A gene for improving the alkali tolerance of cyprinid fish, a recombinant vector containing the gene and application thereof

Abstract

The application discloses a gene for improving the alkali tolerance of cyprinid fishes, a recombinant vector containing the gene and application of the gene, and belongs to the technical field of fish genetic engineering. hba To improve the alkali tolerance of cyprinid fishes, the application constructs a recombinant vector containing a Dali Lake Valenciennes's gibel carp hbb Gene promoter region and CDS region sequence and the recombinant vector containing the gene promoter region and CDS region sequence, the recombinant vector is mixed with Tol2 transposase mRNA respectively, and then microinjection technology is used to inject I-cell period fertilized eggs of cyprinid fishes, and after hatching and culture of the fertilized eggs, it is found that overexpression of the hba gene or hbb the gene can improve the alkali tolerance of cyprinid fishes. The application lays a foundation for developing and cultivating fish germplasm suitable for saline-alkaline water aquaculture.

Description

Technical Field

[0001] This invention belongs to the field of fish genetic engineering technology, specifically relating to a gene that improves the alkali tolerance of cyprinid fish, a recombinant vector containing the gene, and its application. Background Technology

[0002] Low-lying saline-alkali waters are generally characterized by high pH values, high carbonate alkalinity, and imbalances in the proportion of major ions, making them unsuitable for direct use in conventional agriculture and livestock farming, resulting in significant resource idleness. How to effectively develop and utilize saline-alkali water and soil resources is a crucial issue concerning food security and ecological protection. Against this backdrop, cultivating new aquaculture species suitable for saline-alkali water environments is considered a promising approach to resource utilization.

[0003] Varroa var. var. ( Leuciscus waleckii This species belongs to the order Cypriniformes, family Cyprinidae, subfamily Leuciscinae, and genus Leuciscinae. It exists in different geographical populations, including freshwater and alkaline-water populations. The population inhabiting Dali Lake in Inner Mongolia has been able to survive for extended periods in extremely high salinity and alkalinity, becoming the dominant species. Existing research shows that compared to the freshwater population, this alkaline-tolerant population exhibits differential expression of genes related to salinity tolerance, such as those involved in osmotic pressure regulation, acid-base balance, ammonia metabolism, inflammatory responses, immune responses, and myocardial angiogenesis, thus adapting to the high-alkalinity environment. These studies indicate that salinity adaptation in fish is a complex trait involving multiple genes and pathways, and its molecular mechanisms are not yet fully elucidated.

[0004] Hemoglobin (Hb) is a key protein in vertebrate blood responsible for oxygen transport. Besides its basic oxygen-carrying function, hemoglobin and its subunits also play a role in maintaining blood acid-base balance, participating in buffer system formation by binding hydrogen ions. In some fish species, studies have observed that the expression levels of genes encoding hemoglobin subunits change with variations in osmotic pressure or ion environment.

[0005] Nevertheless, current research largely focuses on describing the physiological phenomena of alkali-tolerant fish and screening and identifying their potential regulatory genes. How to translate these basic research findings into breeding techniques that can be practically applied to the genetic improvement of important economic fish species, thereby significantly enhancing the adaptability of target fish to saline-alkali waters, remains a pressing technical challenge in this field, lacking effective solutions. Summary of the Invention

[0006] To improve the alkali tolerance of cyprinid fish, this invention constructs a cultivar containing *Leuciscus vannamei* from Lake Dali. hba Gene promoter region and CDS region sequences and containing hbbRecombinant vectors containing the promoter and CDS regions of a gene were mixed with Tol2 transposase mRNA and injected into I-cell stage fertilized eggs of cyprinid fishes using microinjection. After hatching and culturing the fertilized eggs, overexpression of Tol2 transposase mRNA was observed in cyprinid fishes. hba Gene or hbh Genes can enhance the alkali tolerance of cyprinid fish.

[0007] To solve the above-mentioned technical problems and achieve the corresponding technical effects, the present invention provides the following technical solution: The first objective of this invention is to provide a gene that enhances the alkali tolerance of cyprinid fish, said gene being... hba Gene or hbb Gene, hba The nucleotide sequence of the gene's CDS region is shown in SEQ ID NO.1. hbb The nucleotide sequence of the gene's CDS region is shown in SEQ ID NO.3.

[0008] A second objective of this invention is to provide the application of the above-mentioned gene in improving the alkali tolerance of cyprinid fish.

[0009] A third objective of this invention is to provide a recombinant vector containing the aforementioned genes.

