Method for breeding plectropomus leopardus by using siRNA molecule interfering melanin tyr gene family expression

Abstract

The invention discloses a method for breeding plectropomus leopardus by using an siRNA molecule for interfering melanin tyr gene expression. The sequence of the siRNA molecule is as shown in SEQ ID NO.6-35. The siRNA molecule can interfere the expression of the melanin tyr gene of the skin tissue of the plectropomus leopardus. According to the invention, siRNA sequence design and synthesis are carried out on melanin genes of plectropomus leopardus, such that a powder injection preparation containing siRNA is obtained. The powder injection preparation is dissolved and then injected into the body of the plectropomus leopardus; individuals with melanin fading in the skin tissues of the plectropomus leopardus are screened for parent fish breeding and cross breeding to obtain progeny of the plectropomus leopardus, and the skin tissue characters of the progeny of the plectropomus leopardus are the same as those of the parent fish. By means of the breeding method, melanin in the skin tissue of the plectropomus leopardus can be reduced, stable inheritance can be achieved, a foundation is laid for obtaining an improved variety of the plectropomus leopardus, and the economic value of the plectropomus leopardus is increased.

Description

Technical Field

[0001] The present invention relates to the technical field of biological breeding, and in particular to a method for breeding leopard gill bass by utilizing siRNA molecules that interfere with the expression of melanin tyr gene family. Background Art

[0002] Fish, one of the most diverse groups of vertebrates, are renowned for their rich array of colors and patterns. These vibrant colors not only constitute important phenotypic traits but also directly reflect their unique economic value. The diversity of fish coloration stems from the complex distribution and functional differentiation of pigment cells on their surface, providing an ideal biological model for studying pigment metabolism and color formation mechanisms. Among many fish, the leopard gill perch (Plectropomus leopardus), commonly known as the Eastern Star Grouper, a member of the Serranidae family, boasts a vibrant and eye-catching coloration in the wild: a bright red body with blue and white circular spots. This unique color combination gives it both high ornamental and economic value, making it highly sought after and in high demand. However, in captivity, due to changing environmental conditions, varying nutritional intake, and diverse genetic backgrounds, leopard gill perch often struggle to maintain the vibrant coloration of wild individuals, instead adopting a dull black or brown hue. This color degradation not only affects its market value but also poses a serious threat to aquaculture profitability. The dull body color of the leopard gill bass seriously restricts the improvement of its economic value and has become a key issue that needs to be urgently solved in the current aquaculture field.

[0003] Melanocytes, as a key cell type in the formation of fish body color, have garnered widespread attention. Melanocytes originate from neural crest cells and, under the dual regulation of nerves and hormones, control the distribution and aggregation of melanin granules, resulting in varying shades of body color. The development of animal melanocytes is meticulously regulated by a series of genes involved in melanin synthesis. Tyrosinase (Tyr)-related proteases play a central role in melanin biosynthesis, but the expression patterns of Tyr family genes vary across tissues across species.

[0004] In previous studies, the research team identified five tyr gene family members in leopard gill bass through bioinformatics analysis, namely tyr, tyr-like, tyrp1a, tyrp1b and tyrp2. The five genes are distributed on different chromosomes, and there are multiple conserved regions in the protein structure of all family members. Each family member contains a tyrosinase domain and a complete transmembrane helical domain. The expression levels in the skin tissue of black individuals of leopard gill bass are significantly higher than those in red individuals, and they play an important role in the accumulation of melanin in skin tissue. This shows that the expression level of the tyr gene family is related to body color, and plays an important regulatory role in the process of pigment deposition, promoting the synthesis and deposition of melanin.

[0005] siRNA (small interfering RNA) is a short RNA molecule consisting of approximately 20 to 25 nucleotides. siRNA works by binding to the target gene's mRNA, leading to mRNA degradation or translation inhibition, thereby silencing or inhibiting gene expression. However, the role of tyr gene family members in melanin synthesis in the skin of leopard gill bass remains to be further explored. Therefore, siRNA is needed to further explore the sites where tyr gene family members play a major role in melanin synthesis in leopard gill bass, thereby reducing melanin in the skin of leopard gill bass, laying the foundation for obtaining improved leopard gill bass breeds, and increasing the economic value of leopard gill bass. Summary of the Invention

[0006] The purpose of the present invention is to further explore the sites where tyr gene family members play a major role in the synthesis of melanin in leopard gill perch, so as to reduce the melanin in the skin tissue of leopard gill perch, lay the foundation for obtaining improved varieties of leopard gill perch, and at the same time improve the economic value of leopard gill perch, in order to solve the technical problem: In one aspect, the present invention provides an siRNA molecule, the sequences of which are shown in SEQ ID NO. 6 to SEQ ID NO. 35.

[0007] Another aspect of the present invention provides an application of an siRNA molecule in breeding of leopard gill bass, wherein the sequences of the siRNA molecule are shown in SEQ ID NO.6 to SEQ ID NO.35.

[0008] Furthermore, siRNA molecules can interfere with the expression of the tyr gene family of melanin in the skin tissue of leopard gill bass.

[0009] Furthermore, siRNA molecules can reduce the expression efficiency of the tyr gene family of melanin in the skin tissue of leopard gill bass by 79%~85%, 85%~90% or 90%~99%.

