A primer set for distinguishing germ cells of recipient yellow catfish and three donor catfish and its application

Abstract

The invention discloses a primer set and application for distinguishing reproductive cells of a recipient yellow catfish and three donor catfishes, belonging to the technical field of fish reproductive cell identification. The specific primers of the invention include at least one of the following: a specific primer sequence of a recipient yellow catfish as shown in SEQ ID NO.5 and SEQ ID No.6; a specific primer sequence of a donor yellow catfish as shown in SEQ ID NO.7 and SEQ ID No.8; a specific primer sequence of a donor bigfin catfish as shown in SEQ ID NO.9 and SEQ ID No.10; and a specific primer sequence of a donor longnose catfish as shown in SEQ ID NO.11 and SEQ ID No.12. By using the primer set provided by the invention, reproductive cells of a recipient yellow catfish and donor yellow catfish, bigfin catfish and longnose catfish can be specifically distinguished quickly and accurately from the RNA level by a PCR method. The operation method provided by the invention is simple and easy to operate, and the primer specificity is high, providing an important method for the identification, breeding and reproductive cell transplantation operation of catfish germplasm resources.

Description

Technical Field

[0001] The invention belongs to the technical field of fish germ cell identification, and particularly relates to a primer set for distinguishing germ cells of a recipient yellow catfish and three donor catfishes and an application thereof. Background Art

[0002] Pelteobagrus fulvidraco, Pelteobagrus vachelli, Bigfin catfish and Longnose catfish belong to the family Catfish of the order Siluriformes and are aquaculture species with high economic value among the catfish. Pelteobagrus vachelli, Bigfin catfish and Longnose catfish are high-quality edible fish, which are deeply loved by consumers because of their tender meat, few bones, delicious taste and rich nutrition. Most catfish have obvious sexual dimorphism in growth rate and individual size, with male individuals larger than female individuals. Therefore, cultivating all-male monosexual groups is of great significance to aquaculture. For the cultivation of all-male seedlings, the traditional method is to induce the sex reversal of genetic male fish to pseudo-female fish (XY) through estrogen, and then mate the pseudo-female fish with normal male fish to prepare super male fish (YY). The offspring produced by mating the YY super male fish with normal female fish (XX) are all male fish (XY). However, this process also requires three generations, which seriously restricts the cultivation time of all-male seedlings. Fish reproductive stem cells have the potential to differentiate into gametes of both sexes. After transplantation, they develop into sperm or eggs depending on the gender of the recipient. Therefore, the process of breeding all-male seedlings can be significantly accelerated through reproductive stem cell transplantation technology.

[0003] The initial sexual maturity cycle of Pelteobagrus vachelli, Bigfin Globetail and Longnose Catfish is relatively long, requiring 3-5 years, so the cycle of cultivating new germplasm is relatively long. Pelteobagrus vachelli, Bigfin Globetail and Longnose Catfish belong to the same family of catfish, and are closely related. They are smaller in size, can reach sexual maturity in 1-2 years, and have relatively mature breeding and farming techniques. Using Pelteobagrus vachelli as the recipient and producing gametes and offspring of Pelteobagrus vachelli, Bigfin Globetail and Longnose Catfish through abdominal reproduction can significantly accelerate the process of germplasm creation. For xenotransplantation, whether the donor germ stem cells proliferate, differentiate and develop in the recipient gonads after transplantation and how to detect and distinguish exogenous germ cells are important links in establishing germ stem cell transplantation technology. Summary of the invention

[0004] The purpose of the present invention is to provide a primer set and application for distinguishing the reproductive cells of a recipient yellow catfish and three donor catfishes. The primer set provided by the present invention can quickly, accurately and efficiently distinguish the reproductive cells of the recipient yellow catfish and three donor catfishes. The operation method is simple and easy, the specificity is high, and the market application prospect is huge.

