Method for identifying germ cells of grass carp and carp by using dazl gene and application

Abstract

The invention discloses a method for identifying germ cells of grass carps and carps by using dazl genes. The method comprises the following steps: cloning dazl cDNA sequences of the grass carps and the carps, and performing comparative analysis by using DNAMAN software; designing a universal non-specific primer, a grass carp specific primer and a carp specific primer according to sequence differences; extracting total RNA (Ribonucleic Acid) of testis of 12-month-old grass carp and carp, and reversely transcribing into cDNA Respectively carrying out PCR (Polymerase Chain Reaction) amplification by using three pairs of primers by taking the cDNA as a template; carrying out 1% agarose gel electrophoresis and imaging on the PCR product, and identifying and distinguishing germ cells according to the size of a strip; the method is simple in structure and reasonable in design, high-specificity primers are designed based on small difference of dazl gene sequences for the first time, precise amplification is achieved by combining with optimized annealing temperature (60 DEG C / 65 DEG C), special instruments are not needed, operation is easy and convenient, sensitivity is high, grass carp germ cells in carp gonads after transplantation can be rapidly identified, development of the'belly borrowing reproduction 'technology is supported, and the method is suitable for large-scale popularization and application. The grass carp breeding period is remarkably shortened, and the important industrialization prospect is achieved.

Description

Technical Field

[0001] This invention relates to the field of fish reproductive cell identification technology, specifically a method and application for identifying grass carp and common carp reproductive cells using the dazl gene. Background Technology

[0002] Grass carp (Ctenopharyngodon idella) is an important freshwater aquaculture fish in my country, with a national output of nearly 6.165 million tons in 2024, making it the highest-producing aquaculture fish in the country. It is of great significance for ensuring the supply of high-quality animal protein for residents. In recent years, however, the decline in disease resistance, inconsistent body size, and low feed conversion rate among farmed grass carp have become increasingly prominent, seriously threatening the sustainable development of the industry. Therefore, the need for breeding new varieties is becoming increasingly urgent.

[0003] However, grass carp take 4–5 years to reach sexual maturity, making the traditional breeding cycle extremely long. Fish germ cell transplantation is a cutting-edge technology aimed at overcoming this bottleneck. This technology involves implanting reproductive stem cells from a donor fish into a recipient fish to obtain a chimeric reproductive system and produce gametes or offspring derived from the donor; it is also known as "surrogate reproduction" (Jin et al., 2021). However, research on grass carp "surrogate reproduction" started relatively late, with only reports of using zebrafish to produce grass carp sperm (Ye et al., 2024). Due to the small size of zebrafish, it is difficult to produce large quantities of grass carp sperm on a large scale, and there is no research on using zebrafish to produce mature grass carp eggs.

[0004] The sexual maturity cycle of carp is shorter than that of grass carp, especially for male carp, which typically only need 4-5 months to produce sperm (Cao et al., 2014). Utilizing carp to produce gametes for grass carp can significantly shorten the grass carp breeding cycle. Moreover, using carp as transplant recipients is expected to generate a large number of sperm and eggs, which is of great significance for large-scale grass carp breeding. Research on interspecies germ cell transplantation between grass carp and carp requires analyzing whether the transplanted carp gonads contain grass carp germ cells, but currently, there are no reports on relevant detection techniques. Establishing efficient techniques for identifying and distinguishing between grass carp and carp germ cells is an important technical foundation for utilizing carp to produce mature grass carp gametes and has significant application prospects.

[0005] In the applicant's previous method for identifying eel germ cells after transplantation into carp gonads (Tao et al., Chinese Invention Patent ZL202210411188.3), a species-specific germ cell identification method was reported. Due to the significant differences in gene sequences between eels and carp, primers specifically designed to amplify eel germ cell-specific expression genes in completely different cDNA segments can be used for PCR amplification (Tao et al., 2025). However, the germ cell-specific expression gene sequences of grass carp and carp are highly conserved, making specific primer design difficult. Specific primers need to be found on cDNA fragments with only a few base differences, and different PCR annealing temperatures need to be tried to achieve specific amplification of grass carp and carp germ cell expression genes. Therefore, compared with the technique for identifying eel germ cells in carp gonads, the techniques for identifying grass carp and carp germ cells differ in primer design methods and PCR amplification program optimization. Summary of the Invention

[0006] The purpose of this invention is to provide a method for identifying grass carp and common carp reproductive cells using the dazl gene, in order to solve the problems mentioned in the background art.

[0007] To achieve the above objectives, the present invention provides the following technical solution: a method for identifying grass carp and common carp reproductive cells using the dazl gene, comprising the following steps: S1: Cloning grass carp and common carp dazlcDNA sequences, and comparing and analyzing the dazlcDNA sequences using DNAMAN software; S2: Designing universal non-specific primers, grass carp dazl sequence amplification primers, and common carp dazl sequence amplification primers based on the sequence comparison analysis results; S3: Extracting total RNA from the testes of 12-month-old grass carp and common carp, and reverse transcribing it into cDNA; S4: Using the cDNA obtained in step S3 as a template, performing PCR amplification of the dazl sequence using the non-specific primers, grass carp-specific primers, and common carp-specific primers from step S2, respectively; S5: Electrophoresis of the PCR products from step S4 on a 1% agarose gel, and imaging the electrophoresed agarose gel using a gel imaging system to identify and distinguish the reproductive cells of grass carp and common carp.