[0010] In one embodiment of the present invention, the recombinant vector uses a pDestTol2CG2-Mod plasmid with the cmlc promoter and EGFP reporter gene removed as the vector backbone, and inserts... hba Gene promoter region and CDS region sequences, or insertions hbb The gene promoter region and CDS region sequences were constructed; hba The nucleotide sequence of the gene promoter region is shown in SEQ ID NO.2. hbb The nucleotide sequence of the gene promoter region is shown in SEQ ID NO.4.

[0011] In one embodiment of the present invention, the hba Gene promoter region sequence and hbb The gene promoter region sequence was obtained by amplifying the genomic DNA of *Leuciscus valens* from Lake Dali using it as a template. hba Gene CDS region sequence and hbb The gene CDS region sequence was obtained by amplifying cDNA from the Dali Lake wrasse.

[0012] Further specifying, the genomic DNA is derived from the gill tissue of the Dali Lake wrasse.

[0013] To further specify, the cDNA was obtained by reverse transcription of RNA from the gill tissue of the Dali Lake wrasse.

[0014] In one embodiment of the present invention, for amplification hba The nucleotide sequences of the upstream and downstream primers for the gene promoter region are shown in SEQ ID NO.7 and SEQ ID NO.8, respectively; used for amplification. hba The nucleotide sequences of the upstream and downstream primers for the gene's CDS region sequence are shown in SEQ ID NO.9 and SEQ ID NO.10, respectively; used for amplification. hbb The nucleotide sequences of the upstream and downstream primers for the gene promoter region are shown in SEQ ID NO.11 and SEQ ID NO.12, respectively; used for amplification. hbb The nucleotide sequences of the upstream and downstream primers for the gene CDS region sequence are shown in SEQ ID NO.13 and SEQ ID NO.14, respectively.

[0015] In one embodiment of the present invention, the vector backbone is obtained by amplification using the pDestTol2CG2-Mod plasmid as a template and the upstream primer with the nucleotide sequence shown in SEQ ID NO.5 and the downstream primer with the nucleotide sequence shown in SEQ ID NO.6.

[0016] A fourth objective of this invention is to provide the application of the above-mentioned recombinant expression vector in improving the alkali tolerance of cyprinid fish.

[0017] The fifth objective of this invention is to provide a method for improving the alkali tolerance of cyprinid fish, the method comprising the following steps: constructing the above-mentioned recombinant vector, mixing the recombinant vector with Tol2 transposase mRNA at equal mass concentrations to prepare a mixture sample for microinjection, then injecting the mixture sample into I-cell stage fertilized eggs of cyprinid fish using a microinjection apparatus, and obtaining cyprinid fish with improved alkali tolerance after hatching and culturing the fertilized eggs.

[0018] Further restrictions were imposed, with the concentrations of both the recombinant vector and Tol2 transposase mRNA being 500 ng / μL.

[0019] To further specify, Tol2 transposase mRNA is obtained by sequentially linearizing pCS2-transposase through enzymatic digestion, purification, and reverse transcription.

[0020] Furthermore, the enzyme linearization uses FastDigest Not... I The kit, wherein purification is performed using the sodium acetate method, and reverse transcription is performed using mMESSAGE mMACHINE. TM SP6 kit.

[0021] In one embodiment of the present invention, the carp species include zebrafish and Yellow River carp.

[0022] The beneficial effects of this invention are: This invention constructs a method containing the Dali Lake leuciscus. hba Gene promoter region and CDS region sequences and containing hbb Recombinant vectors containing the promoter and CDS regions of a gene were mixed with Tol2 transposase mRNA and injected into I-cell stage fertilized eggs of cyprinid fishes using microinjection. After hatching and culturing the fertilized eggs, overexpression of Tol2 transposase mRNA was observed in cyprinid fishes. hba Gene or hbh Genes can enhance the alkali tolerance of cyprinid fish. The genes, recombinant vectors, and other materials provided by this invention have significant potential application value in the creation, screening, and breeding of salt-tolerant cyprinid fish, providing support for expanding the aquaculture space for freshwater cyprinid fish and strengthening the effective utilization of saline-alkali waters, especially moderately alkaline waters. Attached Figure Description

