A recombinant plasmid for expressing a heterologous growth hormone gene, a construction method and application thereof in breeding of Ophicephalus argus
Patent Information
- Application Number
- CN202610732831.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2026-05-26
- Publication Date
- 2026-09-25
AI Technical Summary
但目前尚无针对马口鱼的高效Tol2转座子介导生长激素基因表达体系，异源生长激素基因在马口鱼中的适配性、表达效率及促生长效果均未被验证，无法满足马口鱼快速育种的产业需求
高效整合与稳定表达：采用Tol2转座子系统，可实现外源基因在马口鱼基因组中的高效拷贝整合。配合强效的金鱼β-actin启动子，确保了生长激素基因在鱼体全身的稳定、高水平表达。
Smart Images
Figure CN122811279A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of animal genetic engineering and biological breeding technology, specifically relating to a recombinant plasmid expressing a heterologous growth hormone gene to enhance the growth rate of chub, its construction method, and its application in chub breeding. Background Technology
[0002] The Chinese minnow (Siniperca chuatsi) is an important freshwater economic fish species in my country. Due to overfishing, water pollution, and water conservancy projects, its natural resources have declined sharply, leading to a continuous increase in demand for artificial breeding. However, the Chinese minnow suffers from drawbacks such as late sexual maturity (reaching sexual maturity at one winter age), slow growth rate, and small individual size. In southern regions, farmed populations only reach a total length of 95.0-176.5 mm and a body length of 72.0-140.5 mm. These problems directly result in long breeding cycles, low yields, and poor economic benefits, severely restricting the large-scale development of the industry.
[0003] In existing fish breeding technologies, transgenic breeding can precisely accelerate growth by introducing exogenous growth-related genes. Among these, the Tol2 transposon system has become the preferred tool for fish transgenic breeding due to its high integration efficiency and genetic stability. However, there is currently no efficient Tol2 transposon-mediated growth hormone gene expression system for chub. The adaptability, expression efficiency, and growth-promoting effect of heterologous growth hormone genes in chub have not been verified, which cannot meet the industrial demand for rapid breeding of chub.
[0004] Therefore, there is an urgent need in this field for an efficient, stable, and industrially applicable transgenic breeding technology for chub to solve the core problem of its slow growth. Summary of the Invention
[0005] The technical problem to be solved by this invention is to provide a recombinant plasmid expressing a heterologous growth hormone gene, a construction method thereof, and its application in the breeding of chub, thereby realizing the expression of exogenous growth hormone gene. gh The gene is expressed efficiently and stably in the chub, and the positive rate of transgenic chub can reach more than 70%. The body length can increase by 130% and the weight can increase by 150%, which significantly improves the growth rate of chub, significantly shortens the chub farming cycle and increases the yield. It provides a general technical solution for transgenic breeding of economic fish and has high scientific research and industrialization value.
[0006] The technical solution adopted in this invention is as follows: In a first aspect, the present invention provides a recombinant plasmid expressing a heterologous growth hormone gene. The recombinant plasmid uses a plasmid carrying the left arm of the Tol2 transposon, the right arm of the Tol2 transposon, the goldfish β-actin promoter, the enhanced green fluorescent protein gene EGFP-SV40, and a polyA sequence as its backbone. The zebrafish-derived growth hormone gene zebrafish- was inserted into the backbone plasmid. gh Or goldfish-derived growth hormone gene goldfish- gh Furthermore, the growth hormone gene is linked to the EGFP gene via a P2A sequence; The nucleotide sequence of the recombinant plasmid is shown in SEQ ID NO.1 or SEQ ID NO.2.
[0007] Furthermore, the recombinant plasmid is pTol2-β-actin-zebrafish- gh -P2A-EGFP-SV40-polyA, or pTol2-β-actin-EGFP-P2A-goldfish- gh -SV40-polyA.
