WO2022227692A1 - 一种无肌间刺鲫品系及其培育方法 - Google Patents

一种无肌间刺鲫品系及其培育方法 Download PDF

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WO2022227692A1
WO2022227692A1 PCT/CN2022/070608 CN2022070608W WO2022227692A1 WO 2022227692 A1 WO2022227692 A1 WO 2022227692A1 CN 2022070608 W CN2022070608 W CN 2022070608W WO 2022227692 A1 WO2022227692 A1 WO 2022227692A1
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sgrna
crucian carp
intermuscular
bmp6b
bmp6a
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French (fr)
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匡友谊
郑先虎
佟广香
孙志鹏
曹顶臣
闫婷
徐欢
董乐
杨笑星
刘天奇
张潭
张婷婷
孙效文
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中国水产科学研究院黑龙江水产研究所
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Priority to US18/035,797 priority Critical patent/US20240008461A1/en
Priority to EP22757468.8A priority patent/EP4105334A4/en
Publication of WO2022227692A1 publication Critical patent/WO2022227692A1/zh

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Definitions

  • the invention relates to a method for cultivating a crucian carp strain without intermuscular thorns, belonging to the technical field of aquaculture species cultivating methods.
  • Carps are the main farmed fish in China, with an annual output of about 20 million tons, providing nearly 1/3 of the high-quality animal protein for the Chinese people.
  • the carps contain many intermuscular spines, it has caused inconvenience for people in the process of eating, and may even lead to physical damage such as carp.
  • the processing of fish products (such as fish balls, etc.) is also hindered. Therefore, the genetic improvement of intermuscular spines in cyprinids has become one of the important goals of aquatic genetics and breeding in China.
  • Crucian carp is one of the main breeding species in China, with an annual output of about 3 million tons. Its meat is delicious and popular among the people.
  • the intermuscular spines because of the intermuscular spines (the average number of intermuscular spines in different varieties of crucian carp is about 71-84), its quality has been affected, so the intermuscular spines are also an important target of its genetic breeding. Although it has been reported that the number of intermuscular spines in crucian carp can be reduced by cross-breeding and artificial selection, there is no line or variety that reduces the number of intermuscular spines by more than 50%.
  • the present invention proposes a method for cultivating crucian carp with little or no intermuscular spines by knocking out the bmp6 gene.
  • the method for cultivating the crucian carp strain without intermuscular carp of the present invention carries out the cultivation of the crucian carp strain without intermuscular spinach according to the following steps:
  • Knockout target sites were designed for the two copies of bmp6 gene in the crucian carp genome, bmp6a and bmp6b, respectively, and then through two rounds of gene knockout and screening, the F2 generation bmp6a and bmp6b double gene mutation homozygous line was obtained, and then the F2 generation bmp6a was used. Propagated with bmp6b double gene mutation homozygous line to form a new line of crucian carp without intermuscular;
  • the corresponding sgRNAs on bmp6a or bmp6b or bmp6a and bmp6b were mixed with Cas9 protein and microinjected into single-cell embryos of crucian carp, the first round of gene knockout was carried out, and the F0 generation population was constructed, and then the F0 generation population was cultured 3 to 5 PIT labeling and DNA extraction were carried out every month, followed by sequencing to determine the allele and mutation rate of somatic mutation of crucian carp, and F0 generation individuals with a somatic mutation line and a somatic mutation rate of more than 95% were selected as parents to prepare 0-generation fertilized eggs ;
  • the method of the invention obtains the crucian carp species with less than 20 intermuscular spines and no intermuscular spines.
