Substances for regulating carotenoid accumulation in crustaceans and uses thereof

Abstract

The present application belongs to the technical field of aquatic organism breeding, and particularly relates to a substance for regulating carotenoid enrichment in crustaceans and application thereof. The substance for regulating carotenoid enrichment in crustaceans is a CERP1 (Carotenoid enrichment related Protein 1) gene of the crustacean, a protein coded by the CERP1 gene, a substance for regulating expression of the CERP1 gene, and a substance for regulating activity or content of the CERP1 protein. The identified CERP1 gene is a negative regulation gene of the carotenoid enrichment trait, and loss of function of the CERP1 gene can lead to significant increase in content of various carotenoids such as astaxanthin, zeaxanthin, lutein and beta-carotene in the crustacean, and the body color of the crustacean presents red or red-orange, thereby significantly improving the nutritional quality of the aquatic animal. The CERP1 gene has a broad application prospect in improvement and breeding of quality traits of the crustacean.

Description

Technical Field

[0001] This invention belongs to the field of aquatic organism breeding technology, specifically relating to substances that regulate the accumulation of carotenoids in crustaceans and their applications. Background Technology

[0002] Carotenoids are a general term for an important class of natural pigments. Based on their chemical structure, they can be divided into two categories: carotene (containing only carbon and hydrogen, without oxygen, such as β-carotene and lycopene) and lutein (containing oxygen-containing functional groups such as hydroxyl and carboxyl groups, such as lutein, zeaxanthin, and astaxanthin). Carotenoids are mainly synthesized by plants, algae, bacteria, and fungi. Animals cannot synthesize carotenoids, but they can absorb and accumulate them. Carotenoids are a major source of vitamin A in animals and also have antioxidant, immune-regulating, anti-cancer, and anti-aging effects. Therefore, consuming foods high in carotenoids is of great significance to the health of both animals and humans.

[0003] Among carotenoids, astaxanthin (AX) is a ketone carotenoid with strong antioxidant activity and singlet oxygen quenching ability, considered a "super antioxidant." Its antioxidant capacity is more than 10 times that of other carotenoids, and it is currently the only known carotenoid capable of penetrating the blood-brain and blood-retinal barriers. Due to its outstanding antioxidant capacity, it is considered to have great potential in the prevention and treatment of diseases related to reactive oxygen species, such as cancer, neurodegenerative diseases, eye diseases, atherosclerosis, and diabetes. In addition, astaxanthin can enhance immunity, has anti-inflammatory effects, delay aging, and maintain healthy skin.

[0004] Crustaceans include a large number of economically valuable aquaculture animals, primarily shrimp and crabs belonging to the order Decapoda. Statistics show that my country's annual shrimp and crab production exceeds 6 million tons, providing a significant amount of high-quality protein for the Chinese population. In addition, artichokes, cladocerans, and other crustaceans are also important biological feeds for aquaculture, playing a crucial role in ensuring the supply of high-quality aquatic products. Besides providing high-quality protein, crustaceans have a strong ability to absorb and accumulate astaxanthin. Astaxanthin and other carotenoids accumulated through their diet also play an important role in improving human health; therefore, carotenoid content is a key indicator of shrimp and crab quality. Although feeding shrimp and crabs with feed rich in astaxanthin and other carotenoids can increase the carotenoid content, the increase is limited and the application cost is high. Developing new germplasm with significantly increased astaxanthin and other carotenoid content is an important measure to improve the nutritional quality of shrimp and crabs and benefit human health. Summary of the Invention

[0005] The technical problem to be solved by this invention is how to increase the content of carotenoids such as astaxanthin in crustaceans in order to improve the nutritional quality of crustaceans. This invention proposes a substance for regulating the accumulation of carotenoids in crustaceans and its application.

[0006] To solve the above-mentioned technical problems, the present invention adopts the following technical solution: A substance that regulates the accumulation of carotenoids in crustaceans, wherein the substance regulating the accumulation of carotenoids in crustaceans is the CERP1 gene of crustaceans and its encoded protein, a substance that regulates the expression of the CERP1 gene, and a substance that regulates the activity or content of the CERP1 protein.

[0007] The carotenoids mentioned are one or more of astaxanthin, zeaxanthin, lutein, canthaxanthin, and β-carotene.

[0008] The crustaceans mentioned are one or more of the classes Malacostraca (creatures of the order Malacostraca) and Branchiopoda (creatures of the order Branchiopoda). The Malacostraca include species such as *Litopenaeus vannamei*, *Litopenaeus vannamei*, *Litopenaeus japonicus*, *Litopenaeus chinensis*, *Macrobrachium rosenbergii*, *Macrobrachium nipponense*, *Eriocheir sinensis*, *Portunus trituberculatus*, *Muscovy crab*, *Procambarus clarkii*, and *Lobster*. The Branchiopoda include species such as *Artemia* and *Daphnia*.

[0009] Following the above method can increase the carotenoid content by more than 5 times. The increase in carotenoid content also changes the crustacean's body color to red or orange, resulting in a significant improvement in the crustacean's quality traits.

[0010] The amino acid encoded by the CERP1 gene of the crustacean is any one of the proteins with the following amino acid sequence (A1)-A17) of the CERP1 gene of each crustacean, and has a four-transmembrane protein domain and the DUF4728 domain from the pfam database.

