A mutant of mytilus edulis byssal thread adhesive protein and its application and a method for identifying mytilus edulis byssal thread phenotype character

Abstract

The application discloses a mytilus edulis byssal thread adhesion protein mutant and application thereof and a method for identifying mytilus edulis byssal thread phenotype characteristics, and relates to the technical field of biology. The amino acid sequence of the mytilus edulis byssal thread adhesion protein mutant is shown as SEQ ID NO. 4. The application discloses that a non-synonymous mutation in the coding region of the mytilus edulis Mfp3-6 gene can directly affect the functional characteristics of the protein, thereby establishing a clear cause-and-effect relationship between the genotype and the mytilus edulis byssal thread adhesion performance phenotype. The application further provides a mytilus edulis byssal thread adhesion protein mutant, which has excellent adhesion strength characteristics and shows a broad application prospect in the field of functional adhesion materials. The protein mutant can be widely applied to the development of medical glue, dressings and gel materials, and provides important technical support for the research and development of biomimetic adhesion technology and new biological materials, and has good industrialization potential and social value.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a mutant of thick-shelled mussel byssal adhesion protein, its application, and a method for identifying the phenotypic traits of mussel byssal fibers. Background Technology

[0002] Mussels are a core economic species in shellfish aquaculture, but the scarcity of superior germplasm resources restricts the industry's sustainable development. The thick-shelled mussel (Mytilus coruscus) is one of the important farmed mussel species. Current farming strategies overemphasize yield increases while neglecting the selection of stress-resistant traits, leading to a significant decline in byssal adhesion function under summer high-temperature stress, triggering large-scale seasonal seedling shedding events. Therefore, the key to overcoming this bottleneck lies in the genetic improvement of byssal adhesion performance. Thick-shelled mussels secrete byssal proteins through foot glands, which solidify in seawater to form byssal threads that attach to the matrix surface. Currently, various byssal proteins have been identified in mussel byssal threads and foot glands. SNP markers follow Mendelian co-dominant inheritance laws, can distinguish between homozygotes and heterozygotes, provide complete genetic information, and are unaffected by seasonal, developmental stage, and environmental factors. They have the advantages of large numbers, high polymorphism, simple, rapid, and easily automated technology, making them a powerful tool for genetic structure analysis, germplasm resource identification, genetic diversity analysis, and marker-assisted selection breeding. Based on the crucial role of byssal adhesion protein genes in thick-shelled mussels, identifying SNP sites within these genes and screening for molecular markers suitable for byssal adhesion strength provides an important tool for breeding new thick-shelled mussel varieties with strong byssal adhesion. The value of coding region mutations in genetic breeding lies in their ability to directly link genotype and phenotype. Non-synonymous mutations directly alter protein function, establishing a causal relationship between genotype and phenotype, significantly reducing the risk of false positives in breeding.

[0003] This invention aims to identify functional single nucleotide polymorphism (SNP) sites associated with the high adhesion phenotype of thick-shelled mussel byssal threads, and to develop genetic markers for molecular-assisted breeding based on these sites, in order to select new thick-shelled mussel varieties with excellent adhesion traits; it also provides a basis for the development of functional adhesive materials. Summary of the Invention

[0004] The purpose of this invention is to provide a thick-shelled mussel byssal adhesive protein mutant, its applications, and a method for identifying the phenotypic traits of mussel byssal fibers, thereby solving the problems existing in the prior art. The excellent adhesive strength characteristics of this thick-shelled mussel byssal adhesive protein mutant make it show broad application prospects in the field of functional adhesive materials.

[0005] To achieve the above objectives, the present invention provides the following solution:

[0006] This invention provides a mutant of the byssal adhesion protein of thick-shelled mussels, the amino acid sequence of which is shown in SEQ ID NO.4.

[0007] The present invention also provides a gene encoding the above-mentioned thick-shelled mussel byssal adhesion protein mutant.

[0008] Furthermore, the nucleotide sequence of the encoding gene is shown in SEQ ID NO.5.

[0009] The present invention also provides a recombinant expression vector comprising the above-described coding gene.

[0010] The present invention also provides a non-plant host cell comprising the above-described recombinant expression vector.

[0011] The present invention also provides the application of the above-mentioned encoding gene, recombinant expression vector or non-plant host cell in the preparation of the above-mentioned thick-shelled mussel byssal adhesion protein mutant.

[0012] The present invention also provides a primer pair for identifying the adhesion of byssal threads in thick-shelled mussels, comprising an upstream primer with a nucleotide sequence as shown in SEQ ID NO.2 and a downstream primer with a nucleotide sequence as shown in SEQ ID NO.3;

[0013] The primer pair is used to amplify the molecular marker with nucleotide sequence as shown in SEQ ID NO.1; there is an SNP site at the 56th base of the molecular marker, which is a T / C mutation, and the adhesion strength of the CC genotype thick-shelled mussel is greater than that of the CT genotype thick-shelled mussel.