[0023] Figure 1 The image is a map of plasmid pDestTol2CG2-Mod; Figure 2 For the carrier skeleton, hba , hbb The verification results of gene promoter region and CDS region amplification are shown in the figure; where A is the verification result of vector backbone amplification, and B is... hba The verification results of gene promoter region amplification are shown in Figure C. hba The validation results of gene CDS region amplification are shown in the figure, where D represents... hbb Validation results of gene promoter and CDS region amplification; Figure 3 For containing hba A schematic diagram illustrating the construction of a gene recombination vector; Figure 4 For containing hbb A schematic diagram illustrating the construction of a gene recombination vector; Figure 5 To verify using colony PCR hba Gene recombination vectors and hbb The result diagram of the gene recombination vector; where A represents the validation. hba Gene recombination vectors contain hba The result diagram of the gene promoter region, B is for verification. hba Gene recombination vectors contain hba The result diagram of the gene CDS region, C is for validation. hbb Gene recombination vectors contain hbb Resulting diagram of gene promoter region and CDS region; Figure 6 To verify hba or hbbThe results of whether the gene recombinant vector was introduced into zebrafish are shown in the figure; where A is the verification. hba The results of whether the gene recombination vector was introduced into zebrafish are shown in Figure B, where B is the verification result. hbb Image showing the results of whether the gene recombination vector was introduced into zebrafish; Figure 7 To overexpress exogenous hba or hbb The results of alkali tolerance testing in adult zebrafish are shown in the figure; where A is the result of alkali tolerance testing under 20 mmol / L alkalinity, B is the result of alkali tolerance testing under 40 mmol / L alkalinity, C is the result of alkali tolerance testing under 60 mmol / L alkalinity, and D is the result of alkali tolerance testing under 80 mmol / L alkalinity. Figure 8 Overexpression under different concentrations of alkaline stress hba In adult zebrafish genes hba Image showing the results of gene relative expression level detection; Figure 9 Overexpression under different concentrations of alkaline stress hbb In adult zebrafish genes hbb Image showing the results of gene relative expression level detection; Figure 10 To verify hba or hbb The results of whether the gene recombination vector was introduced into Yellow River carp; where A represents the verification. hba The results of whether the gene recombination vector was introduced into Yellow River carp are shown in Figure B, where B is the verification result. hbb Image showing the results of whether the gene recombination vector was introduced into Yellow River carp; Figure 11 To overexpress exogenous hba or hbb The results of alkali tolerance testing in adult Yellow River carp with the gene; where A is the result of alkali tolerance testing under 60 mmol / L alkalinity, B is the result of alkali tolerance testing under 80 mmol / L alkalinity, C is the result of alkali tolerance testing under 100 mmol / L alkalinity, and D is the result of alkali tolerance testing under 120 mmol / L alkalinity. Figure 12 Overexpression under different concentrations of alkaline stress hba In adult Yellow River carp with genes hba Relative expression levels and overexpression of genes hbb In adult Yellow River carp with genes hbb The graph shows the results of the detection of relative gene expression levels; where A represents overexpression. hba In adult Yellow River carp with genes hba The graph shows the results of relative gene expression level detection, with B representing overexpression.hbb In adult Yellow River carp with genes hbb The results of the relative expression level detection of genes. Detailed Implementation

[0024] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific embodiments and accompanying drawings. It should be noted that the embodiments mentioned below are only for explaining the invention and are not intended to limit the scope of the invention. The embodiments mentioned below are only some embodiments of the invention, not all embodiments. In the art, any embodiments obtained by those skilled in the art without creative effort are protected by this invention.

[0025] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. The materials, reagents, culture media, and instruments used are all conventional materials, reagents, culture media, and instruments in the art, and can be obtained commercially by those skilled in the art. Unless otherwise specified, the molecular biology experimental operations involved in this invention, such as PCR amplification, enzyme digestion and ligation, and transformation, are all conventional experimental operations in the art or can be performed according to the product instructions of the corresponding reagents.

[0026] The following examples are used to amplify the vector backbone, hba , hbb Primer information for the gene promoter region and CDS region, as well as amplification product sequence information, are shown in Table 1. Recombinant vectors were constructed using NEBuilder seamless cloning technology. The primer sequences included both specific primer sequences and dangling primer sequences to add homologous arms to the amplification products for sticky end-matching ligation.

[0027] Table 1. Used for amplification vector backbones, hba , hbb Primer information for gene promoter and CDS regions

[0028] Example 1: hba Genes and hbb Application of genes in improving the alkali tolerance of zebrafish 1. Carrier skeleton fragments hba , hbb Amplification of gene promoter and CDS regions Three *Leuciscus variabilis* from Lake Dali were anesthetized with MS-222, and gill tissue was collected. Genomic DNA was extracted using a kit (purchased from Tiangen Biotech). RNA was extracted using the Trizol method and then reverse transcribed into cDNA. The purity and concentration of DNA and cDNA were determined using a K5500C ultra-micro spectrophotometer (Beijing Kai'ao Technology, Beijing).