[0011] A1) The amino acid sequence has the sequence shown in SEQ ID 1 (white shrimp) in the sequence listing; A2) The amino acid sequence has the sequence shown in SEQ ID2 (Japanese giant freshwater prawn) in the sequence listing; A3) The amino acid sequence has the sequence shown in SEQ ID 3 (Macrobrachium rosenbergii) in the sequence listing; A4) The amino acid sequence has the sequence shown in SEQ ID 4 (Litopenaeus vannamei) in the sequence listing; A5) The amino acid sequence has the sequence shown in SEQ ID 5 (penis tiger prawn) in the sequence listing; A6) The amino acid sequence has the sequence shown in SEQ ID 6 (Japanese shrimp) in the sequence listing; A7) The amino acid sequence has the sequence shown in SEQ ID 7 (Chinese white shrimp) in the sequence listing; A8) The amino acid sequence has the sequence shown in SEQ ID 8 (Chinese mitten crab) in the sequence listing; A9) The amino acid sequence has the sequence shown in SEQ ID 9 (swimming crab) in the sequence listing; A10) The amino acid sequence has the sequence shown in SEQ ID 10 (shrimp burrowing crab) in the sequence listing; A11) The amino acid sequence has the sequence shown in SEQ ID 11 (red swamp crayfish) in the sequence listing; The amino acid sequence A12 has the sequence shown in SEQ ID 12 (Procambarus clarkii) in the sequence listing; The amino acid sequence A13 has the sequence shown in SEQ ID 13 (American lobster) in the sequence listing; The amino acid sequence A14 has the sequence shown in SEQ ID 14 (Ornamental Lobster) in the sequence listing; The amino acid sequence A15 has the sequence shown in SEQ ID 15 (large flea) in the sequence listing; The amino acid sequence A16 has the sequence shown in SEQ ID 16 (Artemia salina) in the sequence listing; The amino acid sequence shown in A17)A1)-A16) is obtained by substitution and / or deletion and / or addition of at least one amino acid residue and has the same function, or is a protein that has more than 70% identity with the protein shown in A1)-A16) and has the same function.

[0012] In the above-mentioned proteins, identity refers to the identity of the amino acid sequence. The identity of the amino acid sequence can be determined using homology search sites on the Internet, such as the BLAST calculation on the NCBI homepage. The identity and positives values ​​are obtained through BLAST calculation, and identity greater than 70% means that the positives value is greater than 70%.

[0013] The nucleotide sequence encoding the CERP1 gene of the crustacean is any one of the following (B1-B17): B1) The coding sequence of the coding strand is the cDNA molecule or DNA molecule of SEQ ID 17 (white shrimp) in the sequence listing; B2) The coding sequence of the coding strand is the cDNA molecule or DNA molecule of SEQ ID 18 (Japanese giant freshwater shrimp) in the sequence listing; B3) The coding sequence of the coding strand is the cDNA molecule or DNA molecule of SEQ ID 19 (Macrobrachium rosenbergii) in the sequence listing; B4) The coding sequence of the coding strand is the cDNA molecule or DNA molecule of SEQ ID 20 (Litopenaeus vannamei) in the sequence listing; B5) The coding sequence of the coding strand is the cDNA molecule or DNA molecule of SEQ ID 21 (penalena) in the sequence listing; B6) The coding sequence of the coding strand is the cDNA molecule or DNA molecule of SEQ ID 22 (Japanese shrimp) in the sequence listing; B7) The coding sequence of the coding strand is the cDNA molecule or DNA molecule of SEQ ID 23 (Chinese white shrimp) in the sequence listing; B8) The coding sequence of the coding strand is the cDNA molecule or DNA molecule of SEQ ID 24 (Chinese mitten crab) in the sequence listing; B9) The coding sequence of the coding strand is the cDNA molecule or DNA molecule of SEQ ID 25 (swimming crab) in the sequence listing; B10) The coding sequence of the coding strand is the cDNA or DNA molecule of SEQ ID 26 (scutellaria barbata) in the sequence listing; B11) The coding sequence of the coding strand is the cDNA molecule or DNA molecule of SEQ ID 27 (red swamp crayfish) in the sequence listing; B12) The coding sequence of the coding strand is the cDNA molecule or DNA molecule of SEQ ID 28 (Procambarus clarkii) in the sequence listing; B13) The coding sequence of the coding strand is the cDNA molecule or DNA molecule of SEQ ID 29 (American lobster) in the sequence listing; B14) The coding sequence of the coding strand is the cDNA molecule or DNA molecule of SEQ ID 30 (Ornamental lobster) in the sequence listing; B15) The coding sequence of the coding strand is the cDNA or DNA molecule of SEQ ID 31 (large flea) in the sequence listing; B16) The coding sequence of the coding strand is the cDNA or DNA molecule of SEQ ID 32 (Artemia salina) in the sequence listing; The DNA molecule that has 80% or more identity with the nucleotide sequence defined by B17 and B1-B16 and encodes the protein described above.

[0014] An application of the substance, specifically its use in regulating carotenoid content in crustaceans.

[0015] The CERP1 gene of the crustacean and its encoded protein have any one of the following applications: C1-C5. C1) The application of the protein and / or gene described therein in regulating the accumulation of carotenoids such as astaxanthin in crustaceans; C2) The application of the protein and / or gene described therein in increasing the content of carotenoids such as astaxanthin in crustaceans; C3) Application of the proteins and / or genes described therein in the genetic breeding of crustaceans; C4) Application of the proteins and / or genes described herein in the genetic improvement of quality traits in crustaceans; C5) Application of the proteins and / or genes described therein in the improvement of body color traits in crustaceans.

[0016] A method for cultivating new crustacean germplasm that is significantly enriched in astaxanthin and other carotenoids, wherein the new crustacean germplasm is used to inhibit the activity of the protein and / or the expression level of the gene encoding the protein in the target crustacean.

[0017] The inhibition is achieved by knocking out the gene encoding the protein.

[0018] Gene knockout techniques include CRISPR / Cas9, TALEN technology, and zinc finger nuclease (ZFN) technology. Gene knockdown techniques include siRNA, shRNA, sgRNA, miRNA, or antisense RNA. Alternatively, protein-level knockout can be achieved by blocking CERP1 or its homologous proteins in the target crustacean with antibodies or using inhibitors to inactivate the protein.

[0019] A method for cultivating new germplasm of whiteleg shrimp enriched with astaxanthin and other carotenoids by knocking out the CERP1 gene involves mixing guide RNA with Cas9 protein and injecting it into whiteleg shrimp fertilized eggs. This allows the DNA sequence between the two guide RNA recognition sites in the fifth exon of the CERP1 gene to be recognized and cut, thereby knocking out the CERP1 gene of whiteleg shrimp and obtaining new whiteleg shrimp germplasm enriched with carotenoids.

[0020] The guide RNA has the target sequence of CERP1-gRNA1 as: TCTGTTACTACCATTTAT, and the target sequence of CERP1-gRNA2 as: TAGTGTTCTCTCAATACC.