[0010] Another aspect of the present invention provides a powder injection preparation for interfering with the expression of the tyr gene family in the skin tissue of leopard gill bass. The powder injection preparation comprises siRNA molecules, and the sequences of the siRNA molecules are shown in SEQ ID NO.6 to SEQ ID NO.35.

[0011] Another aspect of the present invention provides a powder injection preparation for interfering with the expression of the tyr gene family of melanin in the skin tissue of leopard gill bass, and the powder injection preparation is used in the breeding of leopard gill bass.

[0012] On the other hand, the present invention provides a leopard gill perch breeding method, comprising the following steps: step A: designing an siRNA sequence using the melanin gene of the leopard gill perch, and synthesizing the siRNA to obtain a powder injection preparation containing the siRNA; step B: dissolving the powder injection preparation containing the siRNA and injecting the resulting mixture into the leopard gill perch; and step C: screening individuals with faded melanin in skin tissue of the leopard gill perch for broodstock cultivation, and performing crossbreeding using the broodstock to obtain leopard gill perch offspring.

[0013] Furthermore, the melanin gene in step A is the tyr gene, and the siRNA sequence is shown as SEQ ID NO.6 to SEQ ID NO.35.

[0014] Furthermore, step B is specifically as follows: step B-1: centrifuging the powder injection preparation containing siRNA, and after centrifugation, dissolving the powder injection preparation containing siRNA using PBS buffer to obtain a dissolved powder injection preparation; step B-2: injecting the dissolved powder injection preparation into the abdominal cavity under the pectoral fin of the leopard gill bass through a syringe, and continuously injecting the dissolved powder injection preparation for at least 4 weeks.

[0015] Furthermore, the melanin in the skin tissue of the leopard gill bass in step C fades by 79% to 85%, 85% to 90%, or 90% to 99%.

[0016] Furthermore, the skin tissue characteristics of the leopard gill bass offspring are the same as those of the parents.

[0017] This patent describes a method for designing and synthesizing siRNA sequences targeting the melanin gene of leopard gill bass to produce a powder injection containing the siRNA. The powder injection is then dissolved and injected into the leopard gill bass. The method then screens individuals with melanin fading in their skin tissue for broodstock breeding and crossbreeding to produce offspring with skin tissue traits identical to those of the broodstock. This breeding method, provided by this patent, can reduce melanin in the skin tissue of leopard gill bass and stabilize its inheritance, laying the foundation for obtaining improved strains of leopard gill bass and increasing their economic value. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] The above content and the following specific embodiments of the present invention will be better understood when read in conjunction with the accompanying drawings. It should be noted that the accompanying drawings are only examples of the technical solutions claimed.

[0019] Figure 1 This is the result of the first siRNA interference efficiency test of the tyr gene family members in the leopard gill bass (wherein, the vertical axis is the relative expression level, and the horizontal axis is the control group (NC) and the experimental group (tyr gene family member siRNA); the letters "a", "b", "b", and "d" are the results of the significance test. If the letters are different in a single experimental group, it means that the p value is <0.05 and there is a significant difference. If the letters are the same in a single experimental group, it means that there is no significant difference); Figure 2 This is the result of the body color phenotype change of the leopard gill bass after siRNA interference of the tyr gene family members; Figure 3 This is the result of the second siRNA interference efficiency test of the tyr gene family members in the leopard gill bass (the vertical axis is the relative expression level, and the horizontal axis is the control group (NC) and the experimental group (tyr gene family member siRNA); "***" represents a very significant difference with a p value < 0.01); Figure 4 Figures show the observation results of paraffin sections of skin tissue of the leopard gill bass control group and the tyr gene family interference group (A and B are the control group, the scale bar of A is 100 μm, B is the enlarged image of the black frame in A, and the scale bar of B is 50 μm; C and D are the interference group; the scale bar of C is 100 μm, D is the enlarged image of the black frame in A, and the scale bar of D is 50 μm; M is melanocyte, and the yellow arrow indicates the location of melanocytes); Figure 5 The results of melanin changes in the skin tissue of leopard gill bass after siRNA interference of tyr gene family members (where A is the statistical result of the number of melanocytes in the skin tissue of leopard gill bass in the control group and interference group, the horizontal axis is the different treatment groups, and the vertical axis is 2 mm 2The number of melanocytes in the skin tissue area, unit is (pieces); B is the statistical graph of melanin content in the skin tissue of leopard gill bass in the control group and the interference group, the horizontal axis is the different treatment groups, the vertical axis is the melanin content, unit is (ng / L); a~e, ab, bc, cd and de contain the same letters, indicating that there is a significant difference between the two, and different letters indicate that there is an extremely significant difference between the two). DETAILED DESCRIPTION

[0020] The detailed features and advantages of the present invention are described in detail below in the specific embodiments, and the content is sufficient to enable any person skilled in the art to understand the technical content of the present invention and implement it accordingly. Based on the description, claims and drawings disclosed in this specification, those skilled in the art can easily understand the relevant purposes and advantages of the present invention.

[0021] All other terms used herein that are not specifically defined in this patent are intended to have the general meanings understood by ordinary technicians in the field to which they belong, and in particular, ordinary technicians in the field can directly and unambiguously determine the meaning of how to implement the technical solution of this patent after reading the claims, description and drawings of this patent.