[0005] In order to achieve the above-mentioned object of the invention, the technical solution adopted by the present invention is:

[0006] The invention provides a yellow catfish dndThe gene has a nucleotide sequence as shown in SEQ ID NO.1.

[0007] The invention provides a primer set for identifying a recipient yellow catfish and three donor catfishes, the primer set comprising a recipient yellow catfish and three donor catfishes dnd Complete cDNA sequence of the gene, specific primers and universal primers; recipient yellow catfish dnd The cDNA sequence of the gene is shown in SEQ ID NO.1; the donor is Pelteobagrus vachelli dnd The cDNA sequence of the gene is shown in SEQ ID NO.2; the donor is Macropterus serratus dnd The cDNA sequence of the gene is shown in SEQ ID NO.3; the donor is Catfish dnd The cDNA sequence of the gene is shown in SEQ ID NO.4.

[0008] Further, the specific primers include the recipient yellow catfish, the donor yellow catfish, the donor bigfin pufferfish, and the donor longnose catfish. dnd At least one of the gene-specific primers:

[0009] Pelteobagrus fulvidraco dnd The gene-specific primer sequences are shown in SEQ ID NO. 5 and SEQ ID No. 6, specifically 5'- GACGTATTAGCAGAGCAATTAACA-3' and 5'- GCAGAAAACAAGTAATTGAGGAA-3';

[0010] Pelteobagrus vachelli dnd The gene-specific primer sequences are shown in SEQ ID NO. 7 and SEQ ID NO. 8, specifically 5'-CCCTGGAGAAGTGGCTTAC-3' and 5'-TGAATTTCCCGCTTCCAGA-3';

[0011] The donor dnd The gene-specific primer sequences are shown in SEQ ID NO.9 and SEQ ID NO.10, specifically 5′-TGCAGATGTTGAATCCACAGA-3′ and 5′-TCTCAACCAAAACTTGAGGTCA-3′;

[0012] The donor catfish dnd The gene-specific primer sequences are shown in SEQ ID NO. 11 and SEQ ID NO. 12, specifically 5′-AGTCCCTGGAGAAGTGGCTTAC-3′ and 5′-GAAATGTCGTACTTTATGAACTCAC-3′;

[0013] The recipient yellow catfish and three donor catfish β- The sequences of the positive control primers for the actin internal reference gene are shown in SEQ ID NO.13 and SEQ ID No.14, specifically 5'-TCCCTGTATGCCTCTGGTCGT-3' and 5'-AAGCTGTAGCCTCTCTCGGTC-3'.

[0014] The invention also provides application of the primer set in identifying the recipient yellow catfish and three donor catfishes.

[0015] Furthermore, the method for identifying the reproductive cells of the recipient yellow catfish and the donor yellow catfish, bigfin catfish, and longnose catfish comprises the following steps:

[0016] (1) Extracting total RNA from gonadal tissues of yellow catfish, yellow catfish, bigfin bream and longnose catfish;

[0017] (2) Reverse transcription of total RNA from gonadal tissues of yellow catfish, yellow catfish, bigfin bream and longnose catfish into total cDNA;

[0018] (3) performing PCR amplification on the total cDNA using primers as shown in SEQ ID No. 5 to SEQ ID No. 14;

[0019] (4) The PCR amplification products were subjected to electrophoresis detection, and the reproductive cells of the recipient yellow catfish and the donor yellow catfish, bigfin bream, and longnose catfish were distinguished based on the electrophoresis results.