[0008] Secondly, the present invention provides a method for constructing the above-mentioned recombinant plasmid. The construction method includes the following steps: S1: Using cDNA reverse-transcribed from total RNA of zebrafish or goldfish as a template, PCR amplification was performed to obtain zebrafish- gh Gene fragments or goldfish- gh Gene fragments; S2: In zebrafish- gh By linking the P2A sequence to the 3' end of the gene, zebrafish- gh -P2A fragment; in goldfish- gh By linking the P2A sequence to the 5' end of the gene, we obtain P2A-goldfish- gh Fragment; S3: Move the zebrafish- gh -P2A fragment or P2A-goldfish- gh The fragment is ligated to the backbone plasmid via seamless cloning to obtain the target recombinant plasmid.
[0009] Furthermore, the primer sequences used for PCR amplification in step S1 are shown in SEQ ID NO.3~SEQ ID NO.6.
[0010] Thirdly, the present invention provides the application of the above-mentioned recombinant plasmid in the preparation of fast-growing chub.
[0011] Fourthly, this invention provides the application of the above-mentioned recombinant plasmid in transgenic breeding of chub.
[0012] Fifthly, the present invention provides a method for improving the growth rate of chub. The method includes the following steps: S1: The recombinant plasmid is mixed with Tol2 transposase mRNA and introduced into wild-type koji fertilized eggs via microinjection; S2: Positive embryos expressing green fluorescence were screened under a fluorescence microscope and cultured to obtain F0 generation fast-growing transgenic chub.
[0013] Further, in step S1, the final concentration of the recombinant plasmid is 50 ng / μL, the final concentration of Tol2 transposase mRNA is 100 ng / μL, and the injection volume for each fertilized egg is 2 nL.
[0014] Furthermore, the temperature of microinjection in step S1 is 28.5℃, and the fertilized eggs are cultured in 1×E3 culture medium after injection.
[0015] The present invention has the following advantages: Efficient integration and stable expression: The Tol2 transposon system enables efficient copy integration of exogenous genes into the chub genome. Combined with the potent goldfish β-actin promoter, it ensures stable and high-level expression of the growth hormone gene throughout the fish.
[0016] Simple to operate and intuitive to screen: Using EGFP as a reporter gene, transgenic positive individuals can be quickly and accurately screened through non-invasive fluorescence observation during the embryonic and juvenile stages, with a positive rate of over 70%, which greatly improves breeding efficiency.
[0017] Significantly promotes growth: Experiments have shown that introducing zebrafish or goldfish-derived... gh After gene therapy, the growth indicators at 30 / 60 / 90 days were significantly better than those of the control group. The body length and weight of the transgenic chub were significantly increased (body length increased by up to 130% and weight increased by up to 150%). At the same time, the expression level of growth hormone in the brain and the triglyceride content in the liver of the transgenic chub were significantly increased. This molecularly confirms that the heterologous growth hormone gene can regulate the growth and metabolism of chub. In other words, this invention has successfully constructed a rapid-growth chub model, effectively solving the defects of slow growth and small size of the naturally occurring chub.
[0018] In summary, this invention provides an efficient genetic engineering solution for transgenic breeding of chub, significantly improving its growth rate. Its technical framework can be directly extended to the rapid breeding of other freshwater economic fish species, providing a standardized and replicable technical solution for aquatic transgenic breeding and significantly improving the economic benefits of aquaculture. Attached Figure Description
[0019] The present invention will be further described below with reference to the accompanying drawings and embodiments.
[0020] Figure 1 Zebrafish in this embodiment of the invention gh Gene gel electrophoresis image; Figure 2 Zebrafish in this embodiment of the invention gh Recombinant plasmid (pTol2-β-actin-zebrafish) gh -P2A-EGFP-SV40 polyA) spectrum; Figure 3 This is a map of the Tol2 transposase in an embodiment of the present invention; Figure 4 Goldfish in this embodiment of the invention gh Gene gel electrophoresis image; Figure 5 Goldfish in this embodiment of the invention gh Recombinant plasmid (pTol2-β-actin-EGFP-P2A-goldfish) gh -SV40 polyA) spectrum; Figure 6 Zebrafish and goldfish are examples of fish used in this invention. gh Comparison of gene-modified 72 hpf white light, green fluorescence and control group images of the genotyped chub. Figure 7 Zebrafish and goldfish are examples of fish used in this invention. gh Comparison of gene-modified 120 hpf white light, green fluorescence and control group.