[0022] Even if there are incomplete descriptions, omissions or ambiguities in the grammar, text, punctuation, graphics, symbols, etc. in the claims, description and drawings of this patent, ordinary technicians in this field can still reach the only correct understanding by reading the claims, description and drawings as a whole without excessive reasoning or experimentation, and effectively exclude various incorrect understandings that are not aimed at achieving the purpose of this patent.

[0023] In this document, unless otherwise specified, all technical features and preferred features mentioned herein can be combined with each other to form a new technical solution.

[0024] In this document, unless otherwise specified, all steps mentioned herein may be performed sequentially or randomly, but are preferably performed sequentially. For example, "the method includes steps (a) and (b)" means that the method may include steps (a) and (b) performed sequentially, or may include steps (b) and (a) performed sequentially. For example, "the method may further include step (c)" means that step (c) may be added to the method in any order, for example, the method may include steps (a), (b), and (c), or may include steps (a), (c), and (b), or may include steps (c), (a), and (b), etc.

[0025] In this document, unless otherwise specified, the terms "include" and "comprising" may be open-ended or closed-ended. For example, "include" and "comprising" may mean that other components not listed may also be included or that only the listed components are included.

[0026] If not specifically stated, the terms "a" and "an" used in this specification mean "at least one".

[0027] The terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of the technical features being referred to. Thus, a feature specified as "first" or "second" may explicitly or implicitly include at least one such feature. In the description of the present invention, "plurality" means at least two, for example, two, three, etc., unless otherwise specifically defined.

[0028] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art of the present invention. The terms used in this specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0029] To make the objectives, technical solutions, and advantages of the present invention more apparent, embodiments of the present invention will be described in further detail below with reference to the accompanying drawings. The experimental methods described in the examples of the present invention are conventional methods unless otherwise specified. The materials, reagents, etc. used in the following examples are all commercially available unless otherwise specified.

[0030] (1) Source of sample materials Leopard gill bass: purchased from Hainan Chenhai Aquatic Products Co., Ltd. (Gancheng Town, Dongfang City, Hainan Province).

[0031] Sequence synthesis: performed by Sangon Biotech (Shanghai) Co., Ltd., hereinafter referred to as Sangon.

[0032] (2) Sources of reagents and consumables

[0033] Table 1 Sources and item numbers of reagents and consumables required for the experiment name Manufacturer Item No. Lipofectamine™ 3000 Reagent (Transfection Reagent) Thermo Fisher L3000015 BlasTaq 2 × qPCR MasterMix Abm G891 Fish Melanin (ML) Kit Shanghai Jingkang Bioengineering 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 Maclean E809056 Xylene Sinopharm 10023428 paraffin Shanghai Test 69019361 embedding cassette 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 ;

[0034] (3) Source of instruments and equipment

[0035] Table 2 Sources and models of instruments and equipment required for the experiment Instrument name Instrument Manufacturer Instrument model Fluorescence quantitative PCR instrument Analytik Jena qTOWER3G paraffin slicer Thermo Fisher Scientific HM325 Film exhibition machine Thermo Fisher Scientific HMS740 Sheet drying machine Thermo Fisher Scientific HMP110 microscope Olympus BX43 -80℃ Refrigerator Haier DW-86L486HC Electric sample grinder TIANGEN OSE-Y30 ;

[0036] Example A siRNA molecule detection and breeding method for interfering with the expression of tyr gene family members of leopard gill bass melanin comprises the following steps.

[0037] S1. Design of interference sites for members of the tyr gene family of the leopard gill bass. The specific steps are as follows: Based on the previous research of the research group, the complete CDS sequences (coding sequence) of five members of the tyr gene family of Leopard gill bass: 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) retrieved from NCBI website were used to perform RNAi sequencing. Designer) for online siRNA sequence design. For each gene, three siRNA sense strand sequences and three siRNA antisense strand sequences were designed, totaling 15 siRNA sequences. These sequences were then submitted to Shanghai Sangon Biotech Co., Ltd. for direct synthesis, resulting in 15 siRNA powder injections.

