Patent Publication Number: US-11643695-B2

Title: Siniperca chuatsi IL-6 gene and detection method of disease-resistant SNP marker thereof

Description:
This application is a Divisional Application of U.S. Ser. No. 16/504,371, filed on Jul. 8, 2019, which claims priority to Chinese Patent Application No.: 201810734995.2, filed on Jul. 6, 2018, which is incorporated by reference for all purposes as if fully set forth herein. 
    
    
     FIELD OF THE INVENTION 
     The present invention relates to the fields of aquiculture and biotechnology, and more particularly to  Siniperca chuatsi  ( S. chuatsi ) IL-6 gene and a detection method of a disease-resistant SNP marker thereof. 
     DESCRIPTION OF THE RELATED ART 
     As a “prestigious” fish with important economic value in China,  S. chuatsi  occupies an important position in freshwater aquaculture in China. However, under the conditions of large-scale artificial breeding in recent years, the genetical characterization decline of  S. chuatsi  is quite common, which leads to the gradual decline of disease resistance, especially the increasingly rampant viral diseases infected by infectious spleen and kidney necrosis virus. And so far, there is no effective prevention method, which causes serious economic losses every year and seriously restricts the sustainable development of the aquaculture industry. 
     The existing fish breeding technique is to select fish based on the apparent traits of fish observed by naked eyes, which makes it impossible to correctly select fish from the perspective of genetic differences closely related to apparent traits. It is not only impossible to carry out early breeding, but also difficult to guarantee the offspring characteristics and the breeding is blind, time-consuming and laborious. 
     Cloning of genes related to disease resistance and screening of disease-resistant germplasm resources by SNP molecular markers are effective means to improve disease resistance of  S. chuatsi  to viruses and other diseases. SNPs, that is, signal nucleotide poly-morphisms. transition, transversion, insertion or deletion of a single base on a gene fragment will lead to SNP polymorphism and disease resistance phenotype differences among individuals. 
     During the disease-resistant genes, IL-6 (interleukin-6) is a cytokine belonging to interleukin, which can promote the proliferation and differentiation of various cells involved in the immune responses, improve their functions, and inhibit cell apoptosis. However, currently the researches on the cloning and functions of fish IL-6 gene are lack, because the fish has low homology of IL-6 gene sequence with the other species, moreover, the transcription and translation process of IL-6 gene is very unstable when subjected to immunostimulation, and thus causes fewer clones of cDNA library of fish cytokines. Accordingly, cloning of cDNA full-length sequence, gDNA full-length sequence of  S. chuatsi  IL-6 gene is of great significance. 
     SUMMARY OF THE INVENTION 
     An object of the invention is to provide a disease-resistant IL-6 gene (interleukin, IL-6) of  S. chuatsi , intended to solve the problem that the fish has low IL-6 gene homology with other species and some fish lack of IL-6 gene. 
     Another object of the invention is to provide a detection method of a disease-resistant SNP marker of  S. chuatsi . The method is based on the IL-6 gene of  S. chuatsi  provided in the invention, wherein single nucleotide polymorphism (SNP) is combined with disease-resistant apparent traits of fish, based on SNP marker (or known as mutation site) of IL-6 of disease-resistant or non-disease-resistant fish, to breed selectively and accurately disease-resistant fish population. The invention can improve the efficiency of breeding of superior breeding, decrease the morbidity and mortality of the breeding fish, and thus increase the economic benefits of fish culture, thereby providing a significant meaning for the progress of fish breeding. 
     The invention utilizes the following technical solutions: 
     In one aspect, the invention provides a  S. chuatsi  IL-6 gene having a cDNA sequence shown in SEQ ID NO: 1. 
     In another aspect, the invention also provides a detection method for a disease-resistant SNP marker based on the above IL-6 gene of  S. chuatsi , comprising the steps of 
     (1) designing a primer for amplifying a disease-resistant SNP locus according to IL-6 gDNA sequence containing intron of the  S. chuatsi  IL-6 gene, and amplifying the  S. chuatsi  IL-6 gene to obtain an amplification product; 
     (2) sequencing the amplification product and finding out SNPs loci relevant to virus disease resistance, and determining the SNP locus according to DNA peak profile. 
     Preferably, in the step (1), the IL-6 gDNA sequence is obtained by designing and amplifying a specific primer for  S. chuatsi  IL-6 gene, and the IL-6 gDNA sequence has a nucleotide sequence shown in SEQ ID NO: 2. 
     Preferably, the specific primer is: 
     