[0021] Figure 8 Zebrafish and goldfish are examples of fish used in this invention. gh Statistical charts of mortality and hatching rate of genotyped chub, including: (A) Statistical chart of mortality rate of chub in different treatment groups at 96 hpf; (B) Statistical chart of hatching rate of chub in different treatment groups at 96 hpf. Figure 9 The zebrafish and goldfish used in this embodiment of the invention gh Comparison of body length and weight of genetically modified zebrafish at 30 / 60 / 90 days with the control group. (AB) Statistical results of body length and weight of different groups of zebrafish at 30 days; (CD) Statistical results of body length and weight of different groups of zebrafish at 60 days; (EF) Statistical results of body length and weight of different groups of zebrafish at 90 days; (G) Statistical results of body length and weight of control and zebrafish at 90 days. gh and goldfish- gh Image of a chub; Figure 10 The brain of a transgenic chub in an embodiment of the present invention. gh Comparison of growth hormone content and liver triglyceride content, including: (A) Standard curve of growth hormone determination; (B) Comparison of growth hormone content in the brain of different groups of chub; (C) Comparison of triglyceride content in the liver of different groups of chub.
[0022] Figure 11This is a schematic diagram illustrating the principle of the Tol2 transposon-mediated transgenic technology for chub fish in this invention. Detailed Implementation
[0023] The following will be combined with the appendix Figure 1-11 The technical solution of the present invention will be clearly and completely described in detail with specific 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. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments used, unless otherwise specified, are all commercially available conventional products.
[0024] Example 1: Transferring zebrafish -gh Creation of the Makou fish strain 1.1 Zebrafish gh Fragment acquisition Total RNA extraction from zebrafish tissue was performed according to the SteadyPure RNA Extraction Kit instructions from Eric Biotechnology. cDNA extraction was performed according to the HiScript II IQRT SuperMix for qPCR (+gDNA wiper) (Vazyme) instructions. Zebrafish tissue samples were obtained via NCBI. gh cDNA sequences were sequenced and primers were designed (Table 1-1). Using the cDNA obtained from the reverse transcription of zebrafish tissue RNA as a template, zebrafish were amplified by PCR to obtain the zebrafish... gh Fragments, such as Figure 1 As shown, the DNA marker is 2000 and the target band length is 696 bp.
[0025] Table 1-1 Primer Sequences
[0026] 1.2 Synthesis of Tol2 backbone plasmid The pTol2-EGFP-SV40 polyA green fluorescent transgenic vector backbone plasmid was created using artificial design and gene company synthesis methods. This plasmid contains two transposon arms of the Tol2 transposon system, includes kozak, and introduces green fluorescent (EGFP) sites between the transposon arms.
[0027] 1.3 Construction of transgenic backbone vector Primers GJ-F and GJ-R (primer sequences shown in Table 1-1) were designed and synthesized according to the sequence. Using 1 µL of the obtained 9.6 ng / µL pTol2-EGFP-SV40 polyA plasmid as a template, 25 µL of whitening enzyme, 2.5 µL each of 10 ng / µL primers GJ-F and GJ-R, and 19 µL of double-distilled water were added to a total volume of 50 µL for PCR amplification (94℃ for 30 s, 58℃ for 10 s, 72℃ for 40 s). After 38 cycles, an extension was performed at 72℃ for 10 min. After gel recovery, the PCR-amplified pTol2-EGFP-SV40 polyA linear backbone vector was obtained, which was broken between pTol2 and EGFP. A goldfish β-actin promoter fragment (provided by the Institute of Life Sciences, Fuzhou University) was then introduced via PCR amplification to obtain the pTol2-β-actin-EGFP-SV40 polyA linear backbone vector for subsequent experiments.