[0038] Among them, 15 siRNA sequences are as follows: tyr-362-siRNA sense strand: 5′-GCAGGAACAUCCUGACCAUTT-3′ (SEQ ID NO. 6); tyr-362-siRNA antisense strand: 5′-AUGGUCAGGAUGUUCCUGCTT-3′ (SEQ ID NO. 7); tyr-639-siRNA sense strand: 5′-GCACCGAGUCUACCUACUUTT-3′ (SEQ ID NO. 8); tyr-639-siRNA antisense strand: 5′-AAGUAGGUAGACUCGGUGCTT-3′ (SEQ ID NO. 9); tyr-1083-siRNA sense strand: 5′-CCAGAGCACCAUGCAUAAUTT-3′ (SEQ ID NO. 10); tyr-1083-siRNA antisense strand: 5′-AUUAUGCAUGGUGCUCUGGTT-3′ (SEQ ID NO. 11); tyrp1a-509-siRNA sense strand: 5′-CCGUGCAGUUUGAGAACAUTT-3′ (SEQ ID NO. 12); tyrp1a-509-siRNA antisense strand: 5′-AUGUUCUCAAACUGCACGGTT-3′ (SEQ ID NO. 13); tyrp1a-931-siRNA sense strand: 5′-GGAAAUGUCAACAGGCCAATT-3′ (SEQ ID NO. 14); tyrp1a-931-siRNA antisense strand: 5′-UUGGCCUGUUGACAUUUCCTT-3′ (SEQ ID NO. 15); tyrp1a-1396-siRNA sense strand: 5′-CCAUUCACUAUGACUGAAATT-3′ (SEQ ID NO. 16); tyrp1a-1396-siRNA antisense strand: 5′-UUUCAGUCAUAGUGAAUGGTT-3′ (SEQ ID NO. 17); tyrp1b-344-siRNA sense strand: 5′-GGAGGAACAUCAUGCAGAUTT-3′ (SEQ ID NO. 18); tyrp1b-344-siRNA antisense strand: 5′-AUCUGCAUGAUGUUCCUCCTT-3′ (SEQ ID NO. 19); tyrp1b-988-siRNA sense strand: 5′-CCACCUUACUACUCCACAUTT-3′ (SEQ ID NO. 20); tyrp1b-988-siRNA antisense strand: 5′-AUGUGGAGUAGUAAGGUGGTT-3′ (SEQ ID NO. 21); tyrp1b-1545-siRNA sense strand: 5′-GGACAAAUCACAGUCCGUUTT-3′ (SEQ ID NO. 22); tyrp1b-1545-siRNA antisense strand: 5′-AACGGACUGUGAUUUGUCCTT-3′ (SEQ ID NO. 23); tyrp2-278-siRNA sense strand: 5′-GCAGGUGUACUGGUAACUUTT-3′ (SEQ ID NO. 24); tyrp2-278-siRNA antisense strand: 5′-AAGUUACCAGUACACCUGCTT-3′ (SEQ ID NO. 25); tyrp2-651-siRNA sense strand: 5′-GCACAGGUAUCAUCUUCUATT-3′ (SEQ ID NO. 26); tyrp2-651-siRNA antisense strand: 5′-UAGAAGAUGAUACCUGUGCTT-3′ (SEQ ID NO. 27); tyrp2-876-siRNA sense strand: 5′-CCUGGUGACUCUUUGUAAUTT-3′ (SEQ ID NO. 28); tyrp2-876-siRNA antisense strand: 5′-AUUACAAAGAGUCACCAGGTT-3′ (SEQ ID NO. 29); tyr-like-815-siRNA sense strand: 5′-GCUCUCAAGCAGAGGACUATT-3′ (SEQ ID NO. 30); tyr-like-815-siRNA antisense strand: 5′-UAGUCCUCUGCUUGAGAGCTT-3′ (SEQ ID NO. 31); tyr-like-1030-siRNA sense strand: 5′-GGAGAUCCUCAGACUGGAUTT-3′ (SEQ ID NO. 32); tyr-like-1030-siRNA antisense strand: 5′-AUCCAGUCUGAGGAUCUCCTT-3′ (SEQ ID NO. 33); tyr-like-1366-siRNA sense strand: 5′-GCAGAAUCUAUGCAUCCUUTT-3′ (SEQ ID NO. 34); tyr-like-1366-siRNA antisense strand: 5'-AAGGAUGCAUAGAUUCUGCTT-3' (SEQ ID NO. 35).

[0039] S2. Detection of the effect of interference sites in the tyr gene family of the leopard gill bass. The specific steps are as follows: 1. Using the above-mentioned synthesized siRNA sequences (SEQ ID NO. 6-35), the sense and antisense strands of each interference site were paired in pairs, and the siRNA sequences were injected into the abdominal cavity under the pectoral fin of the leopard gill bass. The experimental injection period was one month, with one injection per week, for a total of four injections.

[0040] A negative control (NC) and an interference group were set up, with 30 leopard gill bass in each group. The negative control group was injected with a mixture of NC-siRNA powder and transfection reagent; the interference groups were injected with a mixture of tyr-siRNA, tyrp1a-siRNA, tyrp1b-siRNA, tyrp2-siRNA, and tyr-like-siRNA, respectively, and transfection reagent. The injection dose of siRNA powder in each mixture was 1 μg / g, and the injection dose of transfection reagent was 2 μL / fish.

[0041] Among them, the siRNA sequence of the negative control group (NC) is as follows: NC-siRNA sense strand: 5′-UUCUCCGAACGUGUCACGUTT-3′ (SEQ ID NO. 36); NC-siRNA antisense strand: 5′-ACGUGACACGUUCGGAGAATT-3′ (SEQ ID NO. 37).

