GS protein and application of coding gene thereof

Abstract

The invention belongs to the technical field of biology, and particularly relates to a GS protein and application of a coding gene thereof. The GS protein has an amino acid sequence as shown in SEQ ID NO. 1, and the GS protein has a nucleotide sequence as shown in SEQ ID NO. 2. According to the invention, the GS gene and the GS protein are regulated for the first time to correspondingly regulate the new regeneration efficiency of the appendage of the auriculate spirulina, and a new solution can be provided for genetic transformation of species with low regeneration efficiency; moreover, the GS gene can be used as an important molecular mechanism in a new regeneration process of the Mexican spirulina, and a basis is provided for research and application fields of genome synthesis of the Mexican spirulina and customized transformation of the Mexican spirulina.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to the application of a GS protein and its encoding gene. Background Technology

[0002] Mexican axolotl ( Axolotl The hydra is currently the only known tetrapod capable of complete regeneration and possessing four limbs. Its regeneration differs from the amorphous regeneration of hydras and the compensatory regeneration of the liver; the process can be divided into three key stages: wound healing, formation and redevelopment of the regenerating bud base. This process involves the proliferation, differentiation, and tissue reconstruction of the regenerating bud base cells. The core value of this model lies in its ability to maintain a juvenile state throughout life and in the absence of scar tissue formation during the regeneration process.

[0003] Current research on regeneration regulation mechanisms mainly focuses on three major categories of regulatory factors: growth factors, transcription factors, and epigenetic regulatory factors. Growth factors, represented by FGF8 and BMP2, function to activate basal cell proliferation through the MAPK / Smad pathway. Transcription factors Msx1 and Sox2 can maintain cell dedifferentiation. Epigenetic regulatory factors such as HDAC1 and Kdm6b can modify histones to open regeneration gene sites.

[0004] Although the role of energy metabolism (the conversion between glycolysis and oxidative phosphorylation) in regeneration has been reported, there are still many problems, such as the completely unknown regulatory role of nitrogen metabolism in regeneration, the lack of evidence for the association between amino acid metabolism and epigenetic modification, and the fact that glutamine metabolism, although well studied in the field of oncology (Warburg effect), has not been reported in regeneration.

[0005] Glutamine synthase is a core enzyme in nitrogen metabolism, catalyzing the combination of glutamate and ammonia to form glutamine. This process is not only crucial in the nitrogen metabolism cycle between neurons and glial cells, but is also closely related to cell osmotic pressure regulation, anti-oxidative stress, cell proliferation, and repair.

[0006] Catalytic reaction: Glutamate + NH3 + ATP → Glutamine + ADP + Pi Its traditional functions include serving as a nitrogen metabolism center, scavenging toxic ammonia ions, and maintaining nitrogen balance; protecting nerves, exhibiting anti-excitotoxicity properties in astrocytes, and providing amino groups for nucleotide synthesis. Combining theory with this study, it was found that... GS Genes actively participate in the regeneration process: (1) Matching metabolic needs: Regenerated bud bases require a large amount of nitrogen source; (2) Gene regulatory characteristics: Bioinformatics analysis revealed GS Genes undergo significant changes during critical periods of regeneration; (3) Evolutionary conservation: The GS protein sequence is more than 80% conserved in vertebrates.

[0007] Current research on glutamine synthase (GS) genes and their proteins primarily focuses on neurological diseases (such as Alzheimer's disease), with the regenerative field neglecting the crucial role of glutamine metabolism. In regenerative applications, common methods include growth factor therapy and stem cell transplantation, but each has drawbacks such as high cost and the risk of immune rejection. GS If gene and GS protein regenerative therapies are applied clinically, they can reduce costs by targeting endogenous metabolic enzymes based on well-defined molecular mechanisms or avoid immune rejection by reprogramming the host's own cells.

[0008] In-depth research into the regenerative process in the scientific research field reveals that novel functions of glutamine synthase (GS) protein and its encoding gene can significantly influence the interactions of existing signaling pathways, thereby facilitating the development of new drugs and therapies in regenerative medicine.