[0029] Retrieved target gene from the genome sequence of *Leuciscus var. var.* hba and hbb The full sequence, CDS region sequence, and promoter region sequence of ) are included. hba The gene is located at 38,914,015-38,926,226 bp in the positive strand of Chr 1 in the genome of *Leuciscus var. var.*, with a length of 12,212 bp. Its nucleotide sequence is shown in SEQ ID NO. 27. hba The gene's CDS region consists of three exons, is 432 bp in length, and its nucleotide sequence is shown in SEQ ID NO.1. hba The gene promoter region sequence is shown in SEQ ID NO.2; hbb The gene is located on the negative strand of Chr 1 in the genome of *Leuciscus var. varaciana*, ranging from 38,912,030 to 38,912,884 bp, and is 854 bp in length. Its nucleotide sequence is shown in SEQ ID NO. 28. hbb The gene's CDS region consists of three exons, is 444 bp in length, and its nucleotide sequence is shown in SEQ ID NO.3. hbb The gene promoter region sequence is shown in SEQ ID NO.4. The backbone for the amplification vector was designed online using the NEBuilderAssembly Tool (nebuilder.neb.com). hba , hbb Suspended primers for the gene promoter region and CDS region (see Table 1) were synthesized by Kumei Biotechnology Co., Ltd. Genomic DNA was used as a template for amplification. hba and hbb The promoter region sequence was amplified using cDNA as a template. hba and hbb CDS region sequence; obtained from plasmid pDestTol2CG2-Mod using a kit (map shown in figure). Figure 1 Plasmids were extracted from *E. coli* (as shown in the image). Using the plasmids as templates, the vector backbone was amplified using primers Vector-F and Vector-R. The nucleotide sequence of the vector backbone is shown in SEQ ID NO. 29. The specific amplification procedure is as follows: Vector backbone amplification: A 50 μL PCR amplification system was established, comprising 25 μL of 2×Phanta Flash Master Mix (Dye Plus), 2 μL each of primers Vector-F and Vector-R (10 mmol / L), 5 μL of template plasmid pDestTol2CG2-Mod (50 ng / μL), and 16 μL of sterile deionized water. The reaction program was: 95℃ denaturation for 3 min; 95℃ denaturation for 30 s, 58℃ annealing for 45 s, 72℃ extension for 4 min, for 35 cycles; and a final extension at 72℃ for 7 min. The target fragment was 4580 bp, and its nucleotide sequence is shown in SEQ ID NO.29.

[0030] Amplification of the promoter region sequence: A 50 μL PCR amplification system was established, comprising 25 μL of 2×Phanta Flash MasterMix (Dye Plus), 2 μL each of primers hba-pro-F and hba-pro-R (10 mmol / L) or 2 μL each of primers hbb-pro-F and hbb-pro-R (10 mmol / L), 5 μL of template DNA (50 ng / μL), and 16 μL of sterile deionized water. The reaction program was: 95℃ denaturation for 3 min; 95℃ denaturation for 30 s, 58℃ annealing for 45 s, 72℃ extension for 2 min, 35 cycles; and a final extension at 72℃ for 5 min. hba The promoter region amplification product is 1948 bp (nucleotide sequence shown in SEQ ID NO.30). hbb The promoter region amplification product is 2289 bp (nucleotide sequence as shown in SEQ ID NO.33).

[0031] hba Amplification of the CDS region sequence: A 50 μL PCR amplification system was established, including 25 μL of 2×Phanta Flash MasterMix (Dye Plus), 2 μL each of primers hba-cds-F and hba-cds-R (10 mmol / L), 5 μL of template DNA (50 ng / μL), and 16 μL of sterile deionized water. The reaction program was: 95℃ denaturation for 3 min; 95℃ denaturation for 30 s, 52℃ annealing for 45 s, 72℃ extension for 2 min, for 35 cycles; and a final extension at 72℃ for 5 min. The amplified product was 476 bp (nucleotide sequence as shown in SEQ ID NO. 31).

[0032] hbbAmplification of the CDS region sequence: A 50 μL PCR amplification system was established, including 25 μL of 2×Phanta Flash MasterMix (Dye Plus), 2 μL each of primers hbb-cds-F and hbb-cds-R (10 mmol / L), 5 μL of template DNA (50 ng / μL), and 16 μL of sterile deionized water. The reaction program was: 95℃ denaturation for 3 min; 95℃ denaturation for 30 s, 58℃ annealing for 45 s, 72℃ extension for 2 min, for 35 cycles; and a final extension at 72℃ for 5 min. The amplified target fragment was 484 bp (nucleotide sequence as shown in SEQ ID NO. 32).

