A siRNA molecule and a method for reducing the melanin content of the skin tissue of leopard coral grouper

Abstract

The application discloses a method for breeding Ptereleotris radiata by using siRNA molecules interfering with melanin tyr gene expression, wherein the sequence of the siRNA molecules is shown as SEQ ID NO. 6-35, and the siRNA molecules can interfere with the expression of melanin tyr gene in the skin tissue of Ptereleotris radiata. The siRNA sequence is designed and synthesized by using the melanin gene of Ptereleotris radiata, and a powder injection preparation containing the siRNA is obtained. The powder injection preparation is dissolved and injected into the body of Ptereleotris radiata. The individuals with faded melanin in the skin tissue of Ptereleotris radiata are screened out for breeding of parent fish and cross breeding, and the offspring of Ptereleotris radiata is obtained, wherein the skin tissue traits of the offspring are the same as those of the parent fish. By using the breeding method provided by the application, the melanin in the skin tissue of Ptereleotris radiata can be reduced and stably inherited, a foundation for obtaining Ptereleotris radiata varieties is laid, and the economic value of Ptereleotris radiata is improved.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological breeding, and particularly relates to a siRNA molecule and a method for reducing the melanin content of skin tissue of Plectropomus leopardus. BACKGROUND

[0002] Fish, as one of the most diverse vertebrate groups, is known for its rich colors and patterns. These colorful body colors not only constitute important phenotypic traits of fish, but also directly reflect their unique economic value. The diversity of fish body color is derived from the complex distribution and functional differentiation of pigment cells on their body surface, which provides an ideal biological model for scientists to study pigment metabolism and body color formation mechanisms. Among numerous fish species, Plectropomus leopardus, commonly known as the East Star, is a member of the Plectropomus genus in the Serranidae family. Its wild individuals have bright and striking body colors, with a bright red overall color and blue-white circular spots all over the body. This unique combination of body color gives it high ornamental and economic value, making it highly sought after and in high demand on the market. However, during artificial breeding, the body color of P. leopardus often fails to maintain the brightness of wild individuals due to changes in environmental conditions, differences in nutrient intake, and genetic background diversity. Instead, it presents a dull color tone of black or brown. This degradation of body color not only affects its market value but also poses a serious threat to its breeding efficiency. The dull body color of P. leopardus has severely hindered the improvement of its economic value, becoming a key problem that needs to be addressed in the field of aquaculture.

[0003] Melanocyte cells, as the key cell type for fish body color formation, have received extensive attention. Melanocyte cells originate from neural crest cells and, under the dual regulation of nerves and hormones, control the distribution and aggregation of melanin granules to achieve changes in body color depth. The development of animal melanocyte cells is finely regulated by a series of melanin synthesis-related genes. Tyrosinase (tyr) related proteins play a core role in the process of melanin biosynthesis, but tyr family genes have different expression patterns in different tissues among species.

[0004] In the previous study, the research group identified 5 tyr gene family members in Ptereleotris punctatum through bioinformatics analysis, which were tyr, tyr-like, tyrp1a, tyrp1b and tyrp2. The 5 genes were distributed on different chromosomes, and all the family members had multiple conserved regions in the protein structure. Each family member contained a tyrosinase domain and a complete transmembrane helix domain. The expression levels of the tyr gene family in the skin tissue of black Ptereleotris punctatum individuals were significantly higher than those of red individuals, which played an important role in the accumulation of melanin in the skin tissue, indicating that the expression level of the tyr gene family was related to body color and played an important regulatory role in the process of pigment deposition, promoting the synthesis and deposition of melanin.

[0005] siRNA is a short-chain RNA molecule composed of about 20 to 25 nucleotides. The principle of siRNA is to combine with the mRNA of the target gene, leading to the degradation or inhibition of translation of the mRNA, thereby playing a role in gene silencing or gene expression inhibition. However, the function of the tyr gene family members in the synthesis of melanin in the skin tissue of Ptereleotris punctatum still needs further exploration. Therefore, it is necessary to use siRNA to further explore the site of the tyr gene family members that play a major role in the synthesis of melanin in Ptereleotris punctatum, in order to reduce the melanin in the skin tissue of Ptereleotris punctatum, lay a foundation for obtaining good strains of Ptereleotris punctatum, and improve the economic value of Ptereleotris punctatum. SUMMARY

[0006] The purpose of the present application is to further explore the site of the tyr gene family members that play a major role in the synthesis of melanin in Ptereleotris punctatum, in order to reduce the melanin in the skin tissue of Ptereleotris punctatum, lay a foundation for obtaining good strains of Ptereleotris punctatum, and improve the economic value of Ptereleotris punctatum. To solve the technical problem:

[0007] In one aspect, the present application provides a siRNA molecule, the sequence of which is shown in SEQ ID NO. 6-SEQ ID NO. 35.

[0008] In another aspect, the present application provides the application of a siRNA molecule in the breeding of Ptereleotris punctatum, the sequence of which is shown in SEQ ID NO. 6-SEQ ID NO. 35.

[0009] Further, the siRNA molecule can interfere with the expression of the tyr gene family of melanin in the skin tissue of Ptereleotris punctatum.

[0010] Further, the siRNA molecule can reduce the expression efficiency of the melanin tyr gene family in the skin tissue of the leopard grouper by 79-85%, 85-90% or 90-99%.

[0011] In another aspect of the present application, a powder injection for interfering with the expression of the melanin tyr gene family in the skin tissue of the leopard grouper is provided, wherein the powder injection comprises a siRNA molecule, and the sequence of the siRNA molecule is shown in SEQ ID NO. 6-35.

[0012] In another aspect of the present application, the powder injection for interfering with the expression of the melanin tyr gene family in the skin tissue of the leopard grouper is applied in the breeding of the leopard grouper.