[0020] Further, in step (4), the yellow catfish-specific primers were used to amplify the recipient yellow catfish and the donor yellow catfish, bigfin catfish, and longnose catfish gonad cDNA as templates. Only the recipient yellow catfish had the target fragment, while the donor yellow catfish, bigfin catfish, and longnose catfish did not have the target fragment, which was determined to be the recipient yellow catfish germ cell; the yellow catfish-specific primers were used to amplify the recipient yellow catfish and the donor yellow catfish gonad cDNA as templates. Only the donor yellow catfish had the target fragment, and the recipient yellow catfish had the target fragment. There was no target fragment in the recipient yellow catfish, so it was determined to be the germ cell of Pelteobagrus vachelli; the bigfin catfish-specific primers were used to amplify the cDNA of the recipient yellow catfish and the donor bigfin catfish gonad as templates. Only the donor bigfin catfish had the target fragment, while the recipient yellow catfish had no target fragment, so it was determined to be the germ cell of Bigfin Catfish; the longnose catfish-specific primers were used to amplify the cDNA of the recipient yellow catfish and the donor longnose catfish gonad as templates. Only the donor longnose catfish had the target fragment, while the recipient yellow catfish had no target fragment, so it was determined to be the germ cell of Longnose Catfish.

[0021] Furthermore, the PCR amplification system is: 2×Taq Plus Master MIX II (Dye Plus) 10 μL, F and R primers 0.5 μL each, cDNA template 1 μL, ddH2O to make up the system to 20 μL.

[0022] Furthermore, the PCR amplification conditions were as follows: 95°C pre-denaturation for 3 min, 95°C denaturation for 30 s, 54°C (yellow catfish and yellow catfish) / 55°C (bigfin bream and longnose catfish) annealing for 30 s, 72°C extension for 10 s, for a total of 33 cycles, and finally 72°C extension for 10 min.

[0023] The present invention provides a kit for identifying reproductive cells of a recipient yellow catfish and donor yellow catfish, bigfin catfish, and longnose catfish, comprising a PCR reaction solution and primers, wherein the primers include at least one of the following primers (1) to (4):

[0024] (1) The receptor yellow catfish-specific primer sequences shown in SEQ ID NO.5 and SEQ ID No.6;

[0025] (2) donor Pelteobagrus vachelli specific primer sequences as shown in SEQ ID NO.7 and SEQ ID NO.8;

[0026] (3) the donor bigfin bream-specific primer sequences as shown in SEQ ID NO.9 and SEQ ID NO.10;

[0027] (4) The donor long-snout catfish-specific primer sequences as shown in SEQ ID NO.11 and SEQ ID No.12.

[0028] In the kit, the PCR reaction solution includes at least one of 2×Taq Plus Master MIX II (Dye Plus) and ddH2O.

[0029] In the kit, the PCR reaction solution also includes a cDNA template; the cDNA template is a total cDNA formed by reverse transcription of total RNA from gonadal tissues of the recipient yellow catfish and donor yellow catfish, bigfin catfish, and longnose catfish.

[0030] The invention provides use of the kit in germ cell transplantation between a recipient yellow catfish and donor yellow catfish, bigfin bream and longnose catfish.

[0031] In this application, the kit can be used to identify whether the donor germ cells are successfully transplanted into the recipient fish. Preferably, the recipient fish is yellow catfish, and the donor germ cells are yellow catfish, bigfin catfish, and longnose catfish germ cells.

[0032] The present invention has the following beneficial effects: the present invention utilizes germ cell marker genes dnd Specific primers for the recipient yellow catfish and the donor yellow catfish, bigfin catfish, and longnose catfish were designed based on cDNA sequences. The genes of the germ cells of the recipient yellow catfish and the donor yellow catfish, bigfin catfish, and longnose catfish were amplified by PCR, and the germ cells of the recipient yellow catfish and the donor yellow catfish, bigfin catfish, and longnose catfish were specifically detected at the RNA level. Pelteobagrus fulvidraco-specific primers can only amplify and identify the reproductive cells of Pelteobagrus fulvidraco, Pelteobagrus vachelli-specific primers can only amplify and identify the reproductive cells of Pelteobagrus vachelli, Pelteobagrus fulvidraco-specific primers can only amplify and identify the reproductive cells of Pelteobagrus vachelli, Pelteobagrus vachelli-specific primers can only amplify and identify the reproductive cells of Pelteobagrus fulvidraco, and Pelteobagrus longirostris-specific primers can only amplify and identify the reproductive cells of Pelteobagrus longirostris. By comparing and analyzing the donor and recipient specific primers, the reproductive cells of the recipient Pelteobagrus fulvidraco and the donors Pelteobagrus vachelli, Pelteobagrus vachelli, and Pelteobagrus longirostris can be distinguished.