  • Fig. 1 is a typical individual skeletal staining observation contrast chart in embodiment 1, and the arrow points in the figure are intermuscular spines;
  • Fig. 2 is the X-ray diagram of wild-type crucian carp skeleton in embodiment 1, and the arrow points in the figure are intermuscular spines;
  • Fig. 3 is the X-ray image of the mutant tail bone in Example 1, and it can be seen from the figure that there is no intermuscular spine in the tail muscle tissue;
  • Fig. 4 is the X-ray diagram of mutant trunk skeleton in Example 1, and it can be seen from the figure that there is no intermuscular spine in the musculature of the back of the trunk;
  • Figure 5 is a diagram showing the sequencing results of exon 1 of the new line crucian carp bmp6a in Example 1;
  • CAA-1, CAA-2, and CAA-3 are the target sites of exon 1 of bmp6a;
  • WT is a wild type, and the sequence number is in The number is the mutant PIT marker number, and the letter is the mutant allele number;
  • Figure 6 is a diagram showing the sequencing results of exon 1 of the new line of crucian carp bmp6b in Example 1;
  • CAA-7, CAA-8, and CAA-9 are the target sites of exon 1 of bmp6b;
  • WT is a wild type, and the sequence number is in The number is the mutant PIT marker number, and the letter is the mutant allele number;
  • Fig. 7 is the protein sequence comparison diagram of exon 1 of the new line crucian carp bmp6a in Example 1; wherein WT is wild type, the number in the sequence number is the mutant PIT marker number, and the letter is the mutant allele number;
  • Figure 8 is a comparison diagram of exon 1 protein sequences of the new line crucian carp bmp6b in Example 1; wherein WT is a wild type, the number in the sequence number is the mutant PIT marker number, and the letter is the mutant allele number;
  • Fig. 9 is the micro-CT image of wild-type and new line crucian carp bones in Example 1;
  • Figure 10 is the X-ray in vivo observation diagram of wild type, typical individuals with few spines and typical individuals without spines in Example 1, arrows indicate intermuscular spines.
  • the experimental methods used in the following examples are conventional methods unless otherwise specified.
  • the used materials, reagents, methods and instruments, unless otherwise specified, are conventional materials, reagents, methods and instruments in the art, which can be obtained by those skilled in the art through commercial channels.
  • Embodiment 1 The method for cultivating crucian carp strains without intermuscular thorns of the present embodiment:
  • Knockout target sites were designed for the two copies of bmp6 gene in the crucian carp genome, bmp6a and bmp6b, respectively, and then through two rounds of gene knockout and screening, the F2 generation bmp6a and bmp6b double gene mutation homozygous line was obtained, and then the F2 generation bmp6a was used. Propagated with bmp6b double gene mutation homozygous line to form a new line of crucian carp without intermuscular;
  • the corresponding sgRNAs on bmp6a or bmp6b or bmp6a and bmp6b were mixed with Cas9 protein and microinjected into single-cell embryos of crucian carp, the first round of gene knockout was carried out, and the F0 generation population was constructed, and then the F0 generation population was cultured 3 to 5 PIT labeling and DNA extraction were carried out every month, followed by sequencing to determine the allele and mutation rate of somatic mutation of crucian carp, and F0 generation individuals with a somatic mutation line and a somatic mutation rate of more than 95% were selected as parents to prepare 0-generation fertilized eggs ;
  • the DNA extraction and sequencing method is as follows: adding 100 ⁇ L of lysate (the composition of the lysate is proteinase K 0.5 mg /mL, Tris (pH 8.0) 10 mM, KCl 50 mM, 0.3 %Tween 20, 0.3% NP40); the lysis method is: the PCR instrument is set at 55°C for 6h, 98°C for 10min for lysis; after lysis, mix well, centrifuge at 1000-2000rpm for 2min, and take the supernatant as the PCR amplification template.
  • PCR amplification use 2 pairs of primers for PCR amplification, of which 1 pair of primers is the primer of the target site (as shown in Table 2, the primer sequence used to amplify the target fragment and the 5'-end M13 universal primer sequence), and the other pair of primers
  • the primers were index primers (the M13 universal primer sequence and the index base sequence at the 5' end for identifying the sample). PCR amplification was carried out by a 2-step method.