[0033] The PCR products were verified to conform to the target fragment size by 1.5% agarose gel electrophoresis. The verification results are as follows: Figure 2 As shown, this result indicates that the vector backbone fragment was successfully amplified. hba , hbb Gene promoter and CDS regions were extracted. PCR products were purified and recovered using a gel extraction kit. Concentrations were measured using a spectrophotometer and converted to pmol based on fragment size. The calculation method was: 0.05 pmol = DNA concentration ng / μL × X μL × 1000 ÷ number of bases × 650. The concentration of each fragment was adjusted to 0.05 pmol / L.

[0034] 2. Contains hba Gene recombination vectors and those containing hbb Construction of gene recombination vectors The vector backbone fragment, target gene promoter region fragment, and CDS region fragment obtained in step 1 were ligated using the NEBuilder HIFI DNA Assembly Master Mix kit (purchased from New England Biolabs, catalog number E2621) to construct vectors containing DNA backbone fragments, target gene promoter region fragments, and CDS region fragments obtained in step 1. hba Gene recombination vector (construction diagram as shown) Figure 3 As shown, the nucleotide sequence is as shown in SEQ ID NO. 34) and contains hbb Gene recombination vector (construction diagram as shown) Figure 4 As shown in the figure, the nucleotide sequence is shown in SEQ ID NO. 35. The total volume of the ligation system was 20 μL, including 3 μL of mixed fragment (mixed molar ratio of vector backbone: promoter region fragment: CDS region fragment = 1:2:2), 10 μL of NE Builder HiFi DNA Assembly Master Mix, and 7 μL of deionized sterile water. The ligation reaction was carried out at 50°C for 60 min.

[0035] Transform 1 μL of the ligation product into competent E. coli DH5α (Bomaide Biotechnology). Spread the bacterial culture onto LB agar plates containing ampicillin and incubate upside down at 37℃ for 12-16 h to obtain single colonies containing the plasmid. Pick single colonies for colony PCR to detect whether they are recombinant clones containing the complete promoter and CDS regions of the target fragment. The colony PCR amplification system is 25 μL: 12.5 μL of 2×Buffer Mix, 0.5 μL each of forward and reverse primers, and resuspend the picked colonies in 2 μL of sterile water. The remaining volume is made up with sterile deionized water. Detect the PCR product by agarose gel electrophoresis. Plasmids that simultaneously amplify promoter and CDS region bands of the expected size are the target recombinant clones. Figure 5 Further sequencing of the recombinant clone showed that the inserted fragment sequence had a 95%-98% similarity to the reference sequence, confirming it as [the correct sequence]. hba or hbb The recombinant expression vector of the gene was used for positive clone single colony amplification culture, the recombinant plasmid was extracted, the concentration was adjusted to 500 ng / μL, and stored at -20℃ for later use.

[0036] 3. Overexpression hba or hbb Preparation of zebrafish fertilized eggs based on genes E. coli containing the pCS2-transposase plasmid (available on Addgene) were cultured and the plasmid was extracted and processed using FastDigest Not... I After linearization by enzyme digestion using the kit (Thermo Fisher Scientific, catalog number FD0594), the linear plasmid was purified using the sodium acetate method. The specific purification method is as follows: Add 1 / 20 volume of 0.5 mol / L EDTA, 1 / 10 volume of 3 mol / L sodium acetate, and 2 volumes of pre-chilled anhydrous ethanol sequentially to the enzyme digestion product to be purified. After mixing, incubate at -20℃ for 1 h, centrifuge at 12500 rpm for 15 min at 4℃ to recover the precipitate, air dry, and resuspend in enzyme-free water. Determine the concentration and purity of the linearized plasmid DNA using mMESSAGE mMACHINE. TM The SP6 kit (Thermo Fisher Scientific, catalog number AM1340) was used to reverse transcribe Tol2 transposase mRNA. The transcript was purified using the RNeasy® Mini Kit (QIAGEN GmbH, catalog number 74104). The RNA concentration and purity were measured, and the concentration was adjusted to 500 ng / μL. The RNA was then stored at -80℃. Preparation of a mixture sample for microinjection, the mixture sample consisting of... hba or hbbThe recombinant plasmid of the gene was mixed with Tol2 transposase mRNA at a 1:1 mass ratio. Approximately 1 nL of the mixture was injected into zebrafish I-cell stage fertilized eggs using a microinjector. hba Recombinant vectors and hbb Each recombinant vector was injected with 1000 fertilized eggs; 100 fertilized eggs were used as a control group and injected with an equal volume of enzyme-free water containing 0.25% phenol red. The embryos were placed in fully aerated purified water and cultured at 28.5℃. After 72 h of culture, the control group and... ​ Group, ​ The hatching rates of the groups were 80% (80 tails), 30% (300 tails) and 20% (200 tails), respectively, and 75, 180 and 150 individuals were obtained that were able to swim normally and eat.