[0042] 2. Extraction of RNA from Skin Tissue of Leopard Gill Bass The skin tissues of the leopard gill bass injected with siRNA sequences were sampled and RNA was extracted from the skin tissues of the leopard gill bass using the conventional Trizol method. The specific steps are as follows: (1) Take the sample out of the -80℃ freezer sample tube and place it in a 1.5 ml EP tube on ice. Add 300 μL of Trizol reagent according to the size of the skin tissue. Use scissors to cut the tissue into pieces in the Trizol reagent. Then use a grinder to homogenize the sample on ice until no obvious tissue is visible. Add 700 μL of Trizol reagent, mix it upside down, and place it at room temperature for 5 minutes to fully lyse it. (2) Precool the centrifuge to 4°C in advance and centrifuge at 12,000 rpm for 5 min. After centrifugation, separate the sample into two layers and draw 800 μL of the supernatant into a new EP tube. (3) Add 200 μL of chloroform, shake and mix for 15 seconds to fully mix the sample, and let it stand at room temperature for 15 minutes; (4) Centrifuge at 4°C, 12,000 rpm for 20 min. The sample will separate into three layers: aqueous phase, protein phase, and organic phase from top to bottom. (5) Pipette about 400 μL of the top aqueous phase into a new EP tube. Less than 400 μL is acceptable, but the middle protein layer should not be drawn into the tube. (6) Add 500 μL of isopropanol. The isopropanol should be pre-cooled in advance. Mix thoroughly by inverting the tube and place it in a -20°C refrigerator for 2-3 hours to allow it to fully precipitate. (7) Centrifuge at 12,000 rpm for 10 min at 4°C and discard the supernatant. The RNA will precipitate at the bottom of the tube. (8) Add 1 mL of pre-cooled 75% ethanol to wash the precipitate, and shake the EP tube to suspend and wash the precipitate; (9) Centrifuge at 8000 rpm for 5 min at 4°C and discard the supernatant as much as possible; (10) Add 1 mL of pre-cooled anhydrous ethanol and centrifuge at 8000 rpm for 5 min. Discard the supernatant as much as possible. (11) Open the EP tube and dry it in a clean bench at room temperature for 10 minutes to allow the ethanol to evaporate completely; (12) Preheat DEPC water at 56°C and use 35 μL of DEPC water to dissolve the RNA sample; (13) The concentration and OD value of the extracted RNA were detected using a nucleic acid protein quantifier, and the quality of the extracted RNA was detected by 1% agarose gel electrophoresis; Through the above steps, RNA from skin tissue of Rhynchops leopardii was obtained.

[0043] 3. Reverse transcription The RNA from the skin tissue of the leopard gill bass was reverse transcribed using All-In-One 5 × RT MasterMix. The reverse transcription system was as follows: All-In-One 5 × RT MasterMix, 4 μL; total RNA of the sample, 1 μg; RNase-Free H2O to make up to 20 μL; the reverse transcription procedure was as follows: 37℃ for 15 min; 60℃ for 10 min; 95℃ for 3 min; and storage at 4℃ to obtain cDNA from the skin tissue of the leopard gill bass.

[0044] (4) Primer sequence design: Based on the above sequences SEQ ID NO. 1 to 5, the required upstream and downstream primers were designed using Primer Premier 6 software, and the b2m gene was used as the internal reference gene. The 12 primer sequences were directly synthesized by the direct synthesis method at Shanghai Sangon Biotech Co., Ltd. The primer sequences are shown below: b2m-Fw: 5'-CCTTCAGAAACAACTGGCAATT-3' (SEQ ID NO. 38); b2m-Rv: 5'-GGCATATTCCTTAACTTTGGTCC-3' (SEQ ID NO. 39); tyr-Fw: 5'-GACCGTAGCAGAGAGAGATT-3' (SEQ ID NO. 40); tyr-Rv: 5'-CACGAGCACACAGAGAATAC-3' (SEQ ID NO. 41); tyrp1a-Fw: 5'-CAGGTCTTACAAGATGGAAGG-3' (SEQ ID NO. 42); tyrp1a-Rv: 5'-TCAAGAATCTACACATGCTCTC-3' (SEQ ID NO. 43); tyrp1b-Fw: 5'-TCAAGAATCTACACATGCTCTC-3' (SEQ ID NO. 44); tyrp1b-Rv: 5'-TAGACAACGGACTGTGATTTG-3' (SEQ ID NO. 45); tyrp2-Fw: 5'-GGCTTTGAACCGCTGATA-3' (SEQ ID NO. 46); tyrp2-Rv: 5'-CGCTCTCAGGTATGTATAAGTATT-3' (SEQ ID NO. 47); tyr-like-Fw: 5'-CGTGATGACAGAGAGAGGT-3' (SEQ ID NO. 48); tyr-like-Rv: 5'-ATCACAGTTCGGTCCAGT-3' (SEQ ID NO. 49).

[0045] (4) qRT-PCR (real-time fluorescence quantitative PCR) was performed using BlasTaq 2 × qPCR MasterMix (abm) using cDNA from the skin tissue of Leopard gill perch as a template. Each experiment was repeated three times biologically and three times technically.

[0046] The qRT-PCR reaction system 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.

[0047] The qRT-PCR reaction procedure was as follows: pre-denaturation at 95°C for 30 s; denaturation at 95°C for 5 s, annealing at 58°C for 1 min, a total of 40 cycles from denaturation to annealing, and 2 –ΔΔCt The relative expression levels of the target genes (SEQ ID NOs. 1-5) in qRT-PCR were calculated using the qRT-PCR method. SPSS 20.0 software was used to perform one-way analysis of variance and significance test on the relative expression data. When p < 0.05, the differences were considered to be significant.