  • the PCR amplification system in the first step was 10 ⁇ L, including 1 ⁇ L of genomic DNA supernatant, 0.5 ⁇ L of 1 ⁇ M upstream and downstream target primers, 2 ⁇ Dream Taq Master Mix (Thermo Fisher, CA, USA) 5 ⁇ L, supplemented with enzyme-free water to 10 ⁇ L; PCR program is 95°C for 3min; 95°C for 30s, 60°C for 30s, 72°C for 30s, 10 cycles; 72°C for 2min; PCR amplification in the second step is the PCR amplification product in the first step Add 0.5 ⁇ L of 5 ⁇ M upstream and downstream index primers to each, and set the PCR program to 95°C for 2 min; 6 cycles of 95°C for 30s, 58°C for 30s, and 72°C for 30s; 95°C for 30s, 72°C for 30s, 15 cycles; 72°C for 30s 2min.
  • the PCR products were mixed in equal amounts, and the DNA sequencing library was constructed with the TrueSeq library building kit, and the Illumina MiSeq sequencing platform was used for 300bp Pair-End sequencing.
  • the data obtained by high-throughput sequencing first used the method described by Tong et al. [14] to identify amplification sites and samples, and then used the CRISPResso2 [15] program to analyze the read length data of each sample in each PCR product to determine Alleles and mutation rates of somatic mutations.
  • PCR amplification was performed on the samples using the primers shown in Table 2, and the system was set to 25 ⁇ L: 2 ⁇ L of genomic DNA supernatant, 1 ⁇ L of 10 ⁇ M upstream and downstream target primers, 2 ⁇ DreamTaq Master Mix (Thermo Fisher, CA, USA) 12.5 ⁇ L, supplemented with enzyme-free water to 25 ⁇ L; PCR reaction program was 95 °C for 3 min; 95 °C for 30 s, 60 °C for 30 s, 72 °C for 30 s, 35 cycles; 72 °C for 5 min. PCR products were detected by 1.5% agarose gel electrophoresis.
  • Embodiment 2 The difference between this embodiment and Embodiment 1 is that the knockout target site of the bmp6 gene is shown in Table 1.
  • Embodiment 3 The difference between this embodiment and Embodiment 1 or 2 is that the sgRNA upstream primer shown in Table 1 and the sgRNA downstream primer whose sequence is 5'-GATCCGCACCGACTCGGTGCCACTTTTTCAAGTTGATAACGGACTAGCCTTATTTTAACTTGCTATTTCTAGCTCTAAAAC-3' is used to synthesize sgRNA in vitro;
  • the sgRNA synthesis system in vitro was as follows: 10 ⁇ M sgRNA upstream and downstream primers were 2.5 ⁇ L each, 2 ⁇ Dream Taq Master mix (ThermoFisher, CA, USA) 25 ⁇ L, and enzyme-free water was supplemented to 50 ⁇ L; a total of 2 tubes of 100 ⁇ L were amplified; sgRNA synthesis procedure in vitro Denaturation at 95°C for 3 min, followed by 30 cycles of 95°C for 30s, 58°C for 30s, and 72°C for 30s; extension at 72°C for 5
  • the in vitro PCR products of sgRNA were detected by 1.5% agarose gel electrophoresis, and the band was 120bp. After detection, the PCR product was purified and recovered with a PCR product purification and recovery kit (Exygen), and the concentration was determined with a Qubit 3 kit (Thermo Fisher, CA, USA) before use. The recovery concentration of sgRNA PCR product was between 100 and 160 ng/ ⁇ L.
  • the in vitro transcribed sgRNAs were purified and recovered using an RNA purification kit (Qiagen). The concentration of the recovered product was determined by Qubit 3 kit (Thermo Fisher, CA, USA) and stored in a -80°C refrigerator until use. The recovery concentration of in vitro transcribed sgRNA was between 800 and 3000 ng/ ⁇ L.
  • Embodiment 4 The difference between this embodiment and Embodiment 1 or 2 or 3 is that: the method of microinjection is: the sgRNA and Cas9 protein (NEB M0646, MA, USA) synthesized in vitro are in a molar ratio of 3:1 After mixing the concentration ratio, incubate at room temperature for 10 min, then add 25% phenol red, and inject into crucian carp single-cell stage embryos; among them, the target site sgRNAs on each exon are mixed and injected in equal amounts, and each sgRNA is finally injected. The concentration was not lower than 50ng/ ⁇ L, the control group was injected with 25% phenol red, and the injection volume per fertilized egg was 1nL ⁇ 0.02nL. Others are the same as the specific embodiment one or two or three.