[0013] In another aspect of the present application, a method for reducing the expression of the melanin tyr gene family in the skin tissue of the leopard grouper is provided, comprising the following steps: step A, designing a siRNA sequence using the melanin gene of the leopard grouper and synthesizing the siRNA to obtain a powder injection containing the siRNA; step B, dissolving the powder injection containing the siRNA and injecting it into the leopard grouper; and step C, screening the individuals with faded melanin in the skin tissue of the leopard grouper for breeding of the brood fish, using the brood fish for crossbreeding to obtain the offspring of the leopard grouper.

[0014] Further, the melanin gene in step A is the tyr gene, and the sequence of the siRNA is shown in SEQ ID NO. 6-35.

[0015] Further, step B specifically comprises: step B-1, centrifuging the powder injection containing the siRNA, dissolving the powder injection containing the siRNA with PBS buffer after centrifugation to obtain the dissolved powder injection; and step B-2, injecting the dissolved powder injection into the abdominal cavity under the pectoral fin of the leopard grouper using a syringe, and continuously injecting the dissolved powder injection for at least 4 weeks.

[0016] Further, the melanin in the skin tissue of the leopard grouper in step C is faded by 79-85%, 85-90% or 90-99%.

[0017] Further, the skin tissue of the offspring of the leopard grouper is the same as that of the brood fish.

[0018] The application designs and synthesizes siRNA sequences of melanin genes of Plectrypops leopardus, obtains a powder injection containing siRNA, injects the powder injection into the body of Plectrypops leopardus after dissolving, screens individuals with melanin fading in the skin tissue of Plectrypops leopardus for breeding of parent fish and crossbreeding, and obtains offspring of Plectrypops leopardus, the skin tissue traits of which are the same as those of the parent fish. The breeding method provided by the application can reduce melanin in the skin tissue of Plectrypops leopardus and stably inherit, lays a foundation for obtaining a good strain of Plectrypops leopardus, and improves the economic value of Plectrypops leopardus. BRIEF DESCRIPTION OF DRAWINGS

[0019] The above contents of the application and the following detailed embodiments can be better understood when read in conjunction with the accompanying drawings. It should be noted that the drawings are only examples of the claimed technical solutions.

[0020] Figure 1 A detection result graph of the first siRNA interference efficiency of the tyr gene family members of Plectrypops leopardus (wherein the vertical coordinate is the relative expression amount, the horizontal coordinate is the control group (NC) and the experimental group (tyr gene family member siRNA) respectively; the letters "a", "b", "b" and "d" are significant test result graphs, if different letters are used in a single experimental group, it represents that there is a significant difference with p value <0.05, and if the same letter is used in a single experimental group, it represents that there is no significant difference);

[0021] Figure 2 A result graph of the phenotype change of the body color of the population of Plectrypops leopardus after siRNA interference of the tyr gene family members;

[0022] Figure 3 A detection result graph of the second siRNA interference efficiency of the tyr gene family members of Plectrypops leopardus (wherein the vertical coordinate is the relative expression amount, the horizontal coordinate is the control group (NC) and the experimental group (tyr gene family member siRNA) respectively; "***" represents that there is an extremely significant difference with p value <0.01);

[0023] Figure 4 A result graph of the observation of the skin tissue paraffin sections of the control group and the tyr gene family interference group of Plectrypops leopardus (wherein A and B are the control group, the scale of A is 100 μm, B is an enlarged view of the black frame in A, and the scale of B is 50 μm; C and D are the interference group; the scale of C is 100 μm, D is an enlarged view of the black frame in A, and the scale of D is 50 μm; M is a melanin cell, and the yellow arrow points to the position of the melanin cell);

[0024] Figure 5Figure 6 is a result graph of melanin changes in skin tissue after siRNA interference of the members of the tyr gene family of the leopard coral grouper; wherein A is a statistical result of the number of melanin cells in the skin tissue of the leopard coral grouper in the control group and the interference group, the horizontal coordinate is different treatment groups, and the vertical coordinate is the number of melanin cells in 2 mm 2 the skin tissue interval, unit: pieces; B is a statistical graph of melanin content in the skin tissue of the leopard coral grouper in the control group and the interference group, the horizontal coordinate is different treatment groups, and the vertical coordinate is melanin content, unit: ng / L; a-e, ab, bc, cd and de contain the same letters, indicating that there is a significant difference between them, and different letters indicate that there is a very significant difference between them. DETAILED DESCRIPTION

[0025] The detailed features and advantages of the present application are described in detail in the specific embodiments below, which are sufficient for any person skilled in the art to understand the technical content of the present application and to implement it, and according to the description, claims and drawings disclosed in the specification, those skilled in the art can easily understand the related purposes and advantages of the present application.

[0026] All other terms used in this patent are intended to have their general meanings understood by those skilled in the art, especially those skilled in the art who can directly and without doubt determine the meaning of how to implement the technical solutions of the present patent after reading the claims, description and drawings of the present patent.

[0027] Even if the grammar, words, punctuation, graphics, symbols, etc. in the claims, description and drawings of the present patent have not been described in detail, are missing or ambiguous, those skilled in the art can still obtain the only correct understanding without much reasoning or testing by reading the claims, description and drawings as a whole, and effectively exclude all kinds of incorrect understanding methods not aimed at achieving the purpose of the present patent.

[0028] In this text, all technical features and preferred features mentioned in this text can be combined to form new technical solutions if not otherwise specified.

[0029] In the present text, if not otherwise specified, all steps mentioned herein can be performed in sequence or randomly, but preferably in sequence. For example, the method comprises steps (a) and (b) means that the method can comprise steps (a) and (b) in sequence, or steps (b) and (a) in sequence. For example, the method can further comprise step (c) means that step (c) can be added to the method in any sequence, for example, the method can comprise steps (a), (b) and (c), or steps (a), (c) and (b), or steps (c), (a) and (b), etc.

[0030] In the present text, if not otherwise specified, "comprise" and "include" mentioned herein means open or closed. For example, "comprise" and "include" can mean that other components not listed can also be included or contained, or only the listed components can be included or contained.