[0009] therefore, GS The novel functions of genes and GS proteins in the regeneration process are of great significance. Summary of the Invention

[0010] The purpose of this invention is to provide the application of the GS protein and its encoding gene in the preparation of a drug for regulating the regeneration of appendages in the Mexican axolotl. This invention effectively regulates the regenerative capacity of the Mexican axolotl by inhibiting or promoting the activity of the GS protein in the axolotl, thereby verifying... GS Novel functions or applications of genes and GS proteins.

[0011] This invention modulates the activity of GS (gastropods) by administering exogenous small molecule compounds, alone or in combination, in experiments using the Mexican axolotl as the experimental animal. The experimental results show that... GS The gene and its translation product GS (glutamine synthase) have new functions or mechanisms in the process of appendage regeneration.

[0012] In one aspect, this invention proposes the application of a GS protein and its encoding gene in regulating the new formation and regeneration of appendages in the Mexican axolotl. By controlling... GS The expression, activity, or protein content of genes and GS proteins in axolotls can effectively regulate the regeneration process of axolotls.

[0013] In another aspect, the present invention proposes a... GS The use of gene inhibitors in the preparation of drugs that regulate the new growth and regeneration of appendages in the Mexican axolotl.

[0014] Furthermore, the aforementionedGS The gene has a nucleotide sequence as shown in SEQ ID NO.2.

[0015] Furthermore, the GS gene is used to encode the GS protein. Furthermore, the GS protein has the amino acid sequence shown in SEQ ID NO.1. Furthermore, the regulation of new appendage regeneration in the Mexican axolotl refers to promoting or inhibiting the efficiency or ability of new appendage regeneration in the Mexican axolotl. Furthermore, the sources of the Mexican axolotl include Mexican axolotls with pink, black, or yellow body color phenotypes.

[0016] Furthermore, the Mexican axolotl tissue lost during the regeneration of the appendages includes at least one of the palm portion of the four appendages, the fingertip portion of the four appendages, or the tip portion of the tail.

[0017] Furthermore, the aforementioned GS Gene inhibitors include those selected from knockout or silencing. GS At least one of the gene inhibitors.

[0018] Furthermore, the knockout or silence GS Gene inhibitors include at least one selected from siRNA, shRNA, Cre / loxP vector, and CRISPR / Cas9 vector.

[0019] According to an embodiment of the present invention, the promotion or reduction of the Mexican axolotl's... GS The expression and activity of genes and GS proteins are mediated through... GS The gene was generated after contact with the experimental animal, the Mexican axolotl.

[0020] The beneficial effects of this invention are as follows: This invention is the first to propose regulation. GS Genes and GS proteins can correspondingly regulate the new appendage regeneration efficiency of the Mexican axolotl, providing a new solution for genetic transformation of species with low regeneration efficiency; and, GS Genes can serve as an important molecular mechanism in the regeneration process of axolotls, providing a foundation for research and application in the fields of axolotl genome synthesis and customized modification of axolotls. Attached Figure Description

[0021] Figure 1 Figures showing observational records of appendage regeneration after four different treatments in the Mexican axolotl. Detailed Implementation

[0022] In this article, the term "regeneration" refers to the process by which the Mexican axolotl, after losing its appendages, fingertips, and tail, undergoes wound healing, the formation of regenerative buds, and redevelopment, ultimately forming tissues with the same function and shape as the original lost parts.

[0023] According to embodiments of the present invention, the Mexican axolotl tissue lost during regeneration includes, but is not limited to, at least one of the palm portion of the four appendages, the fingertip portion of the four appendages, and the tip portion of the tail.

[0024] It should be noted that when the appendages, fingertips, and tail of the Mexican axolotl are artificially removed, resulting in tissue loss, callus tissue will first form, followed by the formation of regenerative buds. These bud tissues will then redevelop and gradually grow until they form tissues with the same function and shape as the original lost parts.

[0025] According to an embodiment of the present invention, the GS gene has a nucleotide sequence as shown in SEQ ID NO.2.

[0026] It should be noted that the GS gene mentioned in this invention, as understood by those skilled in the art, actually includes any one or both of the complementary double strands. For convenience, although only one strand is given in most cases herein, the other complementary strand is also disclosed. Furthermore, the nucleotide sequences in this invention include DNA or RNA forms; disclosure of one implies that the other is also disclosed.