  • Knockout target sites were designed for the two copies of bmp6 gene in the crucian carp genome, bmp6a and bmp6b, respectively, and then through two rounds of gene knockout and screening, the F2 generation bmp6a and bmp6b double gene mutation homozygous line was obtained, and then the F2 generation bmp6a was used. Propagated with bmp6b double gene mutation homozygous line to form a new line of crucian carp without intermuscular;
  • the corresponding sgRNA on (bmp6a) or (bmp6b) or (bmp6a and bmp6b) was mixed with Cas9 protein and then microinjected into crucian carp single-cell stage embryos, and the first round of gene knockout was performed to construct the F0 generation population, and then the F0 generation Group breeding was carried out for 3 to 5 months for PIT labeling and DNA extraction, and then sequencing was performed to determine the allele and mutation rate of somatic mutation in crucian carp.
  • the DNA extraction and sequencing method is as follows: adding 100 ⁇ L of lysate (the composition of the lysate is proteinase K 0.5mg/mL, Tris (pH8.0) 10mM, KCl 50mM, 0.3 %Tween20, 0.3% NP40); the lysis method is as follows: the PCR instrument is set to 55°C for 6h, 98°C for 10min for lysis; after lysis, mix well, centrifuge at 1000-2000rpm for 2min, and take the supernatant as the PCR amplification template.
  • PCR amplification use 2 pairs of primers for PCR amplification, of which 1 pair of primers is the primer of the target site (as shown in Table 2, the primer sequence used to amplify the target fragment and the 5'-end M13 universal primer sequence), and the other pair of primers
  • the primers were index primers (the M13 universal primer sequence and the index base sequence at the 5' end for identifying the sample).
  • PCR amplification was carried out by a 2-step method.
  • the PCR amplification system in the first step was 10 ⁇ L, including 1 ⁇ L of genomic DNA supernatant, 0.5 ⁇ L of 1 ⁇ M upstream and downstream target primers, and 2 ⁇ Dream Taq Master Mix (Thermo Fisher, CA, USA).
  • PCR program is 95°C for 3min; 95°C for 30s, 60°C for 30s, 72°C for 30s, 10 cycles; 72°C for 2min;
  • PCR amplification in the second step is the PCR amplification product in the first step Add 0.5 ⁇ L of 5 ⁇ M upstream and downstream index primers to each, and set the PCR program to 95°C for 2 min; 6 cycles of 95°C for 30s, 58°C for 30s, and 72°C for 30s; 95°C for 30s, 72°C for 30s, 15 cycles; 72°C for 30s 2min.
  • the PCR products were mixed in equal amounts, and the DNA sequencing library was constructed with the TrueSeq library building kit, and the Illumina MiSeq sequencing platform was used for 300bp Pair-End sequencing.
  • the data obtained by high-throughput sequencing were first used to identify amplification sites and samples using the method described by Tong et al. (Tong et al. 2018), and then each sample was analyzed in each PCR using the CRISPResso2 (Fiume et al. The read length data in the product determines the allele and mutation rate of the somatic mutation.
  • PCR amplification was performed on the samples using the primers shown in Table 2, and the system was set to 25 ⁇ L: 2 ⁇ L of genomic DNA supernatant, 1 ⁇ L of 10 ⁇ M upstream and downstream target primers, 2 ⁇ DreamTaq Master Mix (Thermo Fisher, CA, USA) 12.5 ⁇ L, supplemented with enzyme-free water to 25 ⁇ L; PCR reaction program was 95 °C for 3 min; 95 °C for 30 s, 60 °C for 30 s, 72 °C for 30 s, 35 cycles; 72 °C for 5 min. PCR products were detected by 1.5% agarose gel electrophoresis.