[0037] For those containing ​ or ​ Recombinant plasmid vectors for genes were designed, and cross-fragment detection primers covering the exogenous CDS region and promoter region were developed to detect exogenous genes. ​ The upstream and downstream primer sequences of the gene are shown in SEQ ID NO.15 and SEQ ID NO.16, respectively, with a fragment length of 138 bp; detection of exogenous... ​ The upstream and downstream primer sequences for the gene are shown in SEQ ID NO.17 and SEQ ID NO.18, respectively, with a fragment length of 947 bp. After zebrafish were reared for two months and reached a body length of 2.0-2.5 cm, a small amount of tail fin tissue was harvested to extract genomic DNA. PCR detection was performed, followed by electrophoresis. A positive plasmid vector containing the target fragment was used as a positive control, and individuals not transfected were used as a negative control. The results showed that the exogenous gene target band was amplified in the positive control, while no exogenous gene target band was observed in the control group. Samples in the experimental group that amplified the exogenous target band were considered positive individuals. Statistical results showed: ​ and ​ Among 100 individuals in each group, 37 and 35 zebrafish, respectively, tested positive for the target fragment, with a positive rate of 35-37% (see...). ​ ).

[0038] 4. Overexpression ​ or ​ Verification of alkali tolerance in zebrafish genes For positive zebrafish (TG- ​ TG- ​96-hour acute alkaline stress was applied to healthy 3-month-old zebrafish (WT, control group injected with an equal volume of phenol red) and negative zebrafish (WT). The specific method was as follows: Healthy individuals with a body length of 2.5-3.0 cm were selected as experimental fish. They were acclimatized for one week before the experiment (water temperature 26±1℃, pH 7.0-7.2, alkalinity 1.0-1.5 mmol / L, dissolved oxygen ≥6 mg / L, photoperiod 14L∶10D). Feeding was withheld for 24 hours before the experiment. Based on the preliminary experimental results, acute stress was set with alkalinity gradients of 0, 20, 40, 60, and 80 mmol / L (alkaline water prepared with analytical grade NaHCO3 and aerated). Five experimental fish (5 L of alkaline water) were used in each alkalinity group. During the experiment, continuous aeration and fasting were maintained. Half of the alkaline water of the same concentration was replaced every 24 hours. The condition of the experimental fish was continuously observed, and the number of dead individuals was recorded at 6, 12, 24, 36, 48, 72, and 96 hours. The mortality rate (%) and median lethal concentration (LC50) were calculated. 50 ).

[0039] The results are as follows ​ As shown: No deaths were observed in any group within 96 hours of 20 mmol / L alkaline stress, indicating that zebrafish can live normally at this alkalinity in the short term; under 40 mmol / L alkaline stress, mortality began to appear in the WT group at 72 hours, reaching 50% at 96 hours, while the TG- ​ Group and TG- ​ No deaths occurred in any group within 96 hours; under 60 mmol / L alkaline stress, the 24-hour mortality rate in the WT group was 50%, and the 36-hour mortality rate was 100%, TG- ​ The mortality rate in the group reached 100% within 48 hours, TG- ​ Individual deaths began to occur in the WT group at 36 hours, with a mortality rate of 100% at 72 hours. Under extreme high-alkali stress of 80 mmol / L, the mortality rate in the WT group reached 100% at 6 hours, while in the exogenous gene-introduced group it was delayed to 12 hours. The mortality rates in the WT group and the TG- ​ Group and TG- ​ The 96-hour median lethal concentrations for the three groups were 40 mmol / L, 50 mmol / L, and 50 mmol / L, respectively. These results indicate that the exogenous hemoglobin gene can enhance the alkali tolerance of zebrafish and delay lethality.

[0040] For acute stress 0, 10, 20 、 Fish in the 30 and 40 mmol / L alkalinity groups were sampled at 96 h. Gill tissue was rapidly dissected at low temperature, and total RNA was extracted from each tissue using the TRIzol method, followed by reverse transcription to synthesize cDNA. A design was developed based on the CDS region sequence of the target gene from *Leuciscus vallis*. ​ and ​ qPCR primers for genes, ​The upstream and downstream primer sequences for qPCR of the exogenous gene are shown in SEQ ID NO.19 and SEQ ID NO.20, respectively; ​ The upstream and downstream primer sequences for qPCR of the exogenous gene are shown in SEQ ID NO.21 and SEQ ID NO.22, respectively. Using the actin gene as an internal control, the expression level of the target gene was detected by real-time quantitative PCR, and the relative expression level was calculated using a 22... ( ΔΔCT) The method is used for calculation.