[0031] If not otherwise specified, the term "one" used in the present specification means "at least one".

[0032] The terms "first", "second", are only used for descriptive purposes and cannot be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features. Therefore, the features defined with "first", "second" can explicitly or implicitly include at least one of the features. In the description of the present application, the meaning of "a plurality of" is at least two, for example, two, three, etc., unless otherwise specifically limited.

[0033] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present application belongs. The terms used in the specification of the present application are only for the purpose of describing specific embodiments and are not intended to limit the present application.

[0034] In order to make the purpose, technical solutions and advantages of the present application more clear, the embodiments of the present application will be further described in detail below with reference to the accompanying drawings. The experimental methods described in the embodiments of the present application are all conventional methods, and the materials, reagents, etc. used in the following examples can be obtained from commercial channels unless otherwise specified.

[0035] (1) Source of sample material

[0036] Ptereleotris punctatum: purchased from Hainan Chenhai Aquatic Products Co., Ltd. (Gengcheng Town, Dongfang City, Hainan Province).

[0037] Sequence synthesis: performed by Shengong Biotech (Shanghai) Co., Ltd., hereinafter referred to as Shengong.

[0038] (2) Source of reagent consumables

[0039] Table 1 Source and item number of reagent consumables required for experiment

[0040] Name Manufacturer Part No. Lipofectamine™ 3000 Reagent (Lipofectamine™ 3000 Reagent) Thermo Fisher L3000015 BlasTaq 2 × qPCR MasterMix Abm G891 Melanin (ML) Kit Shanghai Jingant Biological Engineering Co., Ltd. JLC12245 Trizol Reagent Takara T9108 All-In-One 5 × RT MasterMix Abm G592 MS 222 Zhonghong 1-1-03 4% paraformaldehyde Solarbio P1110 Anhydrous ethanol Macklin E809056 Xylene National Pharmaceutical 10023428 Paraffin Shanghai Test 69019361 Embedding Box CITOTEST 31050102W Hematoxylin Solution Nanjing Jiancheng Technology Co., Ltd. D006-1-1 Eosin Solution Nanjing Jiancheng Technology Co., Ltd. D006-1-1 Neutral Resin Solarbio G8590 PBS Buffer Gibco 10010-049 ;

[0041] (3) Source of instruments and equipment

[0042] Table 2 Source and model of instruments and equipment required for experiment

[0043] Instrument Name Instrument Manufacturer Instrument Model Fluorescence Quantitative PCR Instrument Analytik Jena qTOWER3G Paraffin Microtome Thermo Fisher HM325 Slide Stainer Thermo Fisher HMS740 Slide Warmer Thermo Fisher HMP110 Microscope Olympus BX43 -80℃ Freezer Haier DW-86L486HC Electric Sample Grinder TIANGEN OSE-Y30 ;

[0044] Example

[0045] A siRNA molecule detection and breeding method for interfering with the expression of the Ctenochaetus strigosus melanin tyr gene family members includes the following steps.

[0046] S1, design of Ctenochaetus strigosus tyr gene family member interference sites, the specific steps are as follows:

[0047] Based on the previous research of the research group, five members of the Ctenochaetus strigosus tyr gene family retrieved from the website of NCBI were used: tyr gene (GenBank: NC-056467.1, SEQ ID NO. 1), tyrp1a gene (GenBank: NC-056485.1, SEQ ID NO. 2), tyrp1b gene (GenBank: NC-056466.1, SEQ ID NO. 3), tyrp2 gene (GenBank: NC-056472.1, SEQ ID NO. 4) and tyr-like gene (GenBank: NC-056463.1, SEQ ID NO. 5) The complete CDS sequence (coding region sequence, Coding Sequence) was designed for siRNA sequence online using ThermoFisher website (BLOCK-iT™ RNAi Designer). The sense strand sequence of 3 siRNAs and the antisense strand sequence of 3 siRNAs were designed for each gene, a total of 15 siRNA sequences, and were sent to ShangHai Genechem Co., Ltd. for direct synthesis of 15 siRNA sequences by direct synthesis method, and 15 siRNA powder injections were obtained.

[0048] Among them, the 15 siRNA sequences are as follows:

[0049] tyr-362-siRNA sense strand: 5'-GCAGGAACAUCCUGACCAUTT-3' (SEQ ID NO. 6);

[0050] tyr-362-siRNA antisense strand: 5'-AUGGUCAGGAUGUUCCUGCTT-3' (SEQ ID NO. 7);

[0051] tyr-639-siRNA sense strand: 5'-GCACCGAGUCUACCUACUUTT-3' (SEQ ID NO. 8);

[0052] tyr-639-siRNA antisense strand: 5'-AAGUAGGUAGACUCGGUGCTT-3' (SEQ ID NO. 9);

[0053] tyr-1083-siRNA sense strand: 5'-CCAGAGCACCAUGCAUAAUTT-3' (SEQ ID NO. 10);

[0054] tyr-1083-siRNA antisense strand: 5'-AUUAUGCAUGGUGCUCUGGTT-3' (SEQ ID NO. 11);

[0055] tyrplal-509-siRNA sense strand: 5'-CCGUGCAGUUUGAGAACAUTT-3' (SEQ ID NO. 12);

[0056] tyrplal-509-siRNA antisense strand: 5'-AUGUUCUCAAACUGCACGGTT-3' (SEQ ID NO. 13);

[0057] tyrplal-931-siRNA sense strand: 5'-GGAAAUGUCAACAGGCCAATT-3' (SEQ ID NO. 14);

[0058] tyrplal-931-siRNA antisense strand: 5'-UUGGCCUGUUGACAUUUCCTT-3' (SEQ ID NO. 15);

[0059] tyrplal-1396-siRNA sense strand: 5'-CCAUUCACUAUGACUGAAATT-3' (SEQ ID NO. 16);