[0028] 1.4 With homologous arms gh Excerpt Designing a zebrafish with homologous arms - gh -TYB primers (primer sequences are shown in Table 1-1), and zebrafish obtained in Section 1.1 were processed using the PCR method described above. gh The fragment plus the homologous arm, i.e. gh The gene has a portion of the pTol2-EGFP-SV40 polyA backbone and a complete P2A sequence at each end.
[0029] 1.5 gh Construction of transgenic vectors After the gel was recovered, the zebrafish obtained in Section 1.1 were cloned using the pEASY-Basic Seamless Cloning and Assembly Kit. gh The fragment was seamlessly cloned with the pTol2-EGFP-SV40 polyA backbone vector (Table 1-2), and the reaction system is as follows: Table 1-2 Seamless Cloning Reaction System
[0030] Note: *In this system, the amount and ratio of the vector and the insert fragment should be strictly added according to the instructions. The recommended amount is 0.01-0.25 pmols, and the ratio is 1:2. pmols = mass ng / (fraction length bp × 0.65 kDa) After successful cloning, the bacterial culture was sent to Qingke Biotechnology Co., Ltd. for sequencing. The sequencing sequences were then compared with those on SnapGene to observe the effects on zebrafish and goldfish. gh The gene fragment was successfully ligated to the pTol2-EGFP-SV40 polyA backbone fragment. After successful ligation, pTol2-β-actin-zebrafish were obtained using an endotoxin-free plasmid mini-extraction kit (Beijing Tiangen Biotech Co., Ltd.). gh -P2A-EGFP-SV40 polyA plasmid ( Figure 2 (This is used for subsequent experiments.)
[0031] 1.6 Transposon Enzyme Construction A plasmid, designed artificially and synthesized by a gene company, was created that can be directly used for in vitro transcription after enzymatic digestion. This plasmid contains the SP6 transcription initiation sequence, a kozak-containing sequence, and a polyA sequence encoding the Tol2 transposonase optimized with fish codons. After extraction, digestion with EcoRI, and purification, the plasmid can be used as a transcription template for the T7 in vitro transcription kit. Primers were designed to amplify the Tol2 transposon sequence from this plasmid (primer sequences are shown in Table 1-1). Figure 3 As shown. Transcription to transposase RNA was performed using the T7 in vitro transcription kit.
[0032] 1.7 Microinjection The parent fish were placed in a spawning tank in the dark the night before. The eggs were collected approximately 10 minutes after spawning began the following day, and the collected fertilized eggs were placed under a stereomicroscope for embryo injection experiments. In a clean bench, pTol2-β-actin-zebrafish was injected at a final concentration of 50 ng / µL. gh The -P2A-EGFP-SV40 polyA plasmid was mixed with Tol2 transposase RNA at a final concentration of 100 ng / µL for injection. Approximately ~2 nl was injected into each embryo, which was then cultured at 28.5℃. The injected embryos were then cultured in 1×E3 medium, with the medium changed every 12-24 hours. Dead embryos were removed during the medium change. The blank control group consisted of eggs produced at the same time that were not injected and were fed in the same manner.
[0033] A schematic diagram illustrating the principle of Tol2 transposon-mediated transgenic technology in chub is shown below. Figure 11 As shown.
[0034] Example 2: Transferring goldfish -gh Creation of the Makou fish strain 2.1 Goldfish gh The method for obtaining the fragment is the same as in Section 1.1 of Example 1, using NCBI to obtain the goldfish fragment. gh cDNA sequences were sequenced and primers were designed (Table 2-1). Goldfish were obtained by PCR amplification using cDNA reverse transcribed from goldfish tissue RNA as a template. gh Fragments, such as Figure 4 As shown, the target band length is 696 bp.
[0035] Table 2-1 Primer Sequences
[0036] 2.2 Synthesis of Tol2 backbone plasmid The method is the same as in Section 1.2.
[0037] 2.3 Construction of transgenic backbone vector The method is the same as in Section 1.3.