[0033] Therefore, the specific primers of the present invention for distinguishing the germ cells of the recipient yellow catfish and the donor yellow catfish, bigfin catfish and longnose catfish can efficiently, quickly and accurately distinguish the germ cells of the recipient yellow catfish and the donor yellow catfish, bigfin catfish and longnose catfish, and are applied to the detection of germ cell transplantation between the recipient yellow catfish and the donor yellow catfish, bigfin catfish and longnose catfish, and distinguish the chimerism, proliferation and development of the donor germ cells in the gonads of the recipient fish. Specifically, the method of the present invention can directly distinguish the sources of the germ cells of the recipient yellow catfish and the donor yellow catfish, bigfin catfish and longnose catfish by using the gonad tissue as a template; the present invention utilizes the germ cell-specific marker gene dnd As the basis for identification, and according to dnd Specific primers can be designed based on the cDNA sequence of the gene, which can be further used to detect the origin of the reproductive cells of the recipient yellow catfish and the donor yellow catfish, bigfin bream, and longnose catfish.

[0034] The invention provides a primer and method for quickly, accurately and efficiently distinguishing the reproductive cells of the recipient yellow catfish from the donor yellow catfish, bigfin catfish and longnose catfish, which is simple, easy to operate and highly specific, and has important significance in accelerating the cultivation of all-male seedlings of catfish. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 Yellow catfish dnd The full-length cDNA sequence of the gene and its amino acid sequence;

[0036] Figure 2 Pelteobagrus vachelli dnd The full-length cDNA sequence of the gene and its amino acid sequence;

[0037] Figure 3 The donor is a large fin bream dnd The full-length cDNA sequence of the gene and its amino acid sequence;

[0038] Figure 4 The donor is Longnose Catfish dnd The full-length cDNA sequence of the gene and its amino acid sequence;

[0039] Figure 5 The recipient yellow catfish and the donor yellow catfish, bigfin catfish, longnose catfish dnd cDNA sequence of the gene and primer locations;

[0040] Figure 6 The recipient yellow catfish and the donor yellow catfish, bigfin catfish, longnose catfish gonads dnd Agarose gel electrophoresis results of gene PCR amplification products. DETAILED DESCRIPTION

[0041] In order to make the objectives, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with specific embodiments.

[0042] The experimental methods in the following examples are conventional methods unless otherwise specified. The materials, reagents, etc. used in the following examples are commercially available unless otherwise specified.

[0043] Example 1

[0044] The present invention provides a primer set for distinguishing the reproductive cells of a recipient yellow catfish from three donor catfish (donor Pelteobagrus vachelli, Macrofin Ornithorhynchus, and Catfish of the long snout). In this embodiment, PCR technology is used to distinguish the reproductive cells of the recipient yellow catfish from the donor Pelteobagrus vachelli, Macrofin Ornithorhynchus, and Catfish of the long snout.

[0045] 1. A method for distinguishing the reproductive cells of a recipient yellow catfish from those of a donor yellow catfish, a large fin catfish, or a long-snout catfish using PCR technology at the RNA level, comprising the following steps:

[0046] 1) Total RNA was extracted from the gonadal tissues of the recipient yellow catfish and the donor yellow catfish, bigfin bream, and longnose catfish using the RNeasy plus Mini kit.

