gly Arg Pro Asn Leu Asp Tyr Asn Cys
370 375 380
Cys Pro Ile Glu Thr Arg Arg Gly Asn Leu Arg Val Thr Ile Val Arg
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Ala Trp Gly Leu Lys Gly Asp Pro Val Gly Lys Thr Glu Ala Tyr Val
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Lys Met Trp Tyr Gly Gln His Tyr Arg Arg Thr Arg Met Ile Arg Ser
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Asn Ser Pro Arg Trp Asn Ser Asp Tyr Asp Leu Gly Asn Val Tyr Ala
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His Leu Ser Leu Lys Ile Glu Val Trp Asp Glu Asp Val Phe Arg Asp
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Asp Arg Leu Gly Ser Cys Val Arg Asn Leu Arg Gln Gly Ser His Thr
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Phe Ile Cys Ser Val Gln Arg Gly Gly Ile Glu Ile Arg Tyr Ser Leu
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Thr Cys Asp Arg His Leu Thr Gly Asn Gln Cys Gln Asp Tyr Arg Pro
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Val Pro
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<213> Artificial Sequence (Artificial Sequence)
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gaccaactgc caccatactc c 21
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Claims (1)
1. The application of the gene with the nucleotide sequence of SEQ ID NO. 1 as a marker gene for the cynoglossus semilaevis family screening of resisting bacterial diseases.
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