[0038] 2.4 With homologous arms gh Excerpt The method is the same as in Section 1.4. Goldfish with homologous arms - gh -TYB primers (Table 2-1).
[0039] 2.5 gh Construction of transgenic vectors The method is the same as in Section 1.5. Construct pTol2-β-actin-EGFP-P2A-goldfish. gh The -SV40 polyA plasmid is available for use. The nucleotide sequence of this recombinant plasmid is shown in SEQ ID NO.2.
[0040] 2.6 Construction of transposon enzymes The method is the same as in Section 1.6.
[0041] 2.7 Microinjection The method is the same as in Section 1.7.
[0042] Example 3: Transferring zebrafish -gh and goldfish -gh Growth comparison of Makou fish strains and control group 3.1 Fluorescence observation and fluorescence rate of transgenic chub zebrafish and goldfish gh The gene plasmid was transferred into fertilized eggs of *Gnaphalium affine*, with wild-type fertilized eggs (uninjected) serving as a control group. Fluorescence was observed in the eggs at 24 hpf, 48 hpf, and 72 hpf using a fluorescence microscope, and the fluorescence ratio was calculated. Results showed that the transgenic plasmid exhibited green fluorescence in both the transgenic and uninjected eggs at 72 h and 120 h post-injection. gh The fluorescence intensity of the gene plasmid group was weak ( Figure 6 , Figure 7 ).
[0043] Statistical analysis revealed high fluorescence rates in the transgenic genomes with no significant differences (Table 1). The fluorescence rate of the zebrafish gh genome was 72.93% (97 / 133), and that of the goldfish gh genome was 71.01% (98 / 138). The control group showed no fluorescence (Table 3-1). This indicates that the method of the present invention has high transgenic efficiency.
[0044] Table 3-1 Zebrafish and Goldfish gh Fluorescence rate of gene-modified chub
[0045] 3.2 Transfer to a different source gh Mortality and membrane emergence rate of genetically modified chub Following microinjection, thirty eggs of each of the three types were grouped together, with three parallel groups for each type. These eggs were individually reared at 28 °C, and the number of membranes and survivors was recorded every 24 hours. Results showed that by separately analyzing the control group (uninjected) and the zebrafish... gh Gene plasmids and goldfish gh The mortality rate and hatching rate of the 24-96 hpf embryos in the gene plasmid group were found to be significantly different between the microinjection group and the control group. This may be because the injection caused physical damage to the embryos of the chub fish, thus increasing the mortality rate. Figure 8 A, Table 3-2). The hatching rate of transgenome at 48 hpf was significantly different from the control group, possibly because... gh Genes cause accelerated somatic cell growth and development in chub embryos. Figure 8 B, Table 3-3).
[0046] Table 3-2
[0047] Table 3-3
[0048] 3.3 Turn gh Comparison of gene-induced growth indicators in chub Control group, goldfish gh zebrafish gh Three groups of chub were reared under identical conditions. Body length and weight were measured and recorded for each group at 30, 60, and 90 days. The body length and weight of the control group and the transgenic chub at 30, 60, and 90 days were statistically analyzed. Figure 9 (AF, Tables 3-4) The results showed that the body length and weight of the transgenic group were higher than those of the control group, which can also be seen from the comparison of 90-day-old images of the chub. Figure 9 G). At 120 days, the transgenic fish can increase in body length by approximately 130% and in weight by approximately 150% (Table 3-5). This part of the experiment shows that transgenic fish... ghGenes promote the increase in body length and weight of the chub.
[0049] Table 3-4
[0050] Table 3-5
[0051] 3.4 Transfer to a different source gh Genetically modified chub gh Comparison with triglyceride content Fish growth hormone from Nanjing Jiancheng Bioengineering Institute was used. gh ) Enzyme-linked immunosorbent assay kit, according to the instructions, was used to test the control group and zebrafish. gh Turning goldfish gh brain of the mackerel gh Content. The triglyceride content of each group was determined by dissecting the livers of three groups of chub.