[0047] 2) Using PrimeScript™ RT reagent Kit (purchased from Clontech), the total RNA of the gonadal tissues of the recipient yellow catfish and the donor yellow catfish, bigfin catfish, and longnose catfish was reverse transcribed into total cDNA. dnd The cDNA sequence of the gene is shown in SEQ ID NO.1 ( Figure 1 );The donor Pelteobagrus vachelli dnd The cDNA sequence of the gene is shown in SEQ ID NO.2 ( Figure 2 );The donor macrofin bream dnd The cDNA sequence of the gene is shown in SEQ ID NO.3 ( Figure 3 );The donor longnose catfish dnd The cDNA sequence of the gene is shown in SEQ ID NO.4 ( Figure 4 ).

[0048] 3) Use the PCR amplification primers shown in Table 1 ( Figure 5 ),by β -actin was used as an internal reference gene as a positive control, and PCR amplification was performed on the total cDNA of the samples.

[0049] The PCR amplification system was as follows: 2× Taq Plus Master MIX II (Dye Plus) 10 μL, 0.5 μL each of F and R primers, 1 μL of DNA template, and ddH2O to make up the system to 20 μL;

[0050] The PCR amplification conditions were as follows: pre-denaturation at 95 °C for 3 min, denaturation at 95 °C for 30 s, annealing at 54 °C (for yellow catfish and yellow catfish) / 55 °C (for bigfin bream and longnose catfish) for 30 s, extension at 72 °C for 10 s, for a total of 33 cycles, and a final extension at 72 °C for 10 min.

[0051] 4) After the PCR reaction, the PCR products were detected by 1.5% agarose gel electrophoresis. The electrophoresis diagrams of the male and female gonads of the recipient yellow catfish and the donor yellow catfish, bigfin pufferfish, and longnose catfish were amplified by PCR using specific primers ( Figure 6 ), based on the electrophoresis results, the presence or absence of the target band was used to distinguish the germ cells of the recipient yellow catfish and the donor yellow catfish, bigfin bream, and longnose catfish.

[0052] The yellow catfish-specific primers were used to amplify the recipient yellow catfish and the donor yellow catfish, bigfin catfish, and longnose catfish gonad cDNA as templates. Only the recipient yellow catfish had the target fragment, while the donor yellow catfish, bigfin catfish, and longnose catfish did not have the target fragment, which was determined to be the recipient yellow catfish germ cell; the yellow catfish-specific primers were used to amplify the recipient yellow catfish and the donor yellow catfish gonad cDNA as templates. Only the donor yellow catfish had the target fragment, while the recipient yellow catfish had no target fragment. The fragment was determined to be the germ cell of Pelteobagrus vachelli; the bigfin catfish-specific primers were used to amplify the recipient Pelteobagrus and donor Bigfin Catfish gonad cDNA as templates. Only the donor Bigfin Catfish had the target fragment, while the recipient Pelteobagrus did not, so it was determined to be the germ cell of Bigfin Catfish; the longnose catfish-specific primers were used to amplify the recipient Pelteobagrus and donor Longnose Catfish gonad cDNA as templates. Only the donor Longnose Catfish had the target fragment, while the recipient Pelteobagrus did not, so it was determined to be the germ cell of Longnose Catfish.

[0053] Table 1 Specific primers for PCR amplification

[0054]

[0055] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned embodiments, it is still possible for a person skilled in the art to modify the technical solutions described in the aforementioned embodiments, or to replace some of the technical features therein by equivalents. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions claimed to be protected by the present invention.