[0041] Test results as follows ​ and ​ As shown: Under 10 mmol / L alkaline stress, TG- ​ group ​ Relative gene expression levels and TG- ​ group ​ The relative expression levels of genes were significantly higher in the group than in the WT group. P <0.0001). TG- ​ The groups were subjected to alkaline stress at 20 mmol / L and 30 mmol / L. ​ Gene expression levels were not significantly different from the WT group (ns); under 40 mmol / L alkaline stress, ​ The gene expression level was significantly lower than that in the WT group ( P <0.05). TG- ​ Group at 20 mmol / L ( P <0.01) and 30 mmol / L ( P Under coercion of <0.001), ​ The relative expression level of the gene was significantly higher in the group than in the WT group. However, at 40 mmol / L, the expression levels in the two groups became quite similar (ns). These results indicate that the gene carrying... ​ The exogenous expression vector of the gene can maintain high expression over a wider pH range.

[0042] Example 2: ​ Genes and ​ Application of genes in improving the alkali tolerance of Yellow River carp contain ​ Gene recombination vectors and those containing ​ The construction and verification of the gene recombinant vector and the preparation of the microinjection reagent were carried out in the same manner as in Example 1.

[0043] 1. Overexpression ​ or ​ Preparation of Yellow River carp fertilized eggs A mixture containing recombinant plasmid and Tol2 transposase mRNA was introduced into fertilized eggs of the first-cell stage of Yellow River carp using microinjection. After hatching, indoor fry rearing, and culture in a dedicated pond, one-year-old transposable fry were obtained. ​ 399 genetically modified juvenile fish, repost ​ 170 genetically modified juvenile fish were individually tagged, and fin tissue was harvested to detect the integration of the exogenous gene. ​ The upstream and downstream primer sequences for gene integration are shown in SEQ ID NO.23 and SEQ ID NO.24, respectively, with a target fragment size of 303 bp; used for detection. ​ The upstream and downstream primer sequences for gene integration are shown in SEQ ID NO.25 and SEQ ID NO.26, respectively, and the target fragment size is 314 bp. Detection results are shown below. ​ After testing ​ and ​ The groups detected 92 and 43 positive Yellow River carp containing the target fragment, respectively, with positive rates of 23.1% and 25.3%.

[0044] 2. Overexpression ​ or ​ Verification of alkali tolerance in Yellow River carp genes For positive Yellow River carp (TG- ​ and TG- ​ ) and negative-positive Yellow River carp (WT, control group injected with an equal volume of phenol red) were subjected to 96 h of acute alkaline stress treatment. The specific method is as follows: The experimental fish were acclimatized for 1 week before the experiment and fasted for 24 h before the experiment. Based on the preliminary experimental results (Yellow River carp have strong alkalinity tolerance, with no mortality after 7 days of 60 mmol / L stress), the acute stress was set with alkalinity gradients of 60, 80, 100, and 120 mmol / L (alkaline water was prepared with analytical grade NaHCO3 and aerated). Each aquarium (150L) was filled with 5 TG- ​ 5-tailed TG- ​ Five negative individuals (FW) were included in the experiment. During the experiment, the fish were continuously aerated and fasted. Half of the alkaline water of the same concentration was replaced every 24 hours. The condition of the experimental fish was continuously observed, and the number of dead individuals at 6, 12, 24, 36, 48, 72 and 96 hours was recorded. The mortality rate (%) was calculated.

[0045] The results are as follows ​ As shown: Under 80 mmol / L alkaline water stress, the mortality rate of WT rose to 20% after 48 h and remained thereafter, TG- ​ Death occurred 96 hours later, TG- ​ No deaths were observed at 96 h; under 100 mmol / L water reduction stress, the mortality rate of WT reached 100% at 48 h, and TG- ​and TG- ​ All reached 100% survival rate at 96 hours, with a significantly prolonged alkali-tolerant survival time; under 120 mmol / L alkaline water stress, WT mortality reached 100% within 24 hours, and TG- ​ The mortality rate reached 100% after a delay of up to 48 hours, TG- ​ The mortality rate reached 100% after 72 hours. The high alkaline water stress experiment showed that the transgenic Yellow River carp had improved alkali tolerance.