[0021] Specifically as follows: (1) gRNA design and gene editing targeting the CERP1 gene of white shrimp. Based on the CERP1 gene ORF sequence and genomic DNA sequence of *C. prawn*, two gRNAs (CERP1-gRNA1 and CERP1-gRNA2) targeting the fifth exon region were designed and synthesized. The target sequence of CERP1-gRNA1 is: TCTGTTACTACCATTTAT, and the target sequence of CERP1-gRNA2 is: TAGTGTTCTCTCAATACC. The gRNA synthesis method is as follows: using the guide RNA framework plasmid pMD19-gRNA (by ligating “GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTT” into the TA cloning site of the pMD19-T vector (TaKaRa, D102A) to obtain the gRNA framework plasmid pMD19-gRNA) as a template, CERP1-sgRNA1-F: TAATACGACTCACTATAGGTCTGTTACTACCATTTATGTTTTAGAGCTAGAAATAGC Using CERP1-sgRNA2-F: TAATACGACTCACTATAGGTAGTGTTCTCTCAATACCGTTTTAGAGCTAGAAATAGC as the forward primer and gRNA-R: AAAAAAGCACCGACTCGGTGCCA as the reverse primer, PCR amplification of the gRNA template was performed. The PCR template of the guide RNA was then transcribed in vitro to synthesize guide RNAs CERP1-sgRNA1 and CERP1-sgRNA2, respectively. The PCR products of the gRNAs were then transcribed in vitro to synthesize the guide RNAs (CERP1-gRNA1 and CERP1-gRNA2). Subsequently, the two gRNAs were mixed with Cas9 protein and injected into fertilized eggs of *Litopenaeus vannamei*. This mixture allowed the gRNAs to recognize and cleave the DNA sequence between the two guide RNA recognition sites in exon 5 of the CERP1 gene.

[0022] (2) Screening of CERP1 gene-editing mutants. Using CERP1-exon5-F: CCAATTGGTAACCATATCGGACG and CERP1-exon5-R: ATTCAGGCTGACAGTTCATAGCT as primers, gene-edited individuals were amplified by PCR, and then sequenced using the Sanger sequencing method. The sequencing map was viewed using Chromas software, and heterozygous or homozygous individuals with gene editing were screened from it.

[0023] (3) Phenotypic analysis of homozygous CERP1 gene-edited mutants. The biallelic mutants (HomM) obtained above showed significant differences from wild-type individuals at the larval stage. The homozygous mutants were entirely red, while the wild-types were white (Figure 5). Targeted metabolomics analysis of the carotenoid content in the homozygous mutants and wild-type individuals showed that the astaxanthin and β-carotene content in the mutants was significantly increased, with astaxanthin being 18.5 times that of the control.

[0024] Since small shrimp do not contain pigments such as zeaxanthin and lutein, no enrichment of these pigments was detected in the homozygous mutant of the CERP1 gene. However, if the mutant is fed algae powder containing these carotenoids, the related pigments will also be enriched.

[0025] Advantages of this invention: 1. This invention is the first to identify CERP1, a gene that negatively regulates the enrichment of carotenoids in crustaceans. Knocking out or inhibiting this gene can increase the content of carotenoids in crustaceans by more than 5 times, providing a technical solution for the breeding of high-quality crustaceans.

[0026] 2. The technical solution provided by this invention can obtain new crustacean germplasm with astaxanthin and other carotenoid enhancements in only 1-2 generations. By using gene knockout or inhibition technology to knock out the CERP1 gene, the upper limit of carotenoid content in wild populations is broken, and there is no significant impact on the growth, survival and other traits of individuals. It is a precise and efficient solution for improving quality traits.

[0027] 3. The guide RNA targeting the CERP1 gene of white shrimp provided by this invention can efficiently achieve gene editing and rapidly improve the enrichment traits of carotenoids such as astaxanthin in white shrimp. Attached Figure Description

[0028] Figure 1 The images show a comparison of the appearance and quality of the new carotenoid-enriched variety "Kesuhong No. 1" of white shrimp and a wild variety, provided in this embodiment of the invention. Among them, A is a comparison of the appearance of the whole shrimp and muscle of the white shrimp "Kesuhong No. 1" and the common variety; B is the measured value of carotenoids in the muscle of the white shrimp "Kesuhong No. 1" and the common variety.

[0029] Figure 2 The expression differences of the CERP1 gene in the carotenoid-enriched mutant "Kesuhong 1" and wild-type individuals provided in this embodiment of the invention.

[0030] Figure 3The expression differences of the CERP gene in different tissues of the carotenoid-enriched mutant "Kesuhong 1" and wild-type individuals provided in this embodiment of the invention; wherein, Red represents Kesuhong 1, White represents common white shrimp, Epi: epidermal tissue, In: intestinal tissue, Vn: abdominal nerve tissue, Hep: hepatopancreas tissue, Ms: muscle tissue, Gi: gill tissue, St: stomach tissue, Ht: heart tissue, Tg: thoracic nerve tissue, Hc: blood cells, Es: eyestalk tissue.

[0031] Figure 4 The CERP1 gene sequencing results of wild-type and gene-edited individuals of white shrimp provided in the embodiments of the present invention.

[0032] Figure 5 The body color of the CERP1 gene bicelestem mutant of white shrimp provided in this embodiment of the invention is reddish; wherein, Wild represents wild white shrimp; HomM represents the EcCERP1 gene bicelestem mutant.

[0033] Figure 6 The CERP1 gene knockout provided in this embodiment of the invention resulted in a significant increase in the content of astaxanthin and other carotenoids in wild-type individuals.

[0034] Figure 7 The conserved amino acids and domains of the CERP1 gene in crustaceans are provided in the embodiments of the present invention.

[0035] Figure 8 Homologous genes of the CERP1 gene in crustaceans and their phylogenetic tree provided for embodiments of the present invention. Detailed Implementation

[0036] The following examples further illustrate specific embodiments of the present invention. It should be noted that the specific embodiments described herein are merely for illustration and explanation and are not intended to limit the scope of the present invention.

[0037] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0038] Example 1: Discovery and genetic analysis of CERP1, a carotenoid enrichment gene in white shrimp.