[0060] tyrplal-1396-siRNA antisense strand: 5'-UUUCAGUCAUAGUGAAUGGTT-3' (SEQ ID NO. 17);

[0061] tyrp1b-344-siRNA sense strand: 5'-GGAGGAACAUCAUGCAGAUTT-3' (SEQ ID NO. 18);

[0062] tyrp1b-344-siRNA antisense strand: 5'-AUCUGCAUGAUGUUCCUCCTT-3' (SEQ ID NO. 19);

[0063] tyrp1b-988-siRNA sense strand: 5'-CCACCUUACUACUCCACAUTT-3' (SEQ ID NO. 20);

[0064] tyrp1b-988-siRNA antisense strand: 5'-AUGUGGAGUAGUAAGGUGGTT-3' (SEQ ID NO. 21);

[0065] tyrp1b-1545-siRNA sense strand: 5'-GGACAAAUCACAGUCCGUUTT-3' (SEQ ID NO. 22);

[0066] tyrp1b-1545-siRNA antisense strand: 5'-AACGGACUGUGAUUUGUCCTT-3' (SEQ ID NO. 23);

[0067] tyrp2-278-siRNA sense strand: 5'-GCAGGUGUACUGGUAACUUTT-3' (SEQ ID NO. 24);

[0068] tyrp2-278-siRNA antisense strand: 5'-AAGUUACCAGUACACCUGCTT-3' (SEQ ID NO. 25);

[0069] tyrp2-651-siRNA sense strand: 5'-GCACAGGUAUCAUCUUCUATT-3' (SEQ ID NO. 26);

[0070] tyrp2-651-siRNA antisense strand: 5'-UAGAAGAUGAUACCUGUGCTT-3' (SEQ ID NO. 27);

[0071] tyrp2-876-siRNA sense strand: 5'-CCUGGUGACUCUUUGUAAUTT-3' (SEQ ID NO. 28);

[0072] tyrp2-876-siRNA antisense strand: 5'-AUUACAAAGAGUCACCAGGTT-3' (SEQ ID NO. 29);

[0073] tyr-like-815-siRNA antisense strand: 5'-UAGUCCUCUGCUUGAGAGCTT-3' (SEQ ID NO. 31);

[0074] tyr-like-815-siRNA antisense strand: 5'-UAGUCCUCUGCUUGAGAGCTT-3' (SEQ ID NO. 31);

[0075] tyr-like-1030-siRNA antisense strand: 5'-AUCCAGUCUGAGGAUCUCCTT-3' (SEQ ID NO. 33);

[0076] tyr-like-1030-siRNA antisense strand: 5'-AUCCAGUCUGAGGAUCUCCTT-3' (SEQ ID NO. 33);

[0077] tyr-like-1366-siRNA antisense strand: 5'-AAGGAUGCAUAGAUUCUGCTT-3' (SEQ ID NO. 35).

[0078] tyr-like-1366-siRNA antisense strand: 5'-AAGGAUGCAUAGAUUCUGCTT-3' (SEQ ID NO. 35).

[0079] S2, Effect detection of the interference sites of the tyr gene family of Ptereleotris lunula, the specific steps are as follows:

[0080] 1. The siRNA sequences (SEQ ID NO. 6-35) synthesized above are used, the sense strand and the antisense strand of each interference site are paired two by two, and the siRNA sequences are injected through the lower abdominal cavity of the pectoral fin of Ptereleotris lunula, wherein the experimental period of injection is one month, and the injection is performed once a week, a total of 4 times.

[0081] A negative control group (NC) and an interference group, each with 30 tail leopard grouper, were set up. The negative control group was injected with a mixed solution of NC-siRNA powder and transfection reagent; the interference group was injected with a mixed solution of tyr-siRNA, tyrp1a-siRNA, tyrp1b-siRNA, tyrp2-siRNA, tyr-like-siRNA and transfection reagent. The injection dose of siRNA powder in each group was 1 μg / g, and the injection dose of transfection reagent was 2 μL / tail.

[0082] The siRNA sequence of the negative control group (NC) is as follows:

[0083] The positive strand of NC-siRNA is 5'-UUCUCCGAACGUGUCACGUTT-3' (SEQ ID NO. 36);

[0084] The negative strand of NC-siRNA is 5'-ACGUGACACGUUCGGAGAATT-3' (SEQ ID NO. 37).

[0085] 2. Extraction of RNA from the skin tissue of leopard grouper

[0086] The skin tissue of leopard grouper injected with siRNA sequences was sampled, and the RNA of the skin tissue of leopard grouper was extracted by the conventional Trizol method, with the specific steps as follows:

[0087] (1) The sample was taken out from the -80℃ refrigerator sample tube and placed in a 1.5 ml EP tube on ice, 300 μL Trizol reagent was added according to the size of the skin tissue, the tissue was cut into small pieces with scissors first, and then the sample was electrically homogenized on ice until no obvious tissue was observed, 700 μL Trizol reagent was added, mixed well, and incubated at room temperature for 5 min to ensure complete lysis;

[0088] (2) The centrifuge was pre-cooled to 4℃, and centrifuged at 12000 rpm for 5 min. After centrifugation, the sample was divided into two layers, and 800 μL of supernatant was taken into a new EP tube;

[0089] (3) 200 μL of chloroform was added and mixed well for 15 s to ensure complete mixing, and incubated at room temperature for 15 min;

[0090] (4) Centrifuge at 4℃, 12000 rpm for 20 min. At this time, the sample will be divided into three layers, from top to bottom, water phase, protein phase and organic phase;

[0091] (5) Take about 400 μL of the top water phase to a new EP tube, less than 400 μL can be taken, but the middle protein layer cannot be taken;

[0092] (6) Add 500 μL of isopropanol, which is pre-cooled, invert to mix, and then put into a -20℃ refrigerator for 2-3 h to fully precipitate;