[0008] Preferably, in step S5, the electrophoresis voltage is 200V and the electrophoresis time is 20 minutes.

[0009] Preferably, the non-specific primer sequences in step S2 are: dazl-uni-F: 5'-TTTTACCTGAGGGCAAGATG-3', dazl-uni-R: 5'-AACATGTGCCCTGAATGTGAA-3'.

[0010] Preferably, the grass carp-specific primer sequences in step S2 are: dazl-gc-F: 5'-TCAGAGAGTTCTTTGCCAAG-3', dazl-gc-R: 5'-CACACAGACAATGAAGTGCA-3'.

[0011] Preferably, the carp-specific primer sequences in step S2 are: dazl-cc-F: 5'-TGCAAAGGGTATGGTTTCGT-3', dazl-cc-R: 5'-TCTGTGCTTGTGGTTGAGGG-3'.

[0012] Preferably, the PCR amplification program for the non-specific primers in step S4 is as follows: hold at 95°C for 3 minutes; then perform 34 cycles, each cycle including: hold at 95°C for 15 seconds, hold at 60°C for 15 seconds, hold at 72°C for 40 seconds; and finally hold at 72°C for 5 minutes.

[0013] Preferably, the PCR amplification program for grass carp-specific primers and carp-specific primers in step S4 is as follows: hold at 95°C for 3 minutes; then perform 34 cycles, each cycle including: hold at 95°C for 15 seconds, hold at 65°C for 15 seconds, hold at 72°C for 20 seconds; and finally hold at 72°C for 5 minutes.

[0014] This invention provides the application of the aforementioned method in identifying grass carp germ cells transplanted into the gonads of carp.

[0015] Compared with the prior art, the beneficial effects of the present invention are as follows: Compared with the prior art, the advantages and technical effects of the present invention are as follows: The present invention utilizes PCR technology to amplify the dazl gene of grass carp and common carp. Grass carp dazl-specific primers can only recognize grass carp germ cells and amplify the 596bp grass carp dazl fragment, while common carp dazl-specific primers can only recognize common carp germ cells and amplify the 455bp common carp dazl fragment. Universal primers can simultaneously amplify grass carp (1120bp) and common carp (1075bp) dazl fragments as controls. As mentioned above, using agarose gel electrophoresis, grass carp and common carp germ cells can be quickly distinguished based on the size and specificity of the PCR product bands. Combining the advantages of simple and efficient PCR technology and the high specificity of specific primers, this method can accurately identify grass carp germ cells in the gonads of transplanted common carp.

[0016] The dazl gene was amplified by PCR using the three pairs of primers described above, and grass carp and common carp germ cells were rapidly and accurately distinguished based on the product bands; universal primers were used as positive controls during the identification process. The method provided by this invention can be used to detect the success of grass carp-common carp germ cell transplantation, assess germ cell chimerism efficiency, and monitor the developmental process of donor grass carp germ cells in the gonads of recipient common carp. It provides a key detection method for the development of "surrogate reproduction" technology and has significant application value and industrialization prospects. Attached Figure Description

[0017] Figure 1 Image showing the results of dazl cDNA sequence alignment for grass carp and common carp; Figure 2 Agarose gel electrophoresis image of cDNA from the testes of grass carp and common carp after PCR amplification using three pairs of primers. Detailed Implementation

[0018] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0019] In the description of this invention, it should be understood that the terms "center," "longitudinal," "lateral," "length," "width," "thickness," "upper," "lower," "top," "bottom," "inner," "outer," "clockwise," and "counterclockwise," etc., indicate the orientation or positional relationship based on the orientation or positional relationship shown in the accompanying drawings. They are only for the convenience of describing this invention and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation. Therefore, they should not be construed as limitations on this invention.

[0020] In the description of this patent, it should be noted that, unless otherwise explicitly specified and limited, the terms "installation," "connection," and "setting" should be interpreted broadly. For example, they can refer to a fixed connection or setting, a detachable connection or setting, or an integrated connection or setting. Those skilled in the art can understand the specific meaning of the above terms in this patent according to the specific circumstances.

[0021] Furthermore, the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. In the description of this invention, "a number" means two or more, unless otherwise explicitly specified.