[0039] 1.1 Detection of carotenoid content in the red natural mutant of *Sinosaurus spp.* Obtaining white shrimp (Scaenopsis spp.) through breeding Exopalaemon carinicaudaA red mutant was bred and cultivated into a new variety called "Kesuhong No. 1" (GS-01-004-2017) through multiple generations of selective breeding. Tests showed that the astaxanthin content of this variety was 5-10 times that of ordinary wild varieties (Zhang, C. et al. 2018. Isolation and identification of the main carotenoid pigment from a new variety of the ridgetail white prawn Exopalaemon carinicauda. Food Chem 269, 450-454). In order to evaluate whether "Kesuhong No. 1" has the ability to accumulate different carotenoids, a feeding experiment based on algal powder containing multiple carotenoids was carried out. After a two-week feeding period with carotenoids (final concentrations of astaxanthin 400 ppm, lutein 80 ppm, zeaxanthin 40 ppm, canthaxanthin 13 ppm and β-carotene 40 ppm), the carotenoid content in the muscle tissue of wild-type (white) and "Kesuhong No. 1" white shrimp was analyzed by HPLC. Figure 1 The results showed that the content of astaxanthin and other carotenoids in "Kesuhong No. 1" was significantly increased: the astaxanthin content in the muscle tissue of "Kesuhong No. 1" was 6 times that of the wild type, the β-carotene content was 18 times that of the wild type, and the total carotenoid content was 10 times that of the wild type. Meanwhile, zeaxanthin and lutein, which were not detected in the wild type, reached 23 mg / Kg and 4 mg / Kg, respectively, in "Kesuhong No. 1". These results indicate that the red-body mutant individuals have significantly enhanced abilities to accumulate astaxanthin, zeaxanthin, lutein, and β-carotene, with astaxanthin being the main enriched carotenoid.

[0040] 1.2 Construction of carotenoid enrichment trait localization populations and whole-genome resequencing analysis To elucidate the genetic basis of this carotenoid accumulation trait, a hybridization experiment was conducted between wild-type and "Kesuhong No. 1" individuals, and F2 generation families and pseudo-backcross families were further established. The results showed that all F1 offspring from the hybridization of "Kesuhong No. 1" and wild-type white shrimp exhibited a white phenotype, while the F2 generation showed a red to white segregation ratio of approximately 1:3; and the pseudo-backcross offspring showed a 1:1 segregation ratio. These results indicate that the carotenoid accumulation trait is a qualitative trait controlled by a recessive gene.

[0041] To identify key genes controlling carotenoid accumulation, whole-genome resequencing was performed on parents and offspring (n=202) of two backcross families. SNP genotyping was performed using the GTX.CAT acceleration program in GATK software with default parameters. Strict filtering was applied to the obtained SNP loci, specifically: "FS>60.0 || MQ<40.0 || QD<2.0 || SOR>3.0". Further filtering using VCFtools removed SNPs with minor allele frequencies (MAF) below 5% and SNPs with a population genotype deletion rate greater than 10%. A total of 6,111,171 high-quality SNP loci were obtained through this filtering process.

[0042] Using these SNP loci, an ultra-high-density genetic linkage map was constructed using the LepMap3 software with default parameters. The final map contained 126,396 markers, including 46 linkage groups, with an average map distance of 0.011 centimoles (cM), and was highly collinear with the genome map.

[0043] 1.3 Comparative transcriptome analysis of “Kosuhong No. 1” and common white shrimp The materials used for comparative transcriptome sequencing were individuals of the "Kosuhong 1" and common white shrimp preserved in our laboratory. The cephalothorax of 30 "Kosuhong 1" and 30 common white shrimp were collected separately. Ten individuals of "Kosuhong 1" were pooled into one sample, and ten individuals of common white shrimp were pooled into one sample, resulting in three pooled samples of "Kosuhong 1" and three pooled samples of common white shrimp. Total RNA was extracted from these samples using RNAiso Plus (Takara, catalog number 9108) to construct transcriptome sequencing libraries, and high-throughput sequencing was performed using the Illumina platform. The obtained clean data were aligned to the assembled reference genome using Hisat2 software (version 2.2.1, default parameters), and the gene expression differences between "Kosuhong 1" and common white shrimp were analyzed using edgeR (version 3.40.2, default parameters).

[0044] 1.4 Localization of genes for carotenoid enrichment traits To precisely locate the key genes for carotenoid enrichment traits in white shrimp, the transcriptomes of all genes in "Kesuhong No. 1" and common white shrimp were analyzed. Only one gene, CERP1, showed a highly significant difference, with its expression level significantly reduced in "Kesuhong No. 1". Figure 2 Furthermore, one SNP site on this gene (CERP1_SNP1) is completely linked to the astaxanthin enrichment trait (Table 1), indicating that this gene is a key gene for the carotenoid enrichment trait.

[0045] >SEQID33 CERP1_SNP1: AAGCCCAGTCACGTTGCAGAAAAAGTTTCAAGGCACATAAAAATGCTACGCATTTCTTATAAATAGAGAACATGGTTACCAATTATGATATGA[T / A]AAGCACAAAAATTTAAAGTTCGCTTATTCTGCTCTATTCAGGCTG.

[0046] Table 1. Correlation between SNPs on the CERP1 gene and carotenoid enrichment traits. 1.5 The expression of CERP1 in "Kesuhong No. 1" was significantly correlated with astaxanthin enrichment. Further analysis was conducted on the expression of CERP1 in different developmental stages and tissues of the 'Kesuhong 1' shrimp. Epidermis, intestine, abdominal nerves, hepatopancreas, muscle tissue, gills, stomach tissue, heart, thoracic nerves, hemocytosis, and eyestalk tissue were collected from both 'Kesuhong 1' and common white shrimp. Each sample consisted of three tissue samples, with three replicates per tissue from each shrimp species. Total RNA was extracted from the samples using RNAiso Plus (Takara, catalog number 9108). Quantitative real-time PCR was then performed using primers EcCERP1qF: CTATGCGAATGAATAAGATGAGGAG (SEQ ID 34) and EcCERP1qR: CCAGCGTACCAAGTAATACTGAAA (SEQ ID 35) to detect tissue expression of the EcCERP1 gene in both shrimp species. The results showed that, compared to the high expression in common white shrimp, CERP1 expression was extremely low in all tissues of 'Kesuhong 1'. Figure 3 This further confirms the close relationship between this gene and astaxanthin enrichment.