[0093] (7) Centrifuge at 4℃, 12000 rpm for 10 min, and discard the supernatant. At this time, the RNA precipitate is at the bottom of the tube;

[0094] (8) Add 1 mL of pre-cooled 75% ethanol to wash the precipitate, shake the EP tube, and suspend the precipitate for washing;

[0095] (9) Centrifuge at 4℃, 8000 rpm for 5 min, and try to discard the supernatant;

[0096] (10) Add 1 mL of pre-cooled anhydrous ethanol, centrifuge at low temperature 8000 rpm for 5 min, and try to discard the supernatant;

[0097] (11) Open the EP tube, and dry it at room temperature for 10 min in a clean bench to volatilize the ethanol completely;

[0098] (12) Preheat DEPC water at 56℃, use 35 μL of DEPC water to dissolve the RNA sample;

[0099] (13) Use a nucleic acid protein quantifier to detect the concentration and OD value of the extracted RNA, and detect the quality of the extracted RNA by 1% agarose gel electrophoresis;

[0100] Through the above steps, the RNA of the skin tissue of Plectryagus leucopterus is obtained.

[0101] 3. Reverse transcription

[0102] The RNA of the skin tissue of Plectryagus leucopterus is reverse transcribed by using All-In-One 5 × RT MasterMix. The reverse transcription system is: All-In-One 5 × RT MasterMix, 4 μL; total RNA sample, 1 μg; Rnase Free H2O, supplemented to 20 μL; the reverse transcription program is: 37℃ for 15 min; 60℃ for 10 min; 95℃ for 3 min; 4℃ storage, and the cDNA of the skin tissue of Plectryagus leucopterus is obtained.

[0103] (4) Primer sequence design: according to the above-mentioned sequences SEQ ID NO. 1~5, the required upstream and downstream primers were designed by Primer Premier 6 software, and the b2m gene was used as an internal reference gene, and was directly synthesized by Shengong Bioengineering Shanghai (Share) Co., Ltd. using direct synthesis method, and the primer sequences are as follows:

[0104] b2m-Fw: 5'-CCTTCAGAAACAACTGGCAATT-3' (SEQ ID NO. 38);

[0105] b2m-Rv: 5'-GGCATATTCCTTAACTTTGGTCC-3' (SEQ ID NO. 39);

[0106] tyr-Fw: 5'-GACCGTAGCAGAGAGAGATT-3' (SEQ ID NO. 40);

[0107] tyr-Rv: 5'-CACGAGCACACAGAGAATAC-3' (SEQ ID NO. 41);

[0108] tyrp1a-Fw: 5'-CAGGTCTTACAAGATGGAAGG-3' (SEQ ID NO. 42);

[0109] tyrp1a-Rv: 5'-TCAAGAATCTACACATGCTCTC-3' (SEQ ID NO. 43);

[0110] tyrp1b-Fw: 5'-TCAAGAATCTACACATGCTCTC-3' (SEQ ID NO. 44);

[0111] tyrp1b-Rv: 5'-TAGACAACGGACTGTGATTTG-3' (SEQ ID NO. 45);

[0112] tyrp2-Fw: 5'-GGCTTTGAACCGCTGATA-3' (SEQ ID NO. 46);

[0113] tyrp2-Rv: 5'-CGCTCTCAGGTATGTATAAGTATT-3' (SEQ ID NO. 47);

[0114] tyr-like-Fw: 5'-CGTGATGACAGAGAGAGGT-3' (SEQ ID NO. 48);

[0115] tyr-like-Rv: 5'-ATCACAGTTCGGTCCAGT-3' (SEQ ID NO. 49).

[0116] (4) qRT-PCR (quantitative real-time PCR) was performed with the cDNA of the skin tissue of P. leopardus as a template and Blas Taq 2 × qPCR MasterMix (abm company) as a reagent, and each group of experiments was set with three biological replicates and three technical replicates.

[0117] The reaction system of qRT-PCR was as follows: Blas Taq 2 × qPCR Master Mix, 10 μL; Forward Primer (10 μM), 0.4 μL; Reverse Primer (10 μM), 0.4 μL; ddH2O, 7.2 μL; template cDNA (5 ng / μL), 2 μL.

[0118] The reaction procedure of qRT-PCR was as follows: 95 °C pre-denaturation for 30 s; 95 °C denaturation for 5 s, 58 °C annealing for 1 min, and a total of 40 cycles of denaturation and annealing, and the relative expression amount of the target gene (SEQ ID NO. 1-5) in qRT-PCR was calculated by using 2 –ΔΔCt The relative expression amount data were subjected to single factor variance analysis and significance test by using SPSS 20.0 software, and it was considered that there was a significant difference when p < 0.05.

[0119] The first siRNA interference efficiency detection results of the tyr gene family members of P. leopardus are shown in Table 1. Figure 1As shown, the relative expression level of tyr-siRNA-362 was significantly lower than that of the negative control group (NC) and the other two experimental groups (tyr-siRNA-639 and tyr-siRNA-1083); the relative expression level of tyrp1a-siRNA-1396 was significantly lower than that of the negative control group (NC) and the other two experimental groups (tyrp1a-siRNA-509 and tyrp1a-siRNA-931); the relative expression level of tyrp1b-siRNA-344 was significantly lower than that of the negative control group (NC) and the other two experimental groups (tyrp1b-siRNA-188 and tyrp1b-siRNA-344). The relative expression levels of tyr-like-siRNA-651 and tyr-like-siRNA-278 and tyr-like-siRNA-876 were significantly lower than those of the negative control group (NC) and the other two experimental groups (tyr-like-siRNA-815 and tyr-like-siRNA-1366).

[0120] Therefore, subsequent experiments were conducted by synthesizing targeted siRNAs at sites tyr-siRNA-362, tyrp1a-siRNA-1396, tyrp1b-siRNA-344, tyrp2-siRNA-651, and tyr-like-siRNA-1030.