  • sgRNA was synthesized in vitro; the sgRNA in vitro synthesis system was: 10 ⁇ M sgRNA upstream and downstream primers, 2.5 ⁇ L each, 2 ⁇ DreamTaq Master mix (Thermo Fisher, CA, USA) 25 ⁇ L, supplemented with enzyme-free water to 50 ⁇ L; a total of 2 tubes of 100 ⁇ L were amplified; the sgRNA in vitro synthesis program was denaturation at 95 °C for 3 min, followed by 30 cycles of 95 °C for 30 s, 58 °C for 30 s, 72°C for 30s; 72°C extension for 5min; then the amplified product was purified and recovered, followed by in vitro transcription,
  • the in vitro PCR products of sgRNA were detected by 1.5% agarose gel electrophoresis, and the band was 120bp. After detection, the PCR product was purified and recovered with a PCR product purification and recovery kit (Exygen), and the concentration was determined with a Qubit 3 kit (Thermo Fisher, CA, USA) before use. The recovery concentration of sgRNA PCR product was between 100 and 160 ng/ ⁇ L.
  • the in vitro transcribed sgRNAs were purified and recovered using an RNA purification kit (Qiagen). The concentration of the recovered product was determined by Qubit 3 kit (Thermo Fisher, CA, USA) and stored in a -80°C refrigerator until use. The recovery concentration of in vitro transcribed sgRNA was between 800 and 3000 ng/ ⁇ L.
  • the microinjection method is as follows: sgRNA synthesized in vitro and Cas9 protein (NEB M0646, MA, USA) are mixed at a molar concentration ratio of 3:1, incubated at room temperature for 10 min, then added with 25% phenol red, and injected into crucian carp single cells Among them, the target site sgRNAs on each exon were mixed in equal amounts and injected, and the final concentration of each sgRNA was not less than 50ng/ ⁇ L, and the control group was injected with 25% phenol red, and each fertilized grain was The egg injection volume is 1nL ⁇ 0.02nL.
  • the diploid crucian carp used in this example came from the Hulan Test Field of the Heilongjiang Fisheries Research Institute of the Chinese Academy of Fishery Sciences.
  • the broodstock were raised in a 700m2 fish pond, injected with oxytocin (Luteinizing hormone-releasing hormone analog A24 ⁇ g/kg; Dione maleate 1mg/k, gonadotropin chorionic hormone HCG, 100 units/kg) during breeding, and after artificial insemination
  • the fertilized eggs are incubated in a hatching tank of 24cm ⁇ 20cm ⁇ 24cm, and the hatching water temperature is 22°C ⁇ 23°C. After hatching, the larvae were fed with Artemia 4 times/d after the yolk was absorbed.
  • the water flowers are raised in the hatching tank to the size of 2cm ⁇ 3cm, they are moved to the outdoor 500m2 fish pond to raise, and artificial feed is fed during the fingerling culture stage, 2 times/d.