       
         
           
               
               
            
               
                   
                 IL-6-1F: 
               
               
                   
                 (SEQ ID NO: 12) 
               
               
                   
                 5′-CTCAGCATCAGCGGAAACTC-3′; 
               
               
                   
                   
               
               
                   
                 IL-6-1R: 
               
               
                   
                 (SEQ ID NO: 13) 
               
               
                   
                 5′-TGCCCCTGTTGGCCATACTT-3′; 
               
            
           
         
       
     
     Preferably, in the step (1), rapid amplification of cDNA ends (RACE) is performed to clone the  S. chuatsi  IL-6 gene. 
     Preferably, in the step (1), the primer for amplifying disease-resistant SNP loci is: 
     
       
         
           
               
               
            
               
                   
                 IL-6-L1F: 
               
               
                   
                 (SEQ ID NO: 14) 
               
               
                   
                 5′-AACCCAAAGAGGCAGGTGAC-3′; 
               
               
                   
                 IL-6-L1R: 
               
               
                   
                 (SEQ ID NO: 15) 
               
               
                   
                 5′-ACCATCCAATTTCCCTGAGGT-3′. 
               
            
           
         
       
     
     Preferably, in the step (2), multiple sequence alignment is performed on the sequencing results by DNAMAN software and the suspected SNPs loci are found out, and DNA sequencing chromatogram is observed via Chromas software, single peak is a homozygotic SNP locus, and the nested peak is a heterozygous SNP locus. 
     Preferably, the SNP marker is located at the 1744 bp of the gDNA sequence  S. chuatsi  having a nucleotide sequence shown in SEQ ID NO: 2, the base at 1744 bp of  S. chuatsi  susceptible to virus is T, and the base at 1744 bp of antivirus  S. chuatsi  is C, the virus is infectious spleen and kidney necrosis virus. 
     As compared with prior art, the present invention has the following advantages: 
     (1) In the invention, IL-6 cDNA full-length sequence and IL-6 gDNA full-length sequence of  S. chuatsi  are cloned firstly, thereby providing research basis for resistant breeding of fish; 
     (2) In the invention, the SNP locus of mandarin fish IL-6 gene relevant to virus disease resistance is detected to provide a new idea for breeding of  S. chuatsi , this facilitates to promote the genetic breeding process and increase the economic benefits of mandarin fish culture. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         FIG.  1    shows the gel electrophoresis results of segments of  S. chuatsi  IL-6 gene after amplification, wherein (A) agarose gel electrophoresis results of second round PCR of  S. chuatsi  IL-6 gene via cDNA 5′ end RACE, lane 1 represents the target fragment; (B) agarose gel electrophoresis results of second round PCR of  S. chuatsi  IL-6 gene via cDNA 3′ end RACE, lane 1 represents the target fragment; 
         FIG.  2    shows the gel electrophoresis results after PCR amplification of  S. chuatsi  IL-6 gene by adding the primer used by an intron; 
         FIG.  3    shows the gel electrophoresis results after PCR amplification of SNP locus detection of  S. chuatsi  IL-6 gene; 
         FIG.  4    shows the PCR detection results of infectious spleen and kidney necrosis virus (ISKNV) for head kidney tissue of  S. chuatsi , wherein lane {circle around ( 1 )} represents the band after DL500 Mark electrophoresis; both lane {circle around ( 2 )} and lane {circle around ( 3 )} represent a single and bright ISKNV virus nucleotide-specific band (187 bp) obtained by performing PCR amplification and electrophoresis using a template prepared by head kidney tissue homogenate of  S. chuatsi  in the ISKNV virus infection group; the other lanes represent non-specific bands obtained by performing PCR amplification and electrophoresis using a template prepared by head kidney tissue homogenate of  S. chuatsi  in the ISKNV virus infection group; 
         FIG.  5    shows a screen shot of comparison results of IL-6 DNA reverse complementary sequence of 22 groups of  S. chuatsi  samples, wherein the base G is mutated to base A in three samples. 
         FIG.  6    shows the peak profile generated by sequencing IL-6 DNA of a  S. chuatsi  sample, wherein G homozygote is not mutated in IL-6 DNA of  S. chuatsi  sample of this figure; 
         FIG.  7    shows the peak profile generated by sequencing IL-6 DNA of a  S. chuatsi  sample, wherein G heterozygote is not mutated in IL-6 DNA of a  S. chuatsi  sample of this figure. 
         FIG.  8    shows the peak profile generated by sequencing IL-6 DNA of a  S. chuatsi  sample, wherein A homozygote is mutated in IL-6 DNA of  S. chuatsi  sample of this figure. 
     