[0121] (5) Targeted siRNAs synthesized using sites tyr-siRNA-362 (SEQ ID NO.6~7), tyrp1a-siRNA-1396 (SEQ ID NO.16~17), tyrp1b-siRNA-344 (SEQ ID NO.18~19), tyrp2-siRNA-651 (SEQ ID NO.26~27) and tyr-like-siRNA-1030 (SEQ ID NO.32~33) were used to conduct injection experiments on leopard-gill spiny perch. After continuous injection into the abdominal cavity below the pectoral fin of leopard-gill spiny perch for 4 weeks, the changes in body color phenotype of leopard-gill spiny perch were observed.

[0122] The results of changes in body color phenotype in the population after siRNA interference with members of the tyr gene family of leopard gill perch are as follows: Figure 2 As shown, the leopard gill spiny perch injected with siRNA (interference group) showed a significant change in body color phenotype compared to the control group. The body color of the leopard gill spiny perch in the interference group changed significantly from black to light color, while the control group did not show a significant change.

[0123] (6) Further detect the relative expression of the tyr gene family members after continuous injection for 4 weeks under the ventral cavity of the pectoral fin of the C. leopardus.

[0124] The results of the second siRNA interference efficiency detection of the tyr gene family members of the C. leopardus are shown in Table 5. Figure 3 As shown in Table 5, compared with the negative control group (NC), the relative expression of the tyr gene family members after interference was significantly decreased. Among them, the knockdown efficiency of the tyr interference group was 81.7% (i.e., the expression efficiency was reduced by 81.7%), the knockdown efficiency of the tyrp1a interference group was 79.7% (i.e., the expression efficiency was reduced by 79.7%), the knockdown efficiency of the tyrp1b interference group was the highest, which was 98.9% (i.e., the expression efficiency was reduced by 98.9%), the knockdown efficiency of the tyrp2 interference group was 89.3% (i.e., the expression efficiency was reduced by 89.3%), and the knockdown efficiency of the tyr-like interference group was 91.2% (i.e., the expression efficiency was reduced by 91.2%). All reached a high knockdown efficiency, proving that the interference test was successful, and the expression of the melanin tyr gene family could be reduced by injecting siRNA in the C. leopardus.

[0125] S3, cell observation and melanin content detection of the skin tissue of the C. leopardus, the specific steps are as follows:

[0126] S3-1, paraffin-embedded section

[0127] (1) Selection and treatment of sample tissues: 120-day-old C. leopardus with black body color were selected for the experiment. The control group (i.e., without any treatment) selected 9 fish, and the experimental group included five groups, each group selected 9 fish. The five groups were the C. leopardus interfered by siRNA in the above S2 step (i.e., injected with tyr-siRNA-362 (SEQ ID NO. 6-7), tyrp1a-siRNA-1396 (SEQ ID NO. 16-17), tyrp1b-siRNA-344 (SEQ ID NO. 18-19), tyrp2-siRNA-651 (SEQ ID NO. 26-27), and tyr-like-siRNA-1030 (SEQ ID NO. 32-33), respectively). The fish were anesthetized with MS222 and then sampled by vertebral dislocation, and the skin tissue above the lateral line of the C. leopardus was cut and taken, with a size of 1 cm*3 cm, and placed in a 15 mL centrifuge tube to which 4% paraformaldehyde (PFA) had been added, and fixed overnight at 4°C.

[0128] (2) Dehydration: The fixed skin tissue sample is transferred to an ethanol gradient for dehydration, and is dehydrated in 30%, 50%, 70%, 80%, 90% and absolute ethanol, respectively, for 2 hours at each concentration gradient. After dehydration, the sample is permanently stored in absolute ethanol at 4°C.

[0129] (3) Transparency: The skin tissue sample stored in absolute ethanol at 4°C is taken out with tweezers, cut to 5 mm*5 mm, and placed in a mixture of absolute ethanol and xylene at a ratio of 1:1 for 10 minutes. Then, the sample is placed in xylene twice, for 10 minutes the first time and about 5 minutes the second time. The treatment time is determined according to the transparency of the skin tissue, and the sample is treated until it is completely transparent.

[0130] (4) Wax immersion: A new paraffin block is repeatedly melted and solidified three times before use. The embedding machine is turned on in advance, and the temperature is set to 60°C. The completely transparent skin tissue is placed in the embedding machine, and a mixture of xylene and paraffin at a ratio of 1:1 is used to soak the skin tissue for 1 hour. Then, the soaked skin tissue is transferred to pure paraffin for 2 hours.

[0131] (5) Embedding: A layer of paraffin is added to the embedding box and preheated. The wax-immersed skin tissue is placed in the embedding box and flattened, and the same melting point paraffin is added to embed it. The embedding box is placed on a freezing table to cool and solidify. The wax block can be stored in a -20°C refrigerator for long-term preservation.

[0132] (6) Sectioning: The embedded wax block is taken out from the -20°C refrigerator, and a small knife is used to trim the wax block around the skin tissue, so that the paraffin surface where the tissue is located is higher than the surrounding paraffin surface. Then, the trimmed wax block is fixed on a paraffin sectioning machine. The sectioning thickness is adjusted to 5 μm, and automatic single sectioning is used at the beginning, and automatic continuous sectioning is used after the sectioned tissue is not broken.

[0133] (7) Mounting: The mounting machine with added water is preheated to 42°C. A wax ribbon is taken with a brush and laid flat in the mounting machine. After the wax ribbon is completely unfolded, a glass slide is used to collect the section, and the glass slide is placed in a 37°C slide dryer overnight.

[0134] S3-2, HE staining

[0135] (1) Dewaxing: the tissue-containing slide baked in S3-1 above was placed in xylene for twice dewaxing, 5 min each time, after dewaxing, the slide was transferred to a mixture of xylene and anhydrous ethanol 1:1 for 5 min, then transferred to anhydrous ethanol for twice, 5 min each time, then gradient ethanol treatment was performed, the ethanol concentration gradient was 95% concentration~85% concentration~70% concentration~50% concentration~30% concentration in turn, each concentration gradient was treated for 2 min, and then transferred to pure water.