[0047] Example 2: Knocking out the CERP1 gene in white shrimp leads to the accumulation of astaxanthin and other carotenoids in vivo. Using the white shrimp as a representative of crustaceans, gene editing technology was used to delete the function of the CERP1 gene, resulting in new germplasm enriched with astaxanthin and other carotenoids.

[0048] 2.1 Negative regulatory genes for astaxanthin enrichment trait in white shrimp EcCERP1 Guide RNA (gRNA) synthesis a) gRNA framework construction First, based on the structure of the gRNA, the gRNA framework sequence was designed and synthesized: GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTT (SEQ ID 36).

[0049] The above gRNA framework sequence was then ligated into the pMD19-T vector (TaKaRa, D102A) to obtain the gRNA framework plasmid pMD19-gRNA.

[0050] b) Synthesis of gRNA template Use the online tool CRISPRdirect (http: / / crispr.dbcls.jp / ) to select... EcCERP1 Design corresponding gRNA sites of genes EcCERP1 -gRNA1-F and EcCERP1 -gRNA2-F, and simultaneously designed the reverse primer gRNA-R. EcCERP1 -gRNA1-F:TAATACGACTCACTATAGGTCTGTTACTACCATTTATGTTTTAGAGCTAGAAATAGC (SEQID37), EcCERP1 -gRNA2-F: TAATACGACTCACTATAGGTAGTGTTCTCTCAATACCGTTTTAGAGCTAGAAATAGC (SEQID38), gRNA-R: AAAAAAGCACCGACTCGGTGCCA (SEQID39).

[0051] Using the gRNA framework plasmid pMD19-gRNA as a template, respectively with EcCERP1 Using gRNA1-F and EcCERP1-RNA2-F as forward primers and gRNA-R as the reverse primer, PCR amplification was performed using ExTaq polymerase (Takara, RR001A). The PCR cycling program was: 98℃ pre-denaturation for 5 min; 98℃ for 10 sec, 55℃ for 5 sec, 72℃ for 30 sec, 35 cycles; 72℃ extension for 10 min; and incubation at 16℃. After detecting the amplified gRNA template size by agarose gel electrophoresis, the PCR products were sequenced. The remaining PCR products were used as templates for in vitro gRNA transcription.

[0052] 2.2 In vitro transcription and purification of gRNA gRNA was synthesized by in vitro transcription of the PCR product of gRNA using the TranscriptAid T7 High Yield Transcription Kit (Thermo, K0441).

[0053] The in vitro transcription system is shown in Table 2.

[0054] Table 2 In vitro transcription system The transcription system was incubated at 42℃ for 2 h; 2 μL of DNase (QIAGEN, 79254) was added and the mixture was digested at 37℃ for 15 min to remove the DNA template; 2 µL of 0.5M EDTA (pH 8.0) was added and the mixture was incubated at 65℃ for 10 min to terminate the reaction; 1 μL was used for electrophoresis detection. gRNAs EcCERP1-gRNA1 and EcCERP1-gRNA2 were obtained from the digested system using the phenol / chloroform extraction method (Lee S. Toni, et al., Optimization of phenol-chloroformRNA extraction, MethodsX, 2018, 5: 599-608).

[0055] 2.3 Gene Editing Experiments on Wild White Shrimp (Litopenaeus vannamei) Fifteen wild white shrimp of male and female morphology with well-developed gonads were reared together. After the female shrimp laid eggs, the newly laid fertilized eggs were collected.

[0056] Will EcCERP1 -gRNA1 and EcCERP1 -gRNA2 is mixed with commercially available Cas9 protein (GenScript, Z03469, manufactured by Nanjing GenScript Biotech Co., Ltd., or GenScript Biotech Inc.) to make... EcCERP1 -gRNA1, EcCERP1 The final concentrations of both gRNA2 and Cas9 proteins were 1000 ng / μL, and the injection volume per fertilized egg was approximately 0.5 nL.

[0057] The injected embryos were placed in a culture dish containing sterile seawater and incubated on a shaker at room temperature, with the sterile seawater changed three times a day. Gene editing efficiency was assessed when the fertilized eggs developed into adults.

[0058] 2.4 Screening and Phenotypic Identification of Mutants Genomic DNA was extracted from shrimp walkers using lysis buffer microextraction. EcCERP1 The genomic regions flanking the CRISPR target sites of the two sgRNAs of the gene were amplified by PCR.

[0059] The detection primers used are as follows: EcCERP1 -exon5-F: CCAATTGGTAACCATATCGGACG (SEQID40); EcCERP1 -exon5-R:ATTCAGGCTGACAGTTCATAGCT (SEQID41).

[0060] The PCR amplification system is shown in Table 3.

[0061] Table 3 PCR amplification system The PCR amplification conditions are as follows: Pre-denaturation at 94℃ for 5 min; denaturation at 98℃ for 10 sec, annealing at 57℃ for 30 sec, extension at 72℃ for 50 s, 35 cycles; extension at 72℃ for 10 min; hold at 16℃.

[0062] The products obtained from amplifying the target sequence were detected by 1% agarose gel electrophoresis, followed by sequencing using Sanger sequencing. Editing efficiency was calculated, revealing that approximately 21% of the adult EcCERP1 gene showed genetic mutations at the guide RNA recognition site, including 11 biallelic mutants (HomM). Analysis of the 11 biallelic mutants (HomM) obtained through gene editing showed large nucleotide sequence deletions compared to unedited wild-type individuals (WT) (see [link to relevant documentation]). Figure 4 The mutant's body color also changed, turning orange-red. Figure 5 ).

[0063] The carotenoid content in the muscle of the biallelic mutant (HomM-Ms) was measured and compared with that in the muscle of wild white shrimp (Wild-Ms). The results showed that the astaxanthin content in the mutant muscle was significantly higher than that in wild white shrimp. Figure 6 ).