    
    
     DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS 
     The present invention will be explained in more detail below with reference to the drawings and in connection with embodiments. It should be understood that, the exemplary embodiments and description thereof of the invention are used for illustrating the present invention and are not intended to limit the invention 
     I. Clone of IL-6 cDNA Sequence of  S. chuatsi    
     1. Amplification of IL-6 5′ Terminal Sequence of  S. chuatsi    
     (1) Synthesis, Purification and Tailing of the First Strand of cDNA 
     The first strand of cDNA of IL-6 was synthesized for the extracted total RNA using SUPERSCRIPT II RT enzyme and the primer GSP-IL-6-1. RNase Mix was used to perform RNA removal on the synthesized cDNA. Reverse transcription system was: 
     
       
         
           
               
               
               
               
             
               
                   
                   
               
             
            
               
                   
                 GSP-IL-6-1 
                 25  
                 ng 
               
               
                   
                 Template RNA 
                 2  
                 ul 
               
            
           
           
               
               
               
            
               
                   
                 DEPC-treated water 
                 making up to 15.5 μl 
               
               
                   
                   
               
            
           
         
       
     
     This system was incubated at 70° C. for 10 min, and immediately placed on ice to cool for 1 min, and then transient centrifugation was performed to collect liquid. The following system was added sequentially: 
     
       
         
           
               
               
               
             
               
                   
                   
               
             
            
               
                   
                 10 × PCR bbuffer 
                 2.5 μl 
               
               
                   
                 25 nM MgCl 2   
                 2.5 μl 
               
               
                   
                 10 nM dNTP mix 
                   l ul 
               
               
                   
                 0.1M DTT 
                 2.5 ul 
               
               
                   
                   
               
            
           
         
       
     
     The reaction system was mixed gently, centrifuged and incubated at 42° C. for 1 min. 1 μl SUPERSCRIPT II RT was added, mixed evenly and incubated at 42° C. for 50 min, at 70° C. for 15 min and then reaction was stopped. After centrifugation, the resulting product was placed at 37° C., and 1 μl of RNase mix was added to react for 30 min and then purification was performed. poly (c) was added at the purified cDNA terminal using TdT enzyme and dCTP, then the product was stored at a low temperate for use. 
     (2) Quick Amplification of cDNA 5′ Ends 
     The first round PCR amplification was performed on the cDNA to which the dC tail had been added using the primer GSP-IL-6-2. The 5′-RACE system was added in the following order: 
     
       
         
           
               
               
               
             
               
                   
                   
               
             
            
               
                   
                 sterilized, distilled water 
                 31.5 μl  
               
               
                   
                 10 × PCR buffer 
                 5.0 μl 
               
               
                   
                 25 mM MgCl 2   
                 3.0 μl 
               
               
                   
                 10 mM dNTP mix 
                 1.0 μl 
               
               
                   
                 10 μM nested GSP-TLR3-2 
                 2.0 μl 
               
               
                   
                 10 uM Abridged Anchor Primer 
                 2.0 μl 
               
               
                   
                 dC-tailed cDNA 
                 5.0 μl 
               
               
                   
                 Taq DNA polymerase 
                 0.5 μl 
               
               
                   
                 final volume 
                  50 μl 
               
               
                   
                   
               
            
           
         
       