[0048] The results of the first siRNA interference efficiency test of the tyr gene family members of the leopard gill bass are as follows Figure 1 As shown in the figure, 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- The relative expression level of tyrp2-siRNA-651 was significantly lower than that of the negative control group (NC) and the other two experimental groups (tyrp2-siRNA-278 and tyrp2-siRNA-876); the relative expression level of tyr-like-siRNA-1030 was significantly lower than that of the negative control group (NC) and the other two experimental groups (tyr-like-siRNA-815 and tyr-like-siRNA-1366).

[0049] Therefore, we subsequently synthesized targeted siRNAs at the sites tyr-siRNA-362, tyrp1a-siRNA-1396, tyrp1b-siRNA-344, tyrp2-siRNA-651, and tyr-like-siRNA-1030 for formal experiments.

[0050] (5) Targeted siRNAs synthesized at the 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 in an injection experiment on leopard gill bass. After continuous injection into the abdominal cavity under the pectoral fin of leopard gill bass for 4 weeks, the body color phenotype changes of leopard gill bass were observed.

[0051] The results of the body color phenotype changes of the leopard gill bass after siRNA interference of tyr gene family members are as follows Figure 2 As shown in the figure, the body color phenotype of the leopard gill perch after injection of siRNA (interference group) changed significantly compared with the control group. The body color of the leopard gill perch in the interference group changed significantly from black to light color, while there was no obvious change in the control group.

[0052] (6) The above step (4) was further used to detect the changes in the relative expression levels of tyr gene family members after continuous injection into the abdominal cavity under the pectoral fin of leopard gill bass for 4 weeks.

[0053] The results of the second siRNA interference efficiency test of the tyr gene family members of the leopard gill bass are as follows Figure 3 As shown, compared with the negative control (NC), the relative expression levels of all tyr gene family members were significantly decreased after interference. The knockdown efficiency of the tyr interference group was 81.7% (i.e., expression efficiency was reduced by 81.7%), the tyrp1a interference group was 79.7% (i.e., expression efficiency was reduced by 79.7%), and the tyrp1b interference group had the highest knockdown efficiency, at 98.9% (i.e., expression efficiency was reduced by 98.9%). The tyrp2 interference group had a knockdown efficiency of 89.3% (i.e., expression efficiency was reduced by 89.3%), and the tyr-like interference group had a knockdown efficiency of 91.2% (i.e., expression efficiency was reduced by 91.2%). All achieved high knockdown efficiencies, demonstrating the success of the interference experiment and that siRNA injection in leopard gill bass can reduce the expression of the melanin tyr gene family.

[0054] S3. Histocytological observation of the skin of leopard gill bass and detection of melanin content. The specific steps are as follows: S3-1, paraffin-embedded sections (1) Sample tissue selection and processing: 120-day-old black leopard gill perch were selected for the experiment. Nine fish were selected for the control group (i.e., no treatment). There were five experimental groups, with nine fish in each group. The five groups were leopard gill perch that had been subjected to siRNA interference in the above-mentioned 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)). The fish were anesthetized with MS222 and then the vertebrae were broken for sampling. The dorsal skin tissue above the lateral line of the leopard gill perch was cut, and the sample size was 1 cm*3cm, placed in a 15 mL centrifuge tube with 4% paraformaldehyde (PFA) and fixed overnight at 4°C.

[0055] (2) Dehydration and fluid replacement: Transfer the fixed skin tissue samples to an ethanol gradient for dehydration, and dehydrate them in 30% concentration, 50% concentration, 70% concentration, 80% concentration, 90% concentration, and anhydrous ethanol, respectively. Each concentration gradient is placed for 2 hours. After dehydration is completed, immerse in anhydrous ethanol at 4°C for permanent preservation.

[0056] (3) Transparency: Use tweezers to pick up the skin tissue sample stored in anhydrous ethanol at 4°C, cut into 5mm*5mm pieces, and place them in a mixture of anhydrous ethanol and xylene in a ratio of 1:1 for 10 minutes. Then place them in xylene for two more times, the first time for 10 minutes and the second time for about 5 minutes. The treatment time is determined according to the transparency of the skin tissue, until the skin tissue is completely transparent.

[0057] (4) Wax immersion: The new paraffin block should be repeatedly melted and solidified three times before use. The embedding machine should be opened in advance and the temperature should be set to 60°C. The completely transparent skin tissue should be placed in the embedding machine and soaked in a 1:1 mixture of xylene and paraffin for 1 hour. The soaked skin tissue should then be transferred to pure paraffin and soaked for 2 hours.

[0058] (5) Embedding: First add a layer of paraffin wax to the embedding box and preheat it. Place the wax-soaked skin tissue into the embedding box and flatten it. Then add paraffin wax with the same melting point to embed it. Place it on the freezing table to cool and solidify. The wax block can be placed in a -20℃ refrigerator for long-term storage.

[0059] (6) Sectioning: Take the embedded wax block out of the -20℃ freezer and trim the wax block around the skin tissue with a knife so that the paraffin surface where the tissue is located is higher than the surrounding paraffin surface. Then fix the trimmed wax block on the paraffin slicer. When slicing the skin tissue, adjust the slice thickness to 5 μm. Initially, use automatic single cutting. After the sliced ​​tissue is not broken, use automatic continuous cutting.

[0060] (7) Slide spreading: Preheat the slide spreading machine with water added to 42°C in advance, pick up the wax tape with a brush and spread it flat in the slide spreading machine. After the wax tape is fully spread out, use a glass slide to pick up the slide and place it in a 37°C slide drying machine to dry overnight.