[0064] Example 3: CERP1 is a conserved carotenoid enrichment-related gene in crustaceans. 3.1 Identification of CERP1 homologs in other crustaceans Using the amino acid sequence of the CERP1 gene in *Litopenaeus vannamei* as the query sequence, the protein alignment tool Blastp (default parameter) from NCBI was used to identify homologous protein sequences. The results showed that it primarily aligned with genes from crustaceans, including *Litopenaeus vannamei*, *Litopenaeus monodon*, *Litopenaeus japonicus*, *Litopenaeus chinensis*, *Macrobrachium rosenbergii*, *Mammillaria elongata*, *Portunus trituberculatus*, *Muscovy crab*, *Procambarus clarkii*, *Lobster*, *Rhizoctonia clarkii*, *Krill*, and *Amphioxus*, as well as *Artemia* and *Daphnia* from the class Branchiopoda, and copepods and barnacles from the class Maxillopoda. The highest similarity was found with *Macrobrachium rosenbergii* and *Macrobrachium rosenbergii*, both belonging to the same family (Paramyidae), with sequence identity exceeding 80%. Other decapod crustaceans, including Litopenaeus vannamei, Penaeus monodon, Penaeus japonicus, Litopenaeus chinensis, Eriocheir sinensis, Portunus trituberculatus, and Scylla serrata, all share more than 60% CERP1 gene identity with Litopenaeus vannamei, confirming that they are more conserved in the decapod order.

[0065] 3.2 Domain analysis and cluster analysis of CERP1 homologous genes The amino acid sequences encoding other crustacean CERP1 homologs identified by Blastop alignment were subjected to multiple sequence alignment using ClustalW in Bioedit software. Interproscan and SMART software were used for domain prediction. SMART prediction showed that these sequences all contained four transmembrane domains. The multiple sequence alignment results are as follows: Figure 7 As shown, the amino acid sequences of its four transmembrane regions are relatively conserved, all possessing the conserved sequence L(F)LL(M)PYIV. Interproscan prediction results show that all CERP1 genes contain the DUF4728 domain, which annotation indicates is only found in arthropods, and its current function is unknown. However, the results of this patent confirm that the CERP1 gene with the DUF4728 domain plays an important role in the enrichment of carotenoids such as astaxanthin.

[0066] Multiple sequence alignment of the crustacean CERP1 gene was performed using MEGA 7.0, and a phylogenetic tree was constructed. Figure 8 The Japanese giant freshwater prawn and the giant freshwater prawn, which are closely related to the whiteleg shrimp in the infraorder Caridea, cluster together. Following them are the Chinese mitten crab, the swimming crab, and the mud crab from the infraorder Eubrachyura, which cluster together. The Litopenaeus species, including the giant whiteleg shrimp, the Japanese whiteleg shrimp, and the Chinese whiteleg shrimp, cluster together. The spiny lobster and the red-clawed lobster cluster together. Artemia and water fleas from the class Branchiopoda, and barnacles from the class Maxillopoda, are located on an outgroup. Figure 7The sequences of the above genes all have a four-transmembrane protein structure and contain the DUF4728 domain, demonstrating that the gene has a relatively conserved sequence structure and function in crustaceans.

[0067] The above results demonstrate that the CERP1 gene is a conserved "carotenoid enrichment" gene in crustaceans. Therefore, homologous genes of other crustaceans obtained through homology comparison have similar functions. Gene editing and other operations on these homologous genes of other crustaceans will also result in "carotenoid" enrichment. Therefore, the relevant genes also fall within the scope of protection of this patent.

[0068] Sequence List: >SEQID1 XP_068248754.1 uncharacterized protein [Palaemoncarinicauda] Ridgetail white shrimp MPLLRGCCFCYDLKVGSKIIGILSLVGALLNSLTLAGTLVIFVFLGLGADAVNTLSKHLEKAANDYEDEYVDVREDQPPTIMELFLAHITSVTIVLYVLLALCIFMIITSSMLIHGVKNDRRGLLLPFIGQEAVNAIVFLSLFIWILATFGTDTKVIGMAISILLGTLVRGYFALVVFSQYQALGLIRMHEEHSMK.

[0069] >SEQID2 XP_064118386.1 uncharacterized protein LOC135223659 [Macrobrachium nipponense] Japanese prawn MPILSGCCFCFDLKVGAKIIGVLNLIGALMNTLTLAGTLVVFVIMGLGTDTANKLSKTFEAKVDDYEDEYVDVREEQGPNVMEIFLDKISTITIFLYILLAVCILMVITSSMLIHGVKNDRRGLLLPYVCQEVFNVIIFFALFIWILVTFGTDIRVVGTALSMLGAVLLHIYFALVVFSQYQALGLIRMHEEHSMK.

[0070] >SEQID3 XP_066966301.1 uncharacterized protein [Macrobrachium rosenbergii] Giant freshwater prawn MPILSGCCFFFDLKVGTKIIGVLNLIGALVHTLALAGTLVMFVIMGLGADSVNKLSKAFEEKVDDYEDEYVDVREEQGPNIMEIFLDHISTVTIFLYILLAVCILMVITSSMLIHGVKNDRRGLLLPYLCQEAFNIIVFFTLFIWILVIFGTDIRVVGTALSILGAVLLHIYFALVVFSQYQALGLIRMHEEHSMK.

[0071] >SEQ ID 4 XP_069982705.1 uncharacterized protein [Penaeus vannamei] *Penaeus vannamei* MPILSGCCFCFDLKVGSTIIGALNLIGALVNSIMFAILLVMVALFGAGTRKALEDPVFNEDDDYADVKEEQKQAIELVLEHMEAVKLALYILLGVSLLCVVTSSMLIHGVRRKRRGLLLPYIVQEVINIIIFIVLIIGTLVIVGTHQIIVSAVVGVIGGILIHLYFMLVVISQYQALGLIRMHEEISMK.

[0072] >SEQ ID 5 XP_037782533.1 uncharacterized protein LOC119578930 [Penaeus monodon] *Penaeus monodon* MPILSGCCFCFDLKVGSTIIGALNLVGALIYSVMFAILLVMVALFGAGTRKALEDPVFNEDDEYSDVREEQKQIIEVVLEHMEAVKLALYILLGVSLLCVITSSMLIHGVRRKRRGLLLPYIVQEVINIIIFIALTIGALVILGTHRVVVSSVIGVIVGLFVHLYFMLVVISQYQALGLIRMHEEISMK.