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Abstract

提供一种无肌间刺鲫品系培育方法，所述方法针对bmp6基因在鲫基因组中的2个拷贝bmp6a和bmp6b分别设计敲除靶位点，然后通过2轮基因敲除和筛选，获得F2代bmp6a和bmp6b双基因突变纯合系，然后利用F2代bmp6a和bmp6b双基因突变纯合系进行扩繁形成无肌间刺鲫新品系。所述方法可获得肌间刺数量在20枚以下和无肌间刺的鲫品种。

Description

一种无肌间刺鲫品系及其培育方法
本申请要求于2021年04月30日提交中国专利局、申请号为202110478410.7、发明名称为“一种无肌间刺鲫品系培育方法”的中国专利申请的优先权，其全部内容通过引用结合在本申请中。
技术领域
本发明涉及一种无肌间刺鲫品系培育方法，属于水产养殖品种培育方法技术领域。
背景技术
鲤科鱼类是中国的主要养殖鱼类，年产量约2000多万吨，为中国老百姓提供了近1/3的优质动物蛋白。但因为鲤科鱼类含有的肌间刺多，造成了人们在食用过程中的不便，甚至可能导致卡噪子等身体上的伤害，同时鱼肉产品的加工(如鱼丸等)也受到阻碍，因此鲤科鱼类肌间刺的遗传改良成为中国水产遗传育种的重要目标之一。
目前有一些关于肌间刺选育的报道，如徐晓锋等 [1]在草鱼雌核发育的群体中发现一尾生长正常且肌间刺缺失的突变体，但并未获得无或少肌间刺草鱼突变群体的报道；巴西科学家 [2]采用X射线在大盖巨脂鲤中筛选到一个肌间刺缺失的群体，但在后期育种中也未获得少或无肌间刺的群体 [3]。Guo等 [4]通过三角鲂(Megalobrama terminalis)与翘嘴鲌(Culter alburnus)的杂交实验表明，三角鲂♀×翘嘴鲌
Figure PCTCN2022070608-appb-000001
杂交子代的肌间刺数量(127)小于父本(137)，大于母本(124)，但显著性小于团头鲂♀×翘嘴鲌
Figure PCTCN2022070608-appb-000002
的杂交子代肌间刺数量(133)，说明可以通过杂交育种的方法获得少肌间刺数量的品种，并申请了发明专利(公布号CN107347747A)。黎玲等 [5]报道通过人工培育的杂交鲫比野生鲫肌间刺数量少，可通过人工培育和杂交育种方法减少鲫肌间刺数量。鲍宝龙等通过敲除MSTN基因，发明了使肌间刺变粗的方法 [9][10][11][12]。高泽霞等通过敲除斑马鱼scxa基因，获得了肌间刺数量减少70％以上的突变体，但也造成了肋骨等其它骨骼发育不良 [6],[7],[8]，同时也未有在其它鲤科鱼类中进行应用的报道。综上，虽然有一些方法可以减少鲤科鱼类肌间刺的数量，但目前仍未有无肌间刺鲤科鱼类新品种/系出现。
鲫是中国的主要养殖品种之一，年产量约300万吨，其肉质鲜美，深受老百姓喜爱。同时也由于肌间刺(不同品种鲫肌间刺数量平均约71～84)的原因使其品质受到了影响，因此肌间刺也是其遗传育种的重要目标。虽然有报道称可以通过杂交育种和人工选育减少鲫肌间刺数量，但并未有肌间刺数量减少50％以上的品系或品种出现。
参考文献
[1]Xu,X.,Zheng,J.,Qian,Y.&Luo,C.Normally grown and developed intermuscular bone-deficient mutant in grass carp,Ctenopharyngodon idellus.Chin Sci Bulletin Chin Version 60,526(2015).
[2]Perazza,C.A.et al.Lack of intermuscular bones in specimens of Colossoma macropomum:An unusual phenotype to be incorporated into genetic improvementprograms.Aquaculture 472,5760(2017).
[3]Stokstad,E.Tomorrow’s catch.Science 370,902–905(2020).
[4]Guo,H.-H.et al.Comparative analysis of the growth performance and intermuscular bone traits in F1 hybrids of black bream(Megalobrama terminalis)(♀)×topmouth culter(Culter alburnus)(♂).Aquaculture(2018)doi:10.1016/j.aquaculture.2018.03.037.
[5]Li,L.et al.Comparative analysis of intermuscular bones in fish of differentploidies.Sci China Life Sci 56,341–350(2013).
[6]Nie,C.et al.Loss of scleraxis leads to distinct reduction of mineralized intermuscularbone in zebrafish.Aquac Fish 6,169–177(2021).
[7]Kague,E.et al.Scleraxis genes are required for normal musculoskeletal development and for rib growth and mineralization in zebrafish.Faseb J 33,9116–9130(2019).