[0014] The present invention also provides a detection product for identifying the adhesion of byssal threads in thick-shelled mussels, comprising the primer pair described above.

[0015] The present invention also provides the application of the above-mentioned primer pairs or detection products in identifying byssal adhesion, genetic diversity analysis or population structure analysis of thick-shelled mussels.

[0016] This invention also provides a method for identifying the adhesiveness of byssal threads in thick-shelled mussels, comprising the following steps:

[0017] Genomic DNA was extracted from the thick-shelled mussel sample to be tested;

[0018] Using the genomic DNA as a template, PCR amplification was performed using the primer pairs described above to obtain the amplification product. The genotype at the 56th base of the amplification product was obtained by sequencing. Based on the obtained genotype, the byssal adhesion of thick-shelled mussels was identified: the adhesion strength of the CC genotype thick-shelled mussels was greater than that of the CT genotype thick-shelled mussels.

[0019] The present invention discloses the following technical effects:

[0020] This invention, through in-depth research, reveals that non-synonymous mutations of SNPs in the coding region of the Mfp3-6 gene can directly affect the functional properties of the protein, thus establishing a clear causal relationship between genotype and the byssal adhesion performance phenotype of thick-shelled mussels. This discovery provides a theoretical basis for elucidating the molecular mechanism of the superior adhesion performance of thick-shelled mussels and demonstrates that single nucleotide polymorphisms (SNPs) based on the Mfp3-6 gene can serve as efficient screening indicators for the rapid and accurate identification of thick-shelled mussel individuals with excellent adhesion performance. This marker-assisted selection technology not only significantly improves the efficiency and accuracy of breeding but also provides scientific support for the large-scale cultivation of high-quality thick-shelled mussel varieties.

[0021] Furthermore, this invention provides a thick-shelled mussel byssal adhesive protein, whose excellent adhesive strength properties make it a promising candidate for application in the field of functional adhesive materials. This protein can be widely used in the development of medical adhesives, dressings, and gels, providing crucial technical support for the research and development of biomimetic adhesion technologies and novel biomaterials, and possesses significant industrialization potential and social value. Attached Figure Description

[0022] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0023] Figure 1 The figure shows the adhesion strength test results of the byssal adhesion proteins Mfp3-6-1 and Mfp3-6-2 in thick-shelled mussels. Detailed Implementation

[0024] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0025] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0026] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0027] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0028] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0029] The determination procedure for the adhesion of byssal threads in this invention is as follows: The adhesion of mussel byssal threads is determined using a miniature universal testing machine. The proximal end of the byssal thread fiber is separated from the mussel body using a tool, while the byssal disc remains on the culture dish, obtaining a complete byssal thread from the proximal end of the byssal thread fiber to the byssal disc. The distal end of the byssal thread fiber is fixed to the iron support of the testing machine, and the byssal thread is pulled at a speed of 10 mm / min until the byssal disc separates from the culture dish. The instrument data is recorded, which is the adhesion strength.

[0030] Example 1

[0031] This invention obtained three SNP sites (SNP1, SNP2 and SNP3) in the Mfp3-6 gene, a gene containing byssal adhesion protein of thick-shelled mussels, which are related to the adhesion strength of byssal fibers of thick-shelled mussels, through multiple sequence alignment and association analysis, as detailed in Table 1.

[0032] Table 1. Mutation types at SNP sites in the Mfp3-6 gene

[0033]

[0034] The nucleotide sequence (SEQ ID NO.1) of the Mfp3-6 gene is as follows:

[0035] ATGAACAAATTCAGTCTAACAGTTATGCTGGCTTTAGTCCTTAWTGGATTATTTGYCGTGCAGAGTGATGCTGGTTATGATTATTATCCAGGATATAATTCACCATKGCAATACAATGGTTACTATGGC TATAGTGGATACAATGGATATCACGGACGTTATGGCTGGAATAAGGGCTGGAATAACGGTCCATGGGGAGGATCATATTATGGAAACAGAGGCTATCTGTATTAG; where Y represents C or T, W represents T or A, and K represents G or T.

[0036] Example 2

[0037] 1. Genomic DNA was extracted from 150 thick-shelled mussel samples from three populations (Shengsi, Jiaojiang, and Fuding) using the Tiangen Marine Animal Tissue Genome Extraction Kit (TIANGEN, Tiangen Biotech Co., Ltd.). The extracted genomic DNA was tested for quality using 2% agarose gel electrophoresis. After determining the concentration, the DNA was diluted to 50 ng / μL and stored at -20℃ for later use.

[0038] 2. Primer design

[0039] Primers were screened and designed using Primer5 software. The primers designed in Table 2 could stably amplify sequences containing Mfp3-6, and the genotypes of the three SNP sites (SNP1, SNP2, and SNP3) were analyzed.