[0073] >SEQ ID 6 XP_042882200.1 uncharacterized protein LOC122259473 isoform X1 [Penaeus japonicus] *Penaeus japonicus* MPILSGCCFCFDLKVGSTIIGALNLIGALMNSIMFAILLVMVALFGAGTRKVLEDPAFLEDDDYADVKEEQKQILELVLDHMDAVKLALYILLGISLLCVVTSSMLIHGVRRKRRGLLLPYIVQEVINIIVFIALIIGTFVILGTHKVVVSSMIGVIGGILIHLYFMLVVISQYQALGLIRMHEEISMK.

[0074] >SEQ ID 7 XP_047468648.1 uncharacterized protein LOC125024917 [Penaeus chinensis] *Fenneropenaeus chinensis* MNSIVFAILLVMVALFDAGAKKSLDPVFTEDDDYADIREEQKQITEVVLDHMDAVKLALYILLGVSLMCVVTSSMLIHGVRRKRRGLLLPYIVQEVINILVFLALIIGTLVILGTHRIIVSSVLGVVVGLLMHLYFMLVVISQYQALGLIRMHEEISMK.

[0075] >SEQ ID 8 XP_050688139.1 uncharacterized protein LOC126981281 [Eriocheir sinensis] *Eriocheir sinensis* MPLLSNCCFFFDLKVGTKIIGVIKLIGAIMTSLTLAVMLVFLLFVDSSVDLASSIHEVHRNSLKDDDYADVGDQPKQIFDLVKEHLSYVKIAFCIVLTLGLLSVITSSMLIHGVRRDRRGLLLPFITQEVLNIVIFIALDISVAVIFGVPDVLIGLVLGVAGGCIIELYFLLVVISQYQALGVLRMHEEISMK.

[0076] >SEQ ID 9 XP_045112212.1 uncharacterized protein LOC123505155 [Portunus trituberculatus] *Portunus trituberculatus* MPILGSCCICVDLKVGTKIIGILNLIGAIFSSLSTAISLVMVMFLDAGVSTAKVINEIHRSRLNENDDYVDTQDQPKQIMELVQEHLSYVKVALYVMLALALLLIITSSMLIHGVRRDRRGLLLPFIVQQVVNIVVFVTLDIIVLVIFGAHKVIISSVLSMLVGCLIQLYLVLVVVSQYQALGLIRMHEEISMK.

[0077] >SEQ ID 10 XP_063848297.1 uncharacterized protein LOC135093233 [Scylla paramamosain] Scylla paramamosain MGRAAILTGGHQTRAPVGGKRARREVVVGGAATFFTFLTKFRVSLAANMPILGSCCICLDLKVGTKIIGILNLIGAIFSSLSMAISLVMVMFLDAGVSTAKAVNEIHRSRMNDDDYMDTQDQPKQIMELVQEHLSYVKIALYVMLALALLLIITSSMLIHGVRRDRRGLLLPFIVQQLVNIVVFVGLDIAVLVLFGAHKVIIGSVLSMLVGCLIQLYLVLVVVSQYQALGLIRMHEEISMK.

[0078] >SEQ ID 11 KAK8719748.1 hypothetical protein OTU49_013820, partial [Cherax quadricarinatus] Cherax quadricarinatus MPVLSGCCCFDLKVGTTIIGVLNLIVGLANSLAYAGFLVMLVLLSNNRTHIENSFQPTPHPSFDDDDEYLDIPEEDKHLLELIMDNIDLVKIVIYILLGASLLIVITASMLIHGVRQNRRGLLIPFVIQDVINLLLLCAFAVLALVVLGTSMVIVIIVIVIFVVILIKVYFLMVVISQYQALGLIRMHEEISMK.

[0079] >SEQ ID 12 XP_069181455.1 uncharacterized protein [Procambarus clarkii] Procambarus clarkii MTLLKKCCCCCFDLRAGSLAIGVISFLCIGVVQLLVAIYCLLHVEDTKKRLKFENAVTFGIVDPESDAAAYIFFSILLLVAIIYIIAALVLIHGVRTKRRFLLLPWVFFTFLNMVMAFAGGILTLINSHGYSLQIIVGCIVLSISIIWIYFTCVVFSFFQALQNGEYW.

[0080] >SEQ ID 13 XP_042215113.1 uncharacterized protein LOC121861445 [Homarus americanus] American lobster MPVLSGCCFIFDLKVGTKIIGILSLIGALLNSLTFAGVAVMLVFIGTATDSASQLHHFANTELHGSHLNEDDEYTDSWEEQVEGILELIKAHIQMVQILVYLMLAIALVQIVTSSMLIHGVKHSRRGLLLPYLVITTIGILLCIIMGIVMFAVLGNASMIVSSAASMLVGAGLQVYFMLVVFSQYQALGLIRMHEDMSMK.

[0081] >SEQ ID 14 XP_071527344.1 uncharacterized protein [Panulirus ornatus] Ornate spiny lobster MPILSSCCFCLDLRVGTKIIGALKLIGSLMSCIMCACTLVLLMFVGVGIEETRKSRPTPDPLEADDDYIDIREQQENALEWLKDHIDMIKILLYIFLGLSLLLVITTSMLIHGVRRNRQGLLLPFIVQESIMLVVYVLLIILSLVSLGSKMPIIYCDITFVVSFVVDLYFLLVVISQYQALGLIRMHEEISMK.

[0082] >SEQ ID 15 EFX85204.1 hypothetical protein DAPPUDRAFT_300228 [Daphnia pulex] Daphnia pulex MICNKLSKCCCCMSLQTGTKIVGVFSMLGGIGGLALSLAQLTSFINAEYVIGGEILPSSLNQLTGSLVVIAFTASVLVAASKNGKPMLLVPWLVLKSISLFVAIFYSFYFGVSSVMTGDDQGMSYVSVLVIGIVLYSYFWLVVYSYYHELVTGMKKEALINEKC.

[0083] >SEQ ID 16 XP_065579397.1 uncharacterized protein LOC136039522 [Artemia franciscana] Artemia MPLLRRCCCYFPLRRASVTLGVIGFTGSITSLIIVIIGRILVEDVANGVMSLFRKVTDVPYMMGTRHLSESEQEEQEQKLVEYWIDVYKILFIVCFIGMVISCIFSGLMVYGSVKSRKMLLVPWLVLGAINILGLITLVIVNMIYIDLPYNLIVLFLGIFCVSFMIHFWLVVVSFYQVLRDRERLELGGRSSEMKRLNRNY.