[0052] The results showed that the growth hormone content in the brains of the control group and transgenic chub was significantly increased. Figure 10 (AB, Tables 3-6). This study also dissected the liver of the mandarin fish and measured the triglyceride content. The results showed that... gh The triglyceride content in the liver of the group of fish was also significantly higher than that in the control group. Figure 10 C, Table 3-6).
[0053] Table 3-6
[0054] As can be seen, the growth hormone gene derived from zebrafish or goldfish introduced in this invention has been successfully functionally expressed in the fish, producing a biologically active growth hormone protein. This exogenous growth hormone systematically enhances the metabolic level of the transgenic fish by promoting the synthesis and accumulation of hepatic triglycerides, thereby directly driving a significant increase in its body length and weight.
[0055] While specific embodiments of the present invention have been described above, those skilled in the art should understand that the specific embodiments described are merely illustrative and not intended to limit the scope of the present invention. Equivalent modifications and variations made by those skilled in the art in accordance with the spirit of the present invention should be covered within the scope of protection of the claims of the present invention.
Claims
1. A recombinant plasmid expressing a heterologous growth hormone gene, characterized in that: The recombinant plasmid uses a plasmid carrying the left arm of the Tol2 transposon, the right arm of the Tol2 transposon, the goldfish β-actin promoter, the enhanced green fluorescent protein gene EGFP-SV40, and the polyA sequence as its backbone; The zebrafish-derived growth hormone gene zebrafish- was inserted into the backbone plasmid. gh Or goldfish-derived growth hormone gene goldfish- gh Furthermore, the growth hormone gene is linked to the EGFP gene via a P2A sequence; The nucleotide sequence of the recombinant plasmid is shown in SEQ ID NO.1 or SEQ ID NO.
2.
2. The recombinant plasmid according to claim 1, characterized in that: The recombinant plasmid is pTol2-β-actin-zebrafish- gh -P2A-EGFP-SV40-polyA, or pTol2-β-actin-EGFP-P2A-goldfish- gh -SV40-polyA.
3. A method for constructing a recombinant plasmid according to claim 1 or 2, characterized in that, Includes the following steps: S1: Using cDNA reverse-transcribed from total RNA of zebrafish or goldfish as a template, PCR amplification was performed to obtain zebrafish- gh Gene fragments or goldfish- gh Gene fragments; S2: In zebrafish- gh By linking the P2A sequence to the 3' end of the gene, zebrafish- gh -P2A fragment; in goldfish- gh By linking the P2A sequence to the 5' end of the gene, we obtain P2A-goldfish- gh Fragment; S3: Move the zebrafish- gh -P2A fragment or P2A-goldfish- gh The fragment is ligated to the backbone plasmid via seamless cloning to obtain the target recombinant plasmid.
4. The method for constructing the recombinant plasmid according to claim 3, characterized in that: The primer sequences used for PCR amplification in step S1 are shown in SEQ ID NO.3~SEQ ID NO.
6.
5. The application of the recombinant plasmid according to claim 1 or 2 in the preparation of fast-growing chub.
6. The application of the recombinant plasmid according to claim 1 or 2 in transgenic breeding of chub.
7. A method for improving the growth rate of chub, characterized in that: Includes the following steps: S1: Mix the recombinant plasmid described in claim 1 or 2 with Tol2 transposase mRNA and introduce it into wild-type koji fertilized eggs via microinjection; S2: Positive embryos expressing green fluorescence were screened under a fluorescence microscope and cultured to obtain F0 generation fast-growing transgenic chub.
8. The method for increasing the growth rate of chub according to claim 7, characterized in that: In step S1, the final concentration of the recombinant plasmid is 50 ng / μL, the final concentration of Tol2 transposase mRNA is 100 ng / μL, and the injection volume for each fertilized egg is 2 nL.
9. The method for improving the growth rate of chub according to claim 7, characterized in that: The temperature for microinjection in step S1 was 28.5℃, and the fertilized eggs were cultured in 1×E3 culture medium after injection.