     
     The reaction conditions were as follows: pre-denaturation at 94° C. for 2 min, denaturation at 94° C. for 30 s, re-analysis annealing at 55° C. for 30 s, extension at 72° C. for 1 min, 30 cycles, and finally extension at 72° C. for 7 min. 
     The nested second round PCR amplification was performed using the primer GSP-IL-6-3 and abridged universal amplification primer AUAP, and the system was as follows: 
     
       
         
           
               
               
               
             
               
                   
                   
               
             
            
               
                   
                 sterilized, distilled water 
                 33.5 μl  
               
               
                   
                 10 × PCR buffer 
                 5.0 μl 
               
               
                   
                 25 mM MgCl 2   
                 3.0 μl 
               
               
                   
                 10 mM dNTP mix 
                 1.0 μl 
               
               
                   
                 10 μM nested GSP-TLR3-3 
                 1.0 μl 
               
               
                   
                 10 uM AUAP 
                 1.0 μl 
               
               
                   
                 dilution of primary PCR product 
                 5.0 μl 
               
               
                   
                 Taq DNA polymerase 
                 0.5 μl 
               
               
                   
                 final volume 
                  50 μl 
               
               
                   
                   
               
            
           
         
       
     
     The reaction conditions were as follows: pre-denaturation at 94° C. for 2 min, denaturation at 94° C. for 30 s, re-analysis annealing at 59° C. for 30 s, extension at 72° C. for 1 min, 30 cycles, and finally extension at 72° C. for 7 min. 
     Agarose gel electrophoresis (1.2%) was performed on the product of the second round PCR, the target bands were recycled by gel extraction using Gel Extraction Kit (Sangon Shanghai). The purified PCR product was recycled and cloned to the pMD18-T vector (TaKaRa), and positive clones were selected for sequencing. 
     2. Amplification of IL-6 3′ Terminal Sequence of  S. chuatsi    
     (1) Synthesis and Purification of the First Strand of cDNA 
     Reverse transcription was performed on the total extracted RNA using SUPERSCRIPT II RT enzyme and the primer 3′CDS primer A (SMARTer™ RACE cDNA Amplification Kit, Clontech). The used primer was 3′CDS primer A, and the other components and conditions were the same as those of 5′ terminal amplification. 
     (2) Quick Amplification of cDNA Ends 
     The first round PCR amplification was performed using the primers GSP-IL-6-4 and UPM using the cDNA synthesized previously as a template, and the 3′-RACE reaction system was added in the following order (the reaction conditions were the same as those of the 5′ ends amplification): 
     
       
         
           
               
               
               
             
               
                   
                   
               
             
            
               
                   
                 sterilized, distilled water 
                 31.5 μl  
               
               
                   
                 10 × PCR buffer 
                 5.0 μl 
               
               
                   
                 25 mM MgCl 2   
                 3.0 μl 
               
               
                   
                 10 mM dNTP mix 
                 1.0 μl 
               
               
                   
                 10 μM GSP-IL-6-4 
                 2.0 μl 
               
               
                   
                 10 uM UPM 
                 2.0 μl 
               
               
                   
                 dC-tailed cDNA 
                 5.0 μl 
               
               
                   
                 Taq DNA polymerase 
                 0.5 μl 
               
               
                   
                 final volume 
                  50 μl 
               
               
                   
                   
               
            
           
         
       