[0040] Table 2 Primer sequence information

[0041]

[0042] 3. SNP marker amplification detection and data analysis

[0043] Genomic DNA from the thick-shelled mussel was amplified by PCR using the primers shown in Table 2. The PCR amplification system was 25 μL, including: 1 μL template DNA, 0.5 μL forward primer, 0.5 μL reverse primer, 0.5 μL dNTP (10 mM), and 2.5 μL 10×PCR Buffer (containing Mg2+). 2+0.2 μL Taq Plus DNA polymerase (5 U / μL) and 19.8 μL sterile deionized water were added. The PCR amplification program was as follows: 95℃ pre-denaturation for 5 min; 94℃ denaturation for 30 s, 60℃ annealing for 30 s, 72℃ extension for 40 s, for 35 cycles; and 72℃ final extension for 10 min. After PCR amplification, the amplified products were sequenced to obtain the genotype information of each SNP locus of 150 thick-shelled mussels from three populations.

[0044] Genotyping and byssal adhesion strength were detected in 150 thick-shelled mussel samples. The results are shown in Table 3, and the correlation analysis between the genotype of the SNP locus and the byssal adhesion strength trait is shown in Table 4. The results showed that SNP2 had a significant effect on the byssal adhesion strength trait. By comparison, the average adhesion strength of individuals with the CC genotype was significantly greater than that of individuals with the CT genotype (P<0.05). Therefore, the byssal adhesion strength trait of thick-shelled mussels can be screened based on the genotype of the SNP2 locus.

[0045] Table 3. Raw data from genotyping and byssal adhesion strength analysis of 150 thick-shelled mussel samples.

[0046]

[0047]

[0048]

[0049]

[0050]

[0051] Table 4. Correlation analysis of SNP loci genotypes and byssal adhesion strength traits.

[0052]

[0053] Example 3

[0054] 1. Construction of expression vectors for byssal adhesion proteins from different thick-shelled mussels

[0055] Protein expression vectors were constructed for two proteins (Mfp3-6-1 and Mfp3-6-2):

[0056] The amino acid sequence of the Mfp3-6-1 protein (TCT haplotype) is shown in SEQ ID NO.4, and the nucleotide sequence of its encoding gene is shown in SEQ ID NO.5; the amino acid sequence of the Mfp3-6-2 protein (TTT haplotype) is shown in SEQ ID NO.6, and the nucleotide sequence of its encoding gene is shown in SEQ ID NO.7.

[0057] SEQ ID NO.4:

[0058] MNKFSLTVMLALVL I GLF A VQSDAGYDYYPGYNSP L QYNGYYGYSGYNGYHGRYGWNKGWNNGPWGGSYYGNRGYLY。

[0059] SEQ ID NO.5:

[0060] ATGAACAAATTCAGTCTAACAGTTATGCTGGCTTTAGTCCTTATTGGATTATTTGCCGTGCAGAGTGATGCTGGTTATGATTATTATCCAGGATATAATTCACCATTGCAATACAATGGTTACTATGGCTATAGTGGATACAATGGATATCACGGACGTTATGGCTGGAATAAGGGCTGGAATAACGGTCCATGGGGAGGATCATATTATGGAAACAGAGGCTATCTGTATTAG。

[0061] SEQ ID NO.6:

[0062] MNKFSLTVMLALVL I GLF V VQSDAGYDYYPGYNSP L QYNGYYGYSGYNGYHGRYGWNKGWNNGPWGGSYYGNRGYLY。

[0063] SEQ ID NO.7:

[0064] ATGAACAAATTCAGTCTAACAGTTATGCTGGCTTTAGTCCTTATTGGATTATTTGTCGTGCAGAGTGATGCTGGTTATGATTATTATCCAGGATATAATTCACCATTGCAATACAATGGTTACTATGGCTATAGTGGATACAATGGATATCACGGACGTTATGGCTGGAATAAGGGCTGGAATAACGGTCCATGGGGAGGATCATATTATGGAAACAGAGGCTATCTGTATTAG。

[0065] Prokaryotic expression primers were designed based on the coding genes of Mfp3-6-1 and Mfp3-6-2 proteins, and restriction enzyme sites were added. These primers were then ligated into the pEt-28a plasmid (purchased from Hunan Fenghui Biotechnology Co., Ltd.). The plasmid was transformed into E. coli BL21(DE3) competent cells (purchased from Sangon Biotech (Shanghai) Co., Ltd.), plated on LB medium containing ampicillin (100 μg / mL), and cultured overnight. Single colonies were then picked and grown in LB medium. The strains were preserved, and plasmids were extracted and sequenced to confirm the correctness of the expression vector.