[0061] S3-2, HE staining (1) Dewaxing: Place the glass slides with tissue baked in S3-1 above in xylene for dewaxing twice, each time for 5 minutes. After dewaxing, transfer the glass slides to a mixture of xylene and anhydrous ethanol in a ratio of 1:1 for 5 minutes, then transfer them to anhydrous ethanol for treatment twice, each time for 5 minutes, and then perform ethanol gradient treatment. The ethanol concentration gradient is 95% to 85% to 70% to 50% to 30%, and each concentration gradient treatment is 2 minutes. Then transfer the slides to pure water.

[0062] (2) Hematoxylin staining: Place the slide with tissue in hematoxylin solution for about 30 seconds, then rinse with running water for a while until the water becomes colorless and then stop rinsing.

[0063] (3) Dehydration: The slides with tissue washed in (2) were treated with gradient ethanol. The ethanol concentration gradient was 30%~50%~70%~85%, and each gradient treatment lasted 2 minutes.

[0064] (4) Eosin staining: Place the slide with tissue after the ethanol gradient treatment in (3) in the eosin solution for staining for about 30 seconds, and then place it in 95% ethanol solution for washing for 2 minutes.

[0065] (5) Dehydration: Treat with anhydrous ethanol twice, 3 min each time, and then transfer to xylene.

[0066] (6) Sealing: Add three drops of neutral resin to the center of the slide, soak the cover slip in xylene and then seal it, and use pebbles to press the slide to remove bubbles.

[0067] (7) Microscope observation: After the slides with tissue were air-dried, the staining results of the skin tissues of the control group and the experimental group were observed using an Olympus BX43 microscope and photographed.

[0068] The results are as follows Figure 4As shown, melanocytes in the control group were tightly packed around the scales and darker in color, while those in the experimental group became very sparse and lighter in color. Compared to the control group, the number and density of melanocytes in skin tissues expressing members of the tyr gene family decreased after interference, with the most significant results observed when the tyr-like-siRNA-1030 site was interfered with.

[0069] S3-3. Detection of melanin content in skin tissue of leopard gill bass. The specific steps are as follows: Remove the skin tissue sample from S3-1 from the -80°C freezer, cut approximately 5 mm x 5 mm pieces of skin tissue, weigh them, and place them in a 1.5 ml EP tube. Then, add 9 times the volume of PBS buffer to the skin tissue. Use scissors to mince the skin tissue on ice, then use an electric grinder to make a homogenate. Then, use a low-temperature centrifuge at 12,000 rpm for 10 minutes, aspirate the supernatant, and transfer it to a new 1.5 ml EP tube. Use the fish melanin enzyme-linked immunosorbent assay kit to detect melanin content. The specific detection steps are as follows: (1) Dilute the standard: Dilute the standard provided by the kit according to the steps provided; (2) Sample addition: Accurately add 50 μL of the standard sample to each well of the ELISA plate, and then add 50 μL of the sample to be tested to each well. Set up three parallel wells for each sample, and set up a blank well. When adding samples, add the sample to the bottom of the ELISA plate; (3) Incubation: Seal the plate with a sealing film and incubate in a 37°C incubator for 30 min; (4) Liquid preparation: dilute the 30-fold concentrate 30 times with distilled water for later use; (5) Washing: Remove the sealing film, discard the liquid in the wells, then fill each well with washing solution, discard the washing solution after 30 seconds, and repeat the washing five times; (6) Add enzyme: Add 50 μL of enzyme-labeled reagent to each well, and leave it untouched in the blank wells; (7) Incubation: The operation is the same as the above step (3); (8) Washing: The operation is the same as the above step (5); (9) Color development: First add 50 μL of color developer A to each well, then add 50 μL of color developer B, shake gently to mix, wrap the ELISA plate with tin foil, and place it in a 37°C incubator in the dark for 10 min; (10) Termination: Add 50 μL of stop solution to each well to terminate the reaction. You should immediately see the solution change from blue to yellow. (11) Determination: Use the blank well for zero adjustment and measure the absorbance (OD value) of each well at a wavelength of 450 nm. The determination should be carried out within 15 minutes after the addition of the stop solution. Finally, measure the absorbance of each well at a wavelength of 450 nm, with the concentration of the standard as the horizontal axis and the OD value as the vertical axis. Use EXCEL to calculate the regression equation of the standard curve. Substitute the OD value of each sample well into the equation to calculate the concentration of the skin tissue sample.

[0070] The results are as follows Figure 5 As shown in the figure, 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 leopard gill perch in the experimental group was significantly reduced; by analyzing the melanin content in the skin tissue of leopard gill perch, it was found that its trend change was basically consistent with the statistical results of the number of melanocytes. Compared with the control group, the melanin content of the tyr gene family members in the experimental group decreased after interference, among which the results were most significant when the tyr-like-siRNA-1030 site was interfered with.

[0071] By interfering with the tyr gene in leopard gill bass, the melanin in the skin tissue of leopard gill bass is significantly reduced. Therefore, leopard gill bass can be injected with the above-mentioned 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 leopard gill bass broodstock with less melanin in the skin tissue. Subsequently, offspring can be obtained through cross-breeding of broodstock, retaining the trait of less melanin in the skin tissue.