[0046] For acute stress 0, 20 、 Fish in the 40 and 60 mmol / L alkalinity groups were sampled at 96 h. Gill tissue was rapidly dissected at low temperature, and total RNA was extracted from each tissue using the TRIzol method, followed by reverse transcription to synthesize cDNA. A design was developed based on the CDS region sequence of the target gene from *Leuciscus vallis*. ​ and ​ qPCR primers for genes, ​ The upstream and downstream primer sequences for qPCR of the exogenous gene are shown in SEQ ID NO.19 and SEQ ID NO.20, respectively; ​ The upstream and downstream primer sequences for qPCR of the exogenous gene are shown in SEQ ID NO.21 and SEQ ID NO.22, respectively. Using the actin gene as an internal control, the expression level of the target gene was detected by real-time quantitative PCR, and the relative expression level was calculated using a 22... ( ΔΔCT) The method is used for calculation.

[0047] Test results as follows ​ As shown: Under moderate alkali stress of 20 mmol / L, TG- ​ Group ​ Relative gene expression levels and TG- ​ Group ​ The relative expression levels of genes were significantly higher in the group than in the WT group. P <0.0001). As the stress concentration increased to 40 mmol / L, TG- ​ Group ​ Gene expression levels dropped significantly, showing no significant difference from the WT group (ns); while at this time, TG- ​ Group ​ The gene still maintained a high expression level, significantly higher than that of the WT group ( P <0.01). When faced with extreme high-alkali stress of 60 mmol / L, TG- ​ Group ​ Gene expression levels decreased to levels not significantly different from the WT group (ns), while TG- ​ Group ​ Gene expression was significantly suppressed, and significantly lower than that of the WT group (P <0.01), indicating exogenous ​ and ​ The introduction of genes significantly improved the gene transcription level of Yellow River carp under alkaline stress.

[0048] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be defined by the claims.

Claims

1. A gene that enhances the alkali tolerance of cyprinid fish, characterized in that, The gene is hba Gene or hbb Gene, hba The nucleotide sequence of the gene's CDS region is shown in SEQ ID NO.

1. hbb The nucleotide sequence of the gene's CDS region is shown in SEQ ID NO.

3.

2. The application of the gene described in claim 1 in improving the alkali tolerance of cyprinid fish.

3. A recombinant vector containing the gene of claim 1.

4. The recombinant vector according to claim 3, characterized in that, The recombinant vector uses the pDestTol2CG2-Mod plasmid (with the cmlc promoter and EGFP reporter gene removed) as its backbone and inserts... hba Gene promoter region and CDS region sequences, or insertions hbb The gene promoter region and CDS region sequences were constructed; hba The nucleotide sequence of the gene promoter region is shown in SEQ ID NO.

2. hbb The nucleotide sequence of the gene promoter region is shown in SEQ ID NO.

4.

5. The recombinant expression vector according to claim 4, characterized in that, The hba Gene promoter region sequence and hbb The gene promoter region sequence was obtained by amplifying the genomic DNA of *Leuciscus valens* from Lake Dali using it as a template. hba Gene CDS region sequence and hbb The gene CDS region sequence was obtained by amplifying cDNA from the Dali Lake wrasse.

6. The recombinant expression vector according to claim 5, characterized in that, For amplification hba The nucleotide sequences of the upstream and downstream primers for the gene promoter region sequence are shown in SEQ ID NO.7 and SEQ ID NO.8, respectively; For amplification hba The nucleotide sequences of the upstream and downstream primers for the gene's CDS region sequence are shown in SEQ ID NO. 9 and SEQ ID NO. 10, respectively; used for amplification. hbb The nucleotide sequences of the upstream and downstream primers for the gene promoter region sequence are shown in SEQ ID NO.11 and SEQ ID NO.12, respectively; For amplification hbb The nucleotide sequences of the upstream and downstream primers for the gene CDS region sequence are shown in SEQ ID NO.13 and SEQ ID NO.14, respectively.

7. The recombinant expression vector according to claim 4, characterized in that, The vector backbone was obtained by amplification using pDestTol2CG2-Mod plasmid as template and upstream primer with nucleotide sequence as shown in SEQ ID NO.5 and downstream primer with nucleotide sequence as shown in SEQ ID NO.

6.

8. The application of the recombinant expression vector according to any one of claims 3-7 in improving the alkali tolerance of cyprinid fish.

9. A method for improving the alkali tolerance of cyprinid fish, characterized in that, The process includes the following steps: constructing a recombinant vector as described in any one of claims 3-7; mixing the recombinant vector with Tol2 transposase mRNA at equal mass concentrations to prepare a mixture sample for microinjection; then injecting the mixture sample into I-cell stage fertilized eggs of cyprinid fish using a microinjection apparatus; and obtaining cyprinid fish with improved alkali tolerance after hatching and culturing the fertilized eggs.

10. The method according to claim 9, characterized in that, The carp species mentioned include zebrafish and Yellow River carp.

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