[0027] According to an embodiment of the present invention, the GS protein has an amino acid sequence as shown in SEQ ID NO.1.

[0028] It should be noted that, as those skilled in the art will know, according to GS The amino acid sequence of the protein can be obtained using conventional methods or conventional websites or software (such as the online website NCBI (https: / / www.vectorbuilder.cn / tool / codon-optimization.html) etc.) to obtain the amino acid sequence encoding the GS protein (i.e., the one in this invention). GS (Gene translation products), therefore, in addition to SEQ ID NO:2 as defined in this invention, other amino acid sequences having GS proteins are also within the scope of this invention.

[0029] According to embodiments of the present invention, the regulation of new cell regeneration in *Axolotlium gracilis* involves inhibiting or promoting new cell regeneration. The inventors have experimentally discovered that overexpression... GS Genes or Enhancement GS The expression or activity of genes and GS proteins may enhance the regeneration efficiency or ability of axolotls, while knocking out [the gene / protein]... GSGenes or reduction GS The expression or activity of genes and GS proteins may reduce the efficiency or ability of new cell regeneration in axolotls, providing a foundation for studying the metabolic mechanism or signal transduction of glutamine synthase, a core enzyme in nitrogen metabolism, during the new cell regeneration process in axolotls. This holds promise for playing a crucial role in the research and application of axolotl genome synthesis and customized modification. Furthermore, it can also be achieved by controlling... GS The expression or activity of genes or GS proteins can regulate the regeneration efficiency of axolotls, thereby obtaining axolotl individuals or populations with high regeneration efficiency.

[0030] According to an embodiment of the present invention, the source of the axolotl includes at least one of the axolotls of various body color phenotypes such as pink, black, and yellow.

[0031] In another aspect of the invention, the invention proposes a... GS The role of gene inhibitors in regulating the regeneration of new Mexican blunt snouts. As previously known, by controlling... GS The expression and translation of genes and GS proteins in Mexican blunt snouts can effectively regulate the formation and regeneration of these snouts. Therefore, the aforementioned inhibitors can be used to inhibit... GS The expression and translation of genes and GS proteins play a role in regulating the regeneration of new Mexican blunt snouts.

[0032] According to an embodiment of the present invention, the GS Gene inhibitors include those selected from knockout or silencing. GS At least one of the gene inhibitors.

[0033] According to embodiments of the present invention, the function and principle of the experimental reagents and methods used are not included in the patent application content; the experimental reagents and experimental techniques used are merely methods for demonstrating gene function.

[0034] According to an embodiment of the present invention, the knockout or silence GS Gene inhibitors include at least one of siRNA, shRNA, Cre / loxP vector, and CRISPR / Cas9 vector.

[0035] In this paper, "siRNA," "small interfering RNA," "short interfering RNA," and "silencing RNA" all refer to a double-stranded RNA that primarily participates in RNA interference (RNAi) to specifically regulate gene expression. It should be noted that, within the known scope of this invention... GSIn the case of genes and GS proteins, siRNA can be obtained using methods conventional in the art. The obtained siRNA is cloned into an expression vector, and by introducing this expression vector into plant cells, it can be knocked out or silenced. GS Gene.

[0036] In this document, the terms "shRNA" and "short hairpin RNA" both refer to RNA polymerase III transcription terminators consisting of two short inverted repeat sequences separated by a stem-loop sequence, forming a hairpin structure, controlled by the pol III promoter, followed by 5-6 T sequences. It should be noted that, within the known scope of this invention... GS In the case of genes and GS proteins, shRNA can be obtained using methods conventional in the art. The obtained shRNA is cloned into an expression vector, and by introducing this expression vector into Mexican axolotl cells, it can be knocked out or silenced. GS Gene.

[0037] In this paper, the term "CRISPR / Cas9 vector" includes the gene encoding the Cas9 protein and the gene encoding gRNA (or sgRNA). The gene encoding the Cas9 protein and the gene encoding gRNA (or sgRNA) can be located on the same vector or on two separate vectors. When this CRISPR / Cas9 vector is introduced into axolotl cells, it can be knocked out or silenced. GS Genes. It should be noted that, within the known scope of this invention... GS In the case of genes and GS proteins, gRNA (or sgRNA) can be obtained using methods conventional in the art.