     
     The PCR product from the first round amplification was diluted 50 times to perform the second round PCR amplification, the other systems were the same as those of the first round PCR amplification other than the primers GSP-IL-6-5 and UPM, and the reaction conditions were the same as those of 5′ amplification. Agarose gel electrophoresis (1.2%) was performed on the product of the second round PCR, and the target band was recycled by gel extraction, as shown in  FIG.  1   . The purified product was cloned to the pMD18-T vector (TaKaRa), and the positive clones were selected for sequencing, the spliced complete sequence of  S. chuatsi  IL-6 cDNA is shown in SEQ ID NO: 1, and the encoded amino acid sequence of the  S. chuatsi  IL-6 cDNA is shown in SEQ ID NO: 3. 
                     TABLE 1                  Primer Sequences for amplification       of IL-6 gene cDNA                         Primers   Sequences (5′-3′)   Target               GSP-IL-6-1   CTGGGGCACTCCTTCT   IL-6 5′ -       (SEQ ID NO: 4)       Race               GSP-IL-6-2   AACCTGTGGAGACAAGCC   IL-6 5′ -       (SEQ ID NO: 5)       Race               GSP-IL-6-3   CTGAAGTTGGAGTAAGGGCA   IL-6 5′ -       (SEQ ID NO: 6)       Race               3′CDS Primer A   AAGCAGTGGTATCAACGCAG   IL-6 3′-       (SEQ ID NO: 7)   ACTAC   Race               GSP-IL-6-4   CGCCAGCTCCACTACTTCCT   IL-6 3′-       (SEQ ID NO: 8)   TGTCG   Race               GSP-IL-6-5   AAAGGGAGTTCAGAGCAAGT   IL-6 3′-       (SEQ ID NO: 9)   ATGGC   Race               AUAP   GGCCACGCGTCGACTAGTAC   universal       (SEQ ID NO: 10)       5′ -Race               UPM   CTAATACGACTCACTATAGG   universal       (SEQ ID NO: 11)   GC   3′ -Race                    
3. Obtaining of  S. chuatsi  IL-6 gDNA
 
     With reference to the known IL-6 gDNA structure of other fishes, the specific primers were designed for the spliced complete fragment of  S. chuatsi  IL-6 cDNA to amplify the intron of IL-6 gene (table 2), wherein the fragment should contain overlapping regions for facilitating the subsequent sequence assembly. The genomic DNA of  S. chuatsi  was used as a template to perform fragment PCR amplification. The amplification conditions were as follows: pre-denaturation at 94° C. for 5 min, denaturation at 94° C. for 30 s, annealing at 52° C. for 30 s, extension at 72° C. for 1 min, 30 cycles, and finally extension at 72° C. for 7 min. Agarose gel electrophoresis (1.5%) was performed on the PCR product, as shown in  FIG.  2   , single band was selected and recycled by gel extraction, and ligated to the pUmc-T vector using T vector PCR products cloning Kit(TAKARA), the system (10 μL) was stored overnight at 4° C. The positive clones were selected for sequencing. The obtained sequences were spliced using artificial alignment and DNAMAN software to get the full-length sequence of  S. chuatsi  IL-6 gDNA, as shown in SEQ ID NO: 2. 
                     TABLE 2                  Primer sequences for amplifying       the intron of IL-6 gene                             Primers   Primer sequences (5′-3′)                       IL-6-1F   CTCAGCATCAGCGGAAACTC           (SEQ ID NO: 12)                           IL-6-1R   TGCCCCTGTTGGCCATACTT           (SEQ ID NO: 13)                        
4. Detection of Disease-Resistant SNP Marker
 
     (1) Primers (table 3) were designed according to the sequence of  S. chuatsi  IL-6 gDNA. The PCR reaction system is shown in table 4. 
     
       
         
           
               
             
               
                 TABLE 3 
               
             
            
               
                   
               
               
                 Primer sequences for obtaining disease-resistant 
               
               
                 SNP loci by amplifying  S .  chuatsi  IL-6 gene 
               
            
           
           
               
               
            
               
                 Primers 
                 Primer sequences (5′-3′) 
               
               
                   
               
               
                 IL-6- L1F 
                 AACCCAAAGAGGCAGGTGAC 
               
               
                 (SEQ ID NO: 14) 
                   
               
               
                   
               
               
                 IL-6- L1R 
                 ACCATCCAATTTCCCTGAGGT 
               
               
                 (SEQ ID NO: 15) 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 4 
               
             
            
               
                   
               
               
                 PCR reaction system for obtaining disease-resistant SNP  
               
               
                 loci by amplifying  S. chuatsi  IL-6 gene 
               
            
           
           
               
               
               
            
               
                   
                 Components 
                 Volume μl ) 
               
               
                   
                   
               
            
           
           
               
               
               
            
               
                   
                 Template DNA 
                 2 
               
               
                   
                 Primer R ( 10 μl ) 
                 1 
               
               
                   
                 Primer F ( 10 μl ) 
                 1 
               
               
                   
                 2 × Easy Taq PCR Super Mix 
                 25 
               
               
                   
                 ddH 2 O 
                 21 
               
               
                   