[0136] (2) Hematoxylin staining: the tissue-containing slide was placed in a hematoxylin solution for staining for about 30 s, then rinsed with running water for a moment, and stopped rinsing until the water flow was colorless.

[0137] (3) Dehydration: the tissue-containing slide rinsed in (2) was subjected to gradient ethanol treatment, the ethanol concentration gradient was 30% concentration~50% concentration~70% concentration~85% concentration in turn, each gradient was treated for 2 min.

[0138] (4) Eosin staining: the tissue-containing slide subjected to gradient ethanol treatment in (3) was placed in an eosin solution for staining for about 30 s, and then placed in a 95% ethanol solution for 2 min.

[0139] (5) Dehydration: then treated with anhydrous ethanol twice, 3 min each time, and then transferred to xylene.

[0140] (6) Mounting: three drops of neutral resin were added to the center of the slide, the cover glass was soaked in xylene for a moment, then mounted, and pebble pressing was used to remove air bubbles.

[0141] (7) Microscopic observation: after the tissue-containing slide was air-dried, the staining results of the skin tissues of the control group and the experimental group of A. pardalis were observed by an Olympus BX43 microscope and photographed.

[0142] The results are shown in Table 1. Figure 4 As shown in Table 1, the melanocytes of the control group were closely surrounded by scales and had a darker color, while the melanocytes of the experimental group became very sparse and had a lighter color. Compared with the control group, the number of melanocytes in the skin tissue of the tyr gene family members was reduced after interference, and the density was reduced, among which, the result was the most significant when the tyr-like-siRNA-1030 site was interfered.

[0143] S3-3, detection of melanin content in skin tissue of A. pardalis, the specific steps are as follows:

[0144] The skin tissue sample taken in S3-1 was taken out from the -80℃ refrigerator, and skin tissue of about 5 mm*5 mm was cut and weighed, and then put into a 1.5 ml EP tube, then 9 times the volume of PBS buffer was added, and the skin tissue was first cut into pieces on ice using scissors, then an electric grinder was used to grind the skin tissue into a homogenate, then a low-temperature centrifuge was used to centrifuge at 12000 rpm for 10 min, and the supernatant was aspirated and transferred to a new 1.5 ml EP tube. The fish melanin enzyme-linked immunoassay kit was used to detect the melanin content, and the detection steps were as follows:

[0145] (1) Dilute the standard: according to the standard provided by the kit, dilute according to the steps provided;

[0146] (2) Sample addition: accurately add the standard sample to the enzyme-labeled plate, 50 μL per well, then add the sample to be tested, 50 μL per well, set three parallels for each sample, and need to set a blank well, when adding sample, add the sample to the bottom of the enzyme-labeled plate;

[0147] (3) Incubation: after sealing the plate with sealing film, incubate in a 37℃ incubator for 30 min;

[0148] (4) Liquid preparation: dilute the 30 times concentrated solution with distilled water 30 times for standby;

[0149] (5) Washing: remove the sealing film, discard the liquid in the well, then add full of washing solution to each well, treat for 30 s, then discard the washing solution, need to repeat the washing for five times;

[0150] (6) Add enzyme: add enzyme-labeled reagent 50 μL per well, the blank well does not add;

[0151] (7) Incubation: operation as above step (3);

[0152] (8) Washing: operation as above step (5);

[0153] (9) Color development: first add color developing agent A 50 μL per well, then add color developing agent B 50 μL, shake gently, wrap the enzyme-labeled plate with tin foil paper, and place it in a 37℃ incubator to develop color for 10 min in the dark;

[0154] (10) Termination: add 50 μL of termination solution per well to terminate the reaction, at this time, the solution will change from blue to yellow immediately;

[0155] (11) Determination: zero setting was performed with the blank holes, and the absorbance (OD value) of each hole was determined at 450 nm wavelength. The determination should be performed within 15 min after adding the termination solution, and finally the absorbance of each hole was determined at 450 nm wavelength. The concentration of the standard sample was used as the abscissa, and the OD value was set as the ordinate. The regression equation of the standard curve was calculated using EXCEL. The OD value of each sample hole was substituted into the equation, and the concentration of the skin tissue sample could be calculated.

[0156] The results are shown in Table 1. Figure 5 As shown in Table 1, by counting the number of melanocytes in the control group and the experimental group, it was found that compared with the control group, the number of melanocytes in the skin tissue of the experimental group was significantly reduced. By analyzing the melanin content of the skin tissue of the experimental group, it was found that the trend was basically consistent with the statistical results of the number of melanocytes. Compared with the control group, the melanin content of the experimental group was decreased after the interference of the tyr gene family members, and the results were the most significant when the tyr-like-siRNA-1030 site was interfered.

[0157] By interfering with the tyr gene of the A. pardalis, the melanin in the skin tissue of the A. pardalis was significantly reduced. Therefore, the A. pardalis can be injected with tyr-siRNA-362 (SEQ ID NO. 6-7), tyrp1a-siRNA-1396 (SEQ ID NO. 16-17), tyrp1b-siRNA-344 (SEQ ID NO. 18-19), tyrp2-siRNA-651 (SEQ ID NO. 26-27), or tyr-like-siRNA-1030 (SEQ ID NO. 32-33) to obtain A. pardalis brood fish with less melanin in the skin tissue. The subsequent hybridization breeding can obtain the offspring, and the trait of less melanin in the skin tissue is retained.