[0084] >SEQ ID 17 XM_068392653.1 PREDICTED: Palaemon carinicauda uncharacterized protein (LOC137657941), mRNA [Palaemon carinicauda]

[0085] >SEQID18 XM_064262316.1 PREDICTED: Macrobrachium nipponenseuncharacterized LOC135223659 (LOC135223659), mRNA [Japanese giant prawn]

[0086] >SEQID19 XM_067110200.1 PREDICTED: Macrobrachium rosenbergiiun characterized protein (LOC136842612), mRNA [Macrobrachium rosenbergii]

[0087] >SEQID20 c52357 g1 Penaeus vannamei [Litopenaeus vannamei]

[0088] >SEQID21 XM_037926605.1 PREDICTED: Penaeus monodon uncharacterizedLOC119578930 (LOC119578930), mRNA [Penaeus monodon]

[0089] >SEQID22 XM_043026266.1 PREDICTED: Penaeus japonicus uncharacterizedLOC122259473 (LOC122259473), transcript variant X1, mRNA [Japanese shrimp]

[0090] >SEQID23 XM_047612692.1 PREDICTED: Penaeus chinensis uncharacterizedLOC125024917 (LOC125024917), mRNA [Chinese white shrimp]

[0091] >SEQID24 XM_050832182.1 PREDICTED: Eriocheir sinensis uncharacterizedLOC126981281 (LOC126981281), mRNA [Chinese mitten crab]

[0092] >SEQID25 XM_045256277.1 PREDICTED: Portunus trituberculatus uncharacterized LOC123505155 (LOC123505155), mRNA [Portunus trituberculatus]

[0093] >SEQID26 XM_063992227.1 PREDICTED: Scylla paramamosainuncharacterized LOC135093233 (LOC135093233), mRNA [Scylla paramamosainuncharacterized]

[0094] >SEQ ID 27 HACB02026761.1 TSA: Cherax quadricarinatus, contig c34572_g1_i1, transcribed RNA sequence [Red Claw Crawfish]

[0095] >SEQID28 XM_069325354.1 PREDICTED: Procambarus clarkii uncharacterized protein (LOC123749671), mRNA Procambarus clarkii

[0096] >SEQID29 XM_042359179.1 PREDICTED: Homarus americanus uncharacterizedLOC121861445 (LOC121861445), mRNA [American lobster]

[0097] >SEQID30 XM_071671243.1 PREDICTED: Panulirus ornatus uncharacterized protein (LOC139754127), mRNA [Papillonus ornatus]

[0098] >SEQ ID 31 XM_046605515.1 PREDICTED: Daphnia pulex uncharacterized LOC124207879 (LOC124207879), mRNA [Daphnia pulex] ATTTGCAGTTCATTCTTAACTTGCTTGCAGGACGGAGAACGCACATAATCACGCTTATTCCCAAACAGCAACATGATTTGTAATAAACTGAGAAAATGCTGCTGTTGTTTGTCACTCCAAACTGGAACGAAAATTGTAGGACTCCTTTCCATGTTGGGTGGAATTGGTGGCCTTGCTCTCAGTCTGGCTCAAGTGATTTCTATTGTTAACTCCGAATATGTTATTGGAAGTGAGATTCTCCTGTCTTCCCTCGATCGCTTGATCGGTTCGCTTGTTGTTATCGCCTTCACCGCTGCTGTACTAGTCGCCGCATGTAAAAATGGCAAGCCGATGCTCCTAGTTCCCTGGCTAGTATTAAAGTCCATCTCGTTGTTCGTGACCATTTTCTACTCGTTTTACGTCGGTGTCAGCTCTGCGGTGACAGGCGATGATCAAGACATGTCTTACGTTTTTGGCCTCGTTATTATCGGAATTGTCCTTTATTCGTACTTATGGTTGGTGGTCTACAGCTACTACCACGAACTAGTGACGGGAATGAAGAAGGAAGCGCTAGTCAACGAAAAATGTTGATTGAAAAAAAAAGATTGTAAAAGAATAAGATTAGTTTTCTATTCCTATATTTTATTAATTCTTTCTAGCTGTTTTTCTTGCATTTCTTTTTGCATTTGGTTTTCTGCCCGCACACGTTCTCGTTAAGTCTGAATATATTTATTAGAACAAA。

[0099] >SEQ ID 32 ENA|KAK2717017|KAK2717017.1 Artemia franciscana hypothetical protein [Artemia] 。

Claims

1. The application of knocking out the CERP1 gene in crustaceans to increase carotenoid content in crustaceans, characterized by: The amino acid sequence of the CERP1 protein is shown in SEQ ID No. 1; The gene encoding the CERP1 protein is a DNA molecule as shown in SEQ ID No. 17; The crustacean in question is the white shrimp with a spiny tail. The carotenoid is one or both of astaxanthin and β-carotene; The technique used to knock out the CERP1 gene is CRISPR / Cas9 gene editing technology.

2. A method for cultivating carotenoid-enriched crustacean germplasm, characterized in that: The method involves knocking out the CERP1 gene in the target crustacean. The amino acid sequence of the CERP1 protein is shown in SEQ ID No. 1; The gene encoding the CERP1 protein is a DNA molecule as shown in SEQ ID No. 17; The crustacean is the white shrimp with a spiny tail; the carotenoid is one or both of astaxanthin and β-carotene. The technique used to knock out the CERP1 gene is CRISPR / Cas9 gene editing technology.

3. A method for cultivating carotenoid-enriched whiteleg shrimp germplasm by knocking out the CERP1 gene, characterized in that: By mixing guide RNA with Cas9 protein and injecting it into fertilized eggs of white shrimp, the DNA sequence between the two guide RNA recognition sites in the fifth exon of the CERP1 gene can be recognized and cut, thereby knocking out the CERP1 gene of white shrimp and obtaining white shrimp germplasm enriched with carotenoids. The carotenoid is one or both of astaxanthin and β-carotene; The guide RNA includes CERP1-gRNA1 and CERP1-gRNA2. The target sequence of CERP1-gRNA1 is: TCTGTTACTACCATTTAT, and the target sequence of CERP1-gRNA2 is: TAGTGTTCTCTCAATACC.

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