                 Total 
                 50 
               
               
                   
                   
               
            
           
         
       
     
     Steps of PCR amplification were as follows: (1) pre-denaturation at 94° C. for 5 min, (2) denaturation at 94° C. for 30 s, (3) annealing at 52° C. for 30 s, (4) extension at 72° C. for 1 min, 30 cycles, and (5) extension at 72° C. for 10 min, (1) 4° C. end of the reaction. 
     (2) Agarose gel electrophoresis (1.5%) was performed on the PCR product to get the PCR amplification product, as shown in  FIG.  3   , and then immediately sequenced after gel extraction. 
     The multiple sequence alignment was performed on the sequencing results using DNAMAN software, to find out the SNPs loci relevant to the virus disease resistance. Then DNA sequencing chromatogram was observed via Chromas software, single peak was a homozygotic SNP locus, and the nested peak was a heterozygous SNP locus. 
     II Detection Results of Disease-Resistant SNP Marker Based on  S. chuatsi  IL-6 Gene 
     The same population of  S. chuatsi  was bred under the same feeding conditions, and after breeding for about two months, 100 Mandarin fish were randomly selected for challenge experiments from the cultured population. Each fish was intraperitoneally injected with infectious spleen and kidney necrosis virus (ISKNV) (also known as iridescent virus), 10×TCID50 ISKNV. After observing for 10 days, it was observed that the disease symptoms are the same as those of the ISKNV infection in the natural environment. The diseased fish swam slowly on the water surface, or evenly floated on the water surface, the body surface was undamaged and the colour of body was white. The fish gills were ischemic whitish. By means of anatomy, it was found that there were ascites in enterocoelia, the liver, the stomach wall and the intestinal wall were congestive, and there was yellow effusion in intestinal tract. While the disease-resistant fish were normal all the time. The PCR detection indicated that the head kidney of the diseased  S. chuatsi  were ISKNV-positive, this shows that the death was caused by ISKNV infection. 
     9 undiseased fish and 3 diseased fish (marked as E05, H07, A07) were selected, and a small quantity of tail fin was cut and placed in absolute ethyl alcohol and stored at 4° C., respectively. In terms of the detection method of disease-resistant SNP marker as described in above examples, the PCR amplification product was obtained, as shown in  FIG.  3   , after gel extraction, sequencing was performed and multiple sequence alignment was performed by DNAMAN, and the SNP locus associated with virus disease resistance was found at 233 bp of a nucleotide sequence fragment, as shown in  FIG.  5   . 
     The peak profile of suspected mutant base at 233 bp in the above sequence was observed by Chromas software, as shown in  FIG.  6   ,  FIG.  7   , and  FIG.  8   . At the corresponding bases G and A, the peaks are single and there are no impurity peaks. From this, the base at 233 bp is mutated from G to A, that is G233A. Because the mutation was in the Reverse Complement sequence of DNA, that is, a base sequence AGCTCTTTTGCCGTCGACAAGGAA (SEQ ID NO: 16) ( FIG.  5   ) adjacent to 233 bp of a nucleotide sequence fragment, reverse complement to a base sequence adjacent to 1733 bp of IL-6 gDNA sequence (SEQ ID NO: 2.), in the original DNA sequence, SNP locus was at C1744T. The three samples with base mutation (E05, H07, A07) (AGCTCTTTTGCCATCGACAAGGAA (SEQ ID NO: 17) ( FIG.  5   )) all were  S. chuatsi  susceptible to virus. Consequently, the mutation from G to T was generated at 1744 bp of SNP of IL-6 gene of  S. chuatsi  susceptible to virus. The base at 1744 bp of  S. chuatsi  susceptible to virus is T, and the base at 1744 bp of antivirus  S. chuatsi  is C, the virus is infectious spleen and kidney necrosis virus (ISKNV). By means of this method, the antivirus  S. chuatsi  can be distinguished from  S. chuatsi  susceptible to virus. 
     The above preferred embodiments are described for illustration only, and are not intended to limit the scope of the invention. It should be understood, for a person skilled in the art, that various improvements or variations can be made therein without departing from the spirit and scope of the invention, and these improvements or variations should be covered within the protecting scope of the invention.