[0158] Therefore, it can be concluded that the siRNA sequence of the melanin gene of the A. pardalis is designed and synthesized to obtain a powder injection containing siRNA. After the powder injection is dissolved, it is injected into the body of the A. pardalis. The individuals with faded melanin in the skin tissue of the A. pardalis are screened for brood fish breeding and hybridization breeding to obtain the offspring of the A. pardalis, and the skin tissue trait of the offspring is the same as that of the brood fish. The breeding method provided by the patent can reduce the melanin in the skin tissue of the A. pardalis and stably inherit it, which lays a foundation for obtaining the A. pardalis elite and improves the economic value of the A. pardalis.

[0159] In the above description of the exemplary embodiments / implementations of the present patent, various features of the present patent are sometimes grouped together in a single embodiment / implementation or in a figure and described in a common manner, merely for the purpose of convenience and without intention of limiting the disclosed implementations to that which is described in only a single disclosed embodiment / implementation. Additionally, the description and disclosure together with the claims and their equivalents should not be construed as requiring that the disclosed implementations be limited to only those embodiments / implementations expressly described and discloses herein. Rather, the description and disclosure given above in connection with the exemplary embodiments / implementations inherently contain multiple alternatives.

[0160] The terms and expressions used in the present specification are used as terms of description and not of limitation, and there is no intention, in the use of such terms and expressions, of excluding equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the present patent claimed. Thus, it should be understood that although the present patent has been specifically disclosed by preferred embodiments, exemplary embodiments and optional features, modification and variation of the concepts herein disclosed can be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this present patent as defined by the appended claims. The embodiments of the present patent are meant to be examples and do not limit the scope of the present patent to the specific embodiments described. It is therefore contended that such modifications and variations are to be considered as falling within the scope of the present patent.

[0161] The foregoing description of the specific embodiments / implementations of the present patent has been presented for the purposes of illustration and description. It is not intended to be exhaustive or to limit the present patent to the precise form disclosed, and many modifications, variations, alterations, and equivalents can be possible, as will be apparent to one skilled in the art from consideration of the content of this disclosure. It is intended that the disclosed embodiments / implementations cover all such modifications, alterations, permutations, and equivalents. Therefore, it is indicated that the above-described embodiments / implementations are intended to be illustrative only and not restrictive. It is intended that changes and modifications not included in the above-described embodiments / implementations can be made by those skilled in the art without departing from the scope of the present patent. Accordingly, the terms and expressions used throughout this present specification were used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the present patent claimed. Therefore, it should be understood that although the present patent has been specifically disclosed by preferred embodiments, exemplary embodiments and optional features, modification and variation of the concepts herein disclosed can be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this present patent as defined by the appended claims. The embodiments of the present patent are meant to be examples and do not limit the scope of the present patent to the specific embodiments described. It is therefore contended that such modifications and variations are to be considered as falling within the scope of the present patent.

[0162] Furthermore, the scope of the present patent should not be limited to the above-described exemplary embodiments, but rather only be limited by the claims and their equivalents.

Claims

1. The application of an siRNA molecule in reducing melanin content in the skin tissue of the leopard-gill spiny perch, characterized in that, The siRNA molecule has the following sequence pairs: a sequence pair consisting of SEQ ID NO. 6 for the sense strand and SEQ ID NO. 7 for the antisense strand; a sequence pair consisting of SEQ ID NO. 16 for the sense strand and SEQ ID NO. 17 for the antisense strand; a sequence pair consisting of SEQ ID NO. 18 for the sense strand and SEQ ID NO. 19 for the antisense strand; a sequence pair consisting of SEQ ID NO. 26 for the sense strand and SEQ ID NO. 27 for the antisense strand; or a sequence pair consisting of SEQ ID NO. 32 for the sense strand and SEQ ID NO. 33 for the antisense strand.

2. A powder injection formulation for reducing melanin content in the skin tissue of the leopard-gill spiny perch, characterized in that, The powder for injection comprises an siRNA molecule, wherein the siRNA molecule comprises a sequence pair consisting of SEQ ID NO. 6 for the sense strand and SEQ ID NO. 7 for the antisense strand, a sequence pair consisting of SEQ ID NO. 16 for the sense strand and SEQ ID NO. 17 for the antisense strand, a sequence pair consisting of SEQ ID NO. 18 for the sense strand and SEQ ID NO. 19 for the antisense strand, a sequence pair consisting of SEQ ID NO. 26 for the sense strand and SEQ ID NO. 27 for the antisense strand, or a sequence pair consisting of SEQ ID NO. 32 for the sense strand and SEQ ID NO. 33 for the antisense strand.

3. A method for reducing the melanin content in the skin tissue of the leopard-gill spiny perch, characterized in that, Includes the following steps: Step A: Design siRNA sequence pairs using the melanin gene of the leopard-gill spiny perch, and synthesize siRNA to obtain a powder injection formulation containing siRNA; the melanin gene is a tyr gene family, and the siRNA sequence pairs are: a sequence pair consisting of SEQ ID NO.6 for the sense strand and SEQ ID NO.7 for the antisense strand; a sequence pair consisting of SEQ ID NO.16 for the sense strand and SEQ ID NO.17 for the antisense strand; a sequence pair consisting of SEQ ID NO.18 for the sense strand and SEQ ID NO.19 for the antisense strand; a sequence pair consisting of SEQ ID NO.26 for the sense strand and SEQ ID NO.27 for the antisense strand; or a sequence pair consisting of SEQ ID NO.32 for the sense strand and SEQ ID NO.33 for the antisense strand. Step B: Dissolve the powder preparation containing siRNA and inject it into the leopard-gill spiny perch, then culture it.

4. The method for reducing the melanin content in the skin tissue of the leopard-gill spiny perch according to claim 3, characterized in that, Step B specifically involves: Step B-1: Centrifuge the powder injection preparation containing siRNA, and then dissolve the powder injection preparation containing siRNA using PBS buffer to obtain the dissolved powder injection preparation. Step B-2: The dissolved powder preparation is injected into the abdominal cavity below the pectoral fin of the leopard gill spiny perch using a syringe, and the dissolved powder preparation is injected continuously for at least 4 weeks.

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