[0072] This leads to the conclusion that this patent application designs and synthesizes siRNA sequences targeting the melanin gene of leopard gill bass, producing a powder injection containing the siRNA; dissolving the powder injection and injecting it into the leopard gill bass; screening individuals with melanin fading in their skin tissue for broodstock cultivation, and crossbreeding to produce leopard gill bass offspring whose skin tissue traits mirror those of the broodstock. The breeding method provided by this patent can reduce melanin in the skin tissue of leopard gill bass and stabilize its inheritance, laying the foundation for obtaining improved strains of leopard gill bass and increasing its economic value.

[0073] In the above description of exemplary embodiments / embodiments of this patent, various features of this patent are sometimes grouped together in a single embodiment / embodiment or its drawings and description for the purpose of simplifying the disclosure and aiding understanding of one or more of the various inventive aspects. However, except for explicitly stated contrary guidance or obvious technical contradiction or exclusion, the description method of this patent should not be interpreted as reflecting an intention that the claimed invention requires more features than expressly stated in each claim. Instead, the claims reflect that the inventive aspects lie in less than all features of a single aforementioned disclosed embodiment / embodiment. Therefore, the claims following the detailed description are hereby expressly incorporated into this detailed description, with each claim existing independently as a separate embodiment / embodiment of this patent.

[0074] The terms and expressions used in this specification are intended to be illustrative rather than limiting, and in the use of these terms and expressions, there is no intention to exclude any equivalents of the features shown and described or portions thereof, but it should be recognized that various variations are possible within the scope of the protection claimed in this patent. It should therefore be understood that although this patent is specifically disclosed through preferred embodiments, exemplary embodiments, and optional features, those skilled in the art may adopt variations or modifications of the concepts disclosed in this specification, and such variations and modifications are therefore considered to be within the scope of this patent as defined by the appended claims. The specific embodiments given in this specification are examples of useful embodiments of this patent, and it is obvious to those skilled in the art that many variations of the devices, device components, and method steps disclosed in this specification can be used to implement this patent.

[0075] The foregoing description of the specific embodiments fully discloses the general features of this patent so that others can easily modify and / or adapt such specific embodiments for various applications by applying knowledge within the technical scope of the art without undue experimentation and without departing from the general concept of this patent. Therefore, based on the teachings and guidance given herein, it is intended that such adaptations and modifications be included within the meaning and range of equivalents of the disclosed embodiments. It should be understood that the words or terms used herein are for descriptive purposes only and are not intended to be limiting, and thus the words or terms of this specification will be interpreted by those skilled in the art based on the above teachings and guidance.

[0076] Additionally, the scope of this patent should not be limited by any of the above-described exemplary embodiments, but should be defined only in accordance with the following claims and their equivalents.

Claims

1. An application of siRNA molecules in breeding of leopard gill bass, characterized in that, The siRNA molecule can interfere with the expression of the melanin tyr gene in the skin tissue of the leopard gill bass. The sequence of the siRNA molecule is shown in SEQ ID NO.6 to SEQ ID NO.

35.

2. The use of the siRNA molecule according to claim 1 in breeding of leopard gill bass, characterized in that, The siRNA molecule can reduce the expression efficiency of the melanin tyr gene family in the skin tissue of the leopard gill bass by 79% to 85%, 85% to 90% or 90% to 99%.

3. A powder injection preparation for interfering with the expression of the tyr gene family of melanin in the skin tissue of leopard gill bass, characterized in that: The powder injection preparation includes siRNA molecules, and the sequences of the siRNA molecules are shown as SEQ ID NO.6 to SEQ ID NO.

35.

4. Application of the powder injection preparation for interfering with the expression of the tyr gene family in the skin tissue of leopard gill perch as claimed in claim 3 in leopard gill perch breeding.

5. A method for breeding leopard gill bass, characterized in that: The following steps are involved: Step A: Designing siRNA sequences using the melanin gene of the leopard gill bass, and synthesizing the siRNA to obtain a powder injection preparation containing the siRNA; the melanin gene belongs to the tyr gene family, and the siRNA sequences are shown in SEQ ID NO. 6 to SEQ ID NO. 35; Step B: dissolving the powder injection preparation containing siRNA and injecting it into the body of the leopard gill bass; Step C: screening individuals with faded melanin in the skin tissue of the leopard gill perch for broodstock cultivation, and using the broodstock for crossbreeding to obtain the leopard gill perch offspring.

6. The method for breeding leopard gill bass according to claim 5, wherein: The step B is specifically as follows: Step B-1: centrifuging the powder injection preparation containing siRNA, and dissolving the powder injection preparation containing siRNA using PBS buffer after centrifugation to obtain a dissolved powder injection preparation; Step B-2: injecting the dissolved powder injection preparation into the abdominal cavity below the pectoral fin of the leopard gill bass through a syringe, and continuously injecting the dissolved powder injection preparation for at least 4 weeks.

7. The method for breeding leopard gill bass according to claim 5, wherein: The melanin in the skin tissue of the leopard gill bass in the step C fades by 79% to 85%, 85% to 90% or 90% to 99%.

8. The method for breeding leopard gill bass according to claim 7, wherein: The skin tissue characteristics of the leopard gill bass offspring are the same as those of the parent fish.

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