Claims

1. A primer set for identifying reproductive cells of recipient yellow catfish and three donor catfish, characterized in that: The primer set includes the recipient yellow catfish and three donor catfish dnd Complete cDNA sequence of the gene, specific primers and universal primers; recipient yellow catfish dnd The cDNA sequence of the gene is shown in SEQ ID NO.1; Pelteobagrus vachelli dnd The cDNA sequence of the gene is shown in SEQ ID NO.2; the donor is Macropterus serratus dnd The cDNA sequence of the gene is shown in SEQ ID NO.3; the donor is Catfish dnd The cDNA sequence of the gene is shown in SEQ ID NO.4; The specific primers include the recipient yellow catfish, the donor yellow catfish, the donor bigfin pufferfish, and the donor longnose catfish. dnd Gene-specific primers: Pelteobagrus fulvidraco dnd The gene-specific primer sequences are shown in SEQ ID No. 5 and SEQ ID No. 6; Pelteobagrus vachelli dnd The gene-specific primer sequences are shown in SEQ ID NO.7 and SEQ ID NO.8; the donor macrofin bream dnd The gene-specific primer sequences are shown in SEQ ID NO.9 and SEQ ID NO.10; the donor long-nose catfish dnd The gene-specific primer sequences are shown in SEQ ID No. 11 and SEQ ID No. 12; The recipient yellow catfish and three donor catfish β- The sequences of the positive control primers for the actin internal reference gene are shown in SEQ ID NO.13 and SEQ ID No.

14.

2. Use of the primer set described in claim 1 in identifying reproductive cells of recipient yellow catfish and three donor catfish.

3. The use according to claim 2, characterized in that: The application comprises the following steps: (1) Extracting total RNA from gonadal tissues of yellow catfish, yellow catfish, bigfin bream, and longnose catfish; (2) Reverse transcription of total RNA from gonadal tissues of Pelteobagrus fulvidraco, Pelteobagrus vachelli, Macrobrachium brevicaulis, and Catfish was performed into total cDNA; (3) performing PCR amplification on the total cDNA using primers as shown in SEQ ID No. 5 to SEQ ID No. 14; (4) The PCR amplification products were subjected to electrophoresis detection, and the reproductive cells of the recipient yellow catfish and the donor yellow catfish, bigfin bream, and longnose catfish were distinguished based on the electrophoresis results.

4. The use according to claim 3, characterized in that: The identification method in step (4) is as follows: Only the recipient yellow catfish had the target fragment, while the donor yellow catfish, bigfin catfish and longnose catfish did not have the target fragment, which was determined to be the recipient yellow catfish germ cell; Only the donor P. vachelli had the target fragment, while the recipient P. vachelli had no target fragment, which was determined to be the germ cell of P. vachelli. Only the donor giant fin bream had the target fragment, while the recipient yellow catfish had no target fragment, which was determined to be the reproductive cell of giant fin bream; Only the donor longnose catfish had the target fragment, while the recipient yellow catfish did not, and it was determined to be the germ cells of the longnose catfish.

5. The use according to claim 3, characterized in that: The PCR amplification system is: 2×Taq Plus MasterMIX II 10 μL, upstream primer and downstream primer 0.5 μL each, DNA template 1 μL, ddH2O to make up the system to 20 μL.

6. The use according to claim 3, characterized in that: The PCR amplification conditions were as follows: 95°C pre-denaturation for 3 min, 95°C denaturation for 30 s, annealing temperature of 54°C for yellow catfish and yellow catfish / 55°C annealing for 30 s for bigfin puffer and longnose catfish, extension at 72°C for 10 s, for a total of 33 cycles, and finally extension at 72°C for 10 min.

7. A kit for identifying reproductive cells of a recipient yellow catfish and three donor catfish, characterized in that: The kit includes a PCR reaction solution and the following (1)-(4): (1) The receptor yellow catfish-specific primer sequences shown in SEQ ID NO.5 and SEQ ID No.6; (2) donor Pelteobagrus vachelli specific primer sequences as shown in SEQ ID NO.7 and SEQ ID NO.8; (3) the donor bigfin bream-specific primer sequences as shown in SEQ ID NO.9 and SEQ ID NO.10; (4) The donor long-snout catfish-specific primer sequences as shown in SEQ ID NO.11 and SEQ ID No.

12.

8. Use of the kit according to claim 7 in identifying germ cells of a recipient yellow catfish and donor yellow catfish, bigfin bream, and longnose catfish, and in identifying results of germ cell transplantation.

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