[0038] The present invention will be explained below with reference to embodiments. Those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be considered as limiting the scope of the invention. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be obtained commercially.

[0039] Example 1: Regeneration of appendages in Mexican axolotls after loss of appendages 1. Surgical creation of missing appendages in Mexican axolotls Healthy axolotls with a body length of 10 cm were selected and temporarily housed in tap water aerated for at least 24 hours. The correct concentration of reagent was prepared (anesthetic: MS222 solution working concentration 1g / L). The axolotls to be tested were immersed in MS222 solution until they were completely anesthetized. Then, the appendages were removed by using a sterilized scalpel to remove the palm part of both ends or one end of the hind limbs of the axolotls.

[0040] 2. GS Validation of gene or GS protein function in the regeneration of new tissue in Mexican axolotls Prepare the correct concentrations of reagents (1*PBS solution, 2mM MSO, 12mM Met, 4mM MSO, 10mM Gln); administer microinjection or intraperitoneal injection to the Mexican axolotls that have lost some appendages in step 1. The Control group received 0.1 mL of 1*PBS solution, Treatment 1 received 0.1 mL of 2mM MSO solution, Treatment 2 received 0.05 mL of 12mM Met solution and 0.05 mL of 4mM MSO solution, and Treatment 3 received 0.05 mL of 10mM Gln solution and 0.05 mL of 4mM MSO solution. MSO solution was used, with Met and Gln solutions injected one hour prior to the MSO injection. The prepared PBS solution is a commonly used physiological buffer in biological experiments. The prepared Gln solution is the reaction product of the GS enzyme. The prepared MSO solution specifically inhibits GS enzyme activity, and the prepared Met solution alleviates the inhibition of GS enzyme activity caused by the MSO solution. After injection, the images were taken under a microscope and recorded. Observations were taken every two days, repeating the anesthesia steps in step 1. Results are shown in the appendix. Figure 1 .

[0041] The results showed that the regeneration process of appendages in the Mexican axolotl varied across the four treatment groups. Using the regeneration rate in the Control group as a baseline, only the Met group exhibited a significantly faster regeneration rate, validating the effectiveness of controlling the regeneration process. GS The expression and activity of genes and GS proteins can regulate the ability of Mexican axolotls to regenerate new appendages.

Claims

1. Application of GS protein and its encoding gene in the preparation of drugs that regulate the regeneration of appendages in the Mexican axolotl.

2. The application as described in claim 1, characterized in that: The GS protein has the amino acid sequence shown in SEQ ID NO.

1. GS The protein-coding gene has a nucleotide sequence as shown in SEQ ID NO.

2.

3. The application as described in claim 1, characterized in that: The regulation of appendage regeneration in the Mexican axolotl refers to promoting or inhibiting the efficiency or ability of appendage regeneration in the Mexican axolotl.

4. The application as described in claim 1, characterized in that: The sources of the Mexican axolotl include Mexican axolotls with pink, black, or yellow body color phenotypes.

5. The application as described in claim 1, characterized in that: In the regeneration of appendages of the Mexican axolotl, the tissues lost by the Mexican axolotl include at least one of the palm portion of the four appendages, the fingertip portion of the four appendages, or the tip portion of the tail.

6. Application of GS protein and its encoding gene in regulating the process of new limb regeneration in the Mexican axolotl.

7. The application as described in claim 6, characterized in that: By controlling GS The expression, activity, or protein content of genes and GS proteins in axolotl regulates the process of new appendage regeneration in axolotl.

8. GS Application of gene inhibitors in the preparation of drugs that regulate the regeneration of appendages in the Mexican axolotl.

9. The application as described in claim 8, wherein... GS Gene inhibitors include those selected from knockout or silencing. GS At least one of the gene inhibitors.

10. The application as described in claim 9, wherein the knockout or silencing... GS Gene inhibitors include at least one selected from siRNA, shRNA, Cre / loxP vector, and CRISPR / Cas9 vector.

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