[0066] 2. Expression and purification of mussel proteins

[0067] Two recombinant thick-shelled mussel byssal adhesion proteins (Mfp3-6-1 and Mfp3-6-2) were expressed, extracted, and purified using two pre-constructed monoclonal strains, as follows:

[0068] Select correctly identified single colonies and incubate them overnight at 37°C in LB medium. Expand the culture at a 1:1000 ratio. [The text abruptly ends here, likely due to an incomplete sentence or missing information.] 600 When the pH was 0.8, 1 mM isopropyl-β-D-thiogalactoside (IPTG) was added for induction. After induction at 20℃ and 200 rpm for 6 h, 1.0 mL of bacterial culture was collected, the precipitate was resuspended in PBS, and the mixture was sonicated on ice until the solution was clear. After centrifugation at 12000 rpm for 20 min, the supernatant was collected, and the precipitate was resuspended in the same amount of PBS. The expression pattern of the protein was analyzed by SDS-PAGE. After equilibrating the purification column with 10 column volumes of lysis buffer, all the supernatant after membrane separation was transferred into the chromatography column. Impurities were washed with 10 column volumes of wash buffer; finally, the target protein was eluted with 10 column volumes of elution buffer. The protein eluent was placed in a treated dialysis bag and dialyzed for 48 h. After dialysis, the liquid in the dialysis bag was collected and freeze-dried under vacuum to obtain recombinant protein powder.

[0069] LB medium: yeast extract 5 g / L, peptone 10 g / L and sodium chloride 10 g / L.

[0070] 3. Shear force tests were performed on Mfp3-6-1 and Mfp3-6-2 proteins.

[0071] Protein solution preparation: Mfp3-6-1 and Mfp3-6-2 proteins were dissolved in a solution containing 5% (v / v) acetic acid and 150 mM sodium chloride, respectively, to a protein concentration of 100 mg / mL. Approximately 10 μL of the protein solution was applied to the top of a glass plate (approximately 10 mm wide × 10 mm long) and cured at 37°C for 18 hours. A shear test was then performed to test the adhesion strength of the proteins. The shear test was conducted using a universal testing machine with a 100 N load cell and a shear velocity of 10 mm / min. Shear strength was determined by dividing the maximum force by the adhesive area. The shear test results are shown below. Figure 1 The results showed that the adhesion properties of Mfp3-6-1 protein were higher than those of Mfp3-6-2 protein.

[0072] In summary, the non-synonymous mutations in the coding region of the Mfp3-6 gene discovered in this invention can directly alter protein function, establishing a causal relationship between genotype and phenotype. Based on this, the SNP sites of the Mfp3-6 gene can be used to screen for superior adhesion performance in thick-shelled mussels, thereby providing technical support for the breeding of superior thick-shelled mussel varieties and molecular marker-assisted selection.

[0073] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A mutant of Mytilus edulis byssal thread adhesive protein, characterized in that, The amino acid sequence is shown as SEQ ID NO.

4.

2. A coding gene of the Mytilus edulis byssal thread adhesion protein mutant according to claim 1.

3. The genetic code according to claim 2, wherein, The nucleotide sequence of the coding gene is shown as SEQ ID NO.

5.

4. A recombinant expression vector, characterized in that, The coding gene according to claim 2 or 3.

5. A non-plant host cell, wherein, The recombinant expression vector according to claim 4.

6. Use of the coding gene according to claim 2 or 3, the recombinant expression vector according to claim 4 or the non-plant host cell according to claim 5 in the preparation of the Mytilus edulis byssal thread adhesion protein mutant according to claim 1.

7. A primer pair for identifying the byssus adhesion of Mytilus edulis, characterized by, The upstream primer comprising the nucleotide sequence shown as SEQ ID NO. 2 and the downstream primer comprising the nucleotide sequence shown as SEQ ID NO. 3; The primer pair is used for amplifying the molecular marker comprising the nucleotide sequence shown as SEQ ID NO. 1; the SNP site at the 56th base of the molecular marker is a T / C mutation, and the adhesion strength of the Mytilus edulis with CC genotype is greater than that of the Mytilus edulis with CT genotype.

8. A test product for discriminating the byssus adhesion of Mytilus edulis, characterized by, The primer pair according to claim 7.

9. Use of the primer pair according to claim 7 or the detection product according to claim 8 in identifying the byssal thread adhesion of Mytilus edulis.

10. A method of discriminating the adhesion of Mytilus edulis byssus, characterized by, The steps comprise: extracting the genomic DNA of the Mytilus edulis sample to be detected; using the primer pair according to claim 7 to perform PCR amplification on the genomic DNA as the template to obtain an amplification product, sequencing the genotype at the 56th base of the amplification product, and identifying the byssal thread adhesion of Mytilus edulis according to the obtained genotype: the adhesion strength of the Mytilus edulis with CC genotype is greater than that of the Mytilus edulis with CT genotype.

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