[0022] Example This invention uses primers and PCR technology to distinguish between the reproductive cells of grass carp and common carp. The specific technical steps are as follows: 1. Primer design Grass carp and common carp dazl cDNA sequences were obtained by PCR cloning, and sequence alignment analysis was performed using DNAMAN software (Lynnon Biosoft, USA). Figure 1 Based on the comparison results, three pairs of primers were designed: Non-specific primers: dazl-uni-F:5'-TTTTACCTGAGGGCAAGATG-3' dazl-uni-R:5'-AACATGTGCCCTGAATGTGAA-3' Grass carp specific primers: dazl-gc-F:5'-TCAGAGAGTTCTTTGCCAAG-3' dazl-gc-R:5'-CACACAGACAATGAAGTGCA-3' Carp-specific primers: dazl-cc-F:5'-TGCAAAGGGTATGGTTTCGT-3' dazl-cc-R:5'-TCTGTGCTTGTGGTTGAGGG-3' 2. Sample preparation Three 12-month-old grass carp and three 12-month-old common carp were dissected to obtain testicular tissue. Total RNA was extracted using Trizol reagent, and the RNA was reverse transcribed into cDNA according to the reverse transcription kit instructions.

[0023] 3. PCR amplification Using cDNA as a template, amplification was performed using three pairs of primers. Reaction volume (20 μL): 10 μL 2×Taq PCRMaster Mix Upstream primer (10 μM) 0.5 μL Downstream primer (10 μM) 0.5 μL 1 μL of cDNA template ddH2O 7 μL PCR amplification procedure: Non-specific primers: Hold at 95℃ for 3 min; then perform 34 cycles (95℃ for 15 s, 60℃ for 15 s, 72℃ for 40 s); finally hold at 72℃ for 5 min, and end at 12℃. Grass carp-specific primers and carp-specific primers: hold at 95℃ for 3 min; then perform 34 cycles (95℃ for 15 s, 65℃ for 15 s, 72℃ for 20 s); finally hold at 72℃ for 5 min, and end at 12℃.

[0024] 4. Electrophoresis detection The PCR products were electrophoresed in a 1% agarose gel at 200 V for 20 min.

[0025] 5. Results Analysis Observe using a gel imaging system ( Figure 2 ): Non-specific primers: Grass carp amplified a 1120 bp band, and common carp amplified a 1075 bp band; Grass carp-specific primers: only grass carp showed a 596 bp band, while carp showed no amplification; Carp-specific primers: Only carp showed a 455 bp band, while grass carp showed no amplification.

[0026] Based on the presence and size of the stripes, it is possible to accurately identify whether a sample contains grass carp or common carp reproductive cells.

Claims

1. A method for identifying germ cells of grass carp and common carp using the dazl gene, characterized in that: Includes the following steps: S1: The dazlcDNA sequences of grass carp and common carp were cloned and compared and analyzed using DNAMAN software; S2: Based on the results of sequence comparison analysis, design universal non-specific primers for amplifying the dazl sequence of grass carp and common carp, specific primers for amplifying the dazl sequence of grass carp, and specific primers for amplifying the dazl sequence of common carp; S3: Extract total RNA from the testes of 12-month-old grass carp and common carp and reverse transcribe it into cDNA. S4: Using the cDNA obtained in step S3 as a template, PCR amplification of the dazl sequence was performed using the non-specific primers, grass carp-specific primers, and carp-specific primers from step S2, respectively; S5: The PCR products from step S4 were electrophoresed with 1% agarose gel, and the agarose gel after electrophoresis was imaged using a gel imaging system to identify and distinguish the reproductive cells of grass carp and carp.

2. The method according to claim 1, characterized in that: In step S5, the electrophoresis voltage is 200V and the electrophoresis time is 20 minutes.

3. The method according to claim 1, characterized in that: The non-specific primer sequences in step S2 are: dazl-uni-F: 5'-TTTTACCTGAGGGCAAGATG-3', dazl-uni-R: 5'-AACATGTGCCCTGAATGTGAA-3'.

4. The method according to claim 1, characterized in that: The grass carp-specific primer sequences in step S2 are: dazl-gc-F: 5'-TCAGAGAGTTCTTTGCCAAG-3', dazl-gc-R: 5'-CACACAGACAATGAAGTGCA-3'.

5. The method according to claim 1, characterized in that: The carp-specific primer sequences in step S2 are: dazl-cc-F: 5'-TGCAAAGGGTATGGTTTCGT-3', dazl-cc-R: 5'-TCTGTGCTTGTGGTTGAGGG-3'.

6. The method according to claim 1, characterized in that: The PCR amplification program for the non-specific primers in step S4 is as follows: hold at 95°C for 3 minutes; then perform 34 cycles, each cycle including: hold at 95°C for 15 seconds, hold at 60°C for 15 seconds, hold at 72°C for 40 seconds; and finally hold at 72°C for 5 minutes.

7. The method according to claim 1, characterized in that: The PCR amplification program for grass carp-specific primers and carp-specific primers in step S4 is as follows: hold at 95°C for 3 minutes; then perform 34 cycles, each cycle including: hold at 95°C for 15 seconds, hold at 65°C for 15 seconds, hold at 72°C for 20 seconds; and finally hold at 72°C for 5 minutes.

8. The use of the method according to any one of claims 1-7 in identifying grass carp germ cells transplanted into carp gonads.

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