[8]CN110684777A(华中农业大学，公开日2020.01.14)；
[9]CN111560401A(上海海洋大学，公开日2020.08.21)；
[10]CN111549030A(上海海洋大学，公开日2020.08.18)；
[11]CN111549031A(上海海洋大学，公开日2020.08.18)；
[12]CN111500581A(上海海洋大学，公开日2020.08.07)；
[13]Chen,Z.et al.De novo assembly of the goldfish(Carassius auratus) genome and the evolution of genes after whole-genome duplication.Sci Adv 5,eaav0547(2019).
[14]Tong,G.et al.De novo assembly and characterization of the Hucho taimen transcriptome.Ecol Evol 8,12711285(2018).
[15]Fiume,M.et al.CRISPResso2 provides accurate and rapid genome editing sequence analysis.Nat Biotechnol 37,224–226(2019).
发明内容
为对鲫肌间刺进行遗传改良，本发明提出了一种通过基因敲除bmp6基因培育少或无肌间刺鲫的方法。
本发明无肌间刺鲫品系培育方法按以下步骤进行无肌间刺鲫品系培育：
针对bmp6基因在鲫基因组中的2个拷贝bmp6a和bmp6b分别设计敲除靶位点，然后通过2轮基因敲除和筛选，获得F2代bmp6a和bmp6b双基因突变纯合系，然后利用F2代bmp6a和bmp6b双基因突变纯合系进行扩繁形成无肌间刺鲫新品系；
其中，用bmp6a或bmp6b或bmp6a和bmp6b上对应的sgRNA与Cas9蛋白混合后显微注射鲫单细胞期胚胎，进行第一轮基因敲除，构建F0代群体，然后F0代群体养殖3～5个月进行PIT标记和DNA提取，再进行测序确定鲫个体体细胞突变的等位基因及突变率，选取体细胞突变系且体细胞突变率为95％以上的F0代个体作为亲本制备0代受精卵；
用bmp6a或bmp6b或bmp6a和bmp6b上对应的sgRNA与Cas9蛋白混合后显微注射0代受精卵，进行第二轮基因敲除，构建F1代群体，然后F1代群体养殖3～5个月进行PIT标记和DNA提取，再进行测序确定鲫个体体细胞突变的等位基因及突变率，选取体细胞bmp6a和bmp6b双基因突变系且体细胞突变率为95％以上的F1代作为亲本进行繁殖，构建F2代，再从F2代中选取bmp6a和bmp6b双基因突变纯合系。
本发明方法获得肌间刺数量在20枚以下和无肌间刺的鲫品种。
因鲫基因组经历了第4轮基因组复制事件 [13]，因此bmp6基因在鲫基因组中存在2个拷贝。
附图说明
图1是实施例1中典型个体骨骼染色观察对比图，图中箭头所指为肌 间刺；
图2是实施例1中野生型鲫骨骼X光射线图，图中箭头所指为肌间刺；
图3是实施例1中突变体尾部骨骼X光射线图，由图中可见尾部肌肉组织中无肌间刺存在；
图4是实施例1中突变体躯干部骨骼X光射线图，由图中可见躯干部背部肌肉组织中无肌间刺存在；
图5是实施例1中新品系鲫bmp6a的1号外显子测序结果图；其中CAA-1、CAA-2、CAA-3为bmp6a 1号外显子靶位点；WT为野生型，序列编号中数字为突变体PIT标记号，字母为突变等位基因编号；
图6是实施例1中新品系鲫bmp6b的1号外显子测序结果图；其中CAA-7、CAA-8、CAA-9为bmp6b 1号外显子靶位点；WT为野生型，序列编号中数字为突变体PIT标记号，字母为突变等位基因编号；
图7是实施例1中新品系鲫bmp6a的1号外显子蛋白序列对比图；其中WT为野生型，序列编号中数字为突变体PIT标记号，字母为突变等位基因编号；
图8是实施例1中新品系鲫bmp6b的1号外显子蛋白序列对比图；其中WT为野生型，序列编号中数字为突变体PIT标记号，字母为突变等位基因编号；
图9为实施例1中野生型和新品系鲫骨骼的micro-CT图；
图10为实施例1中野生型、少刺典型个体和无刺典型个体X射线活体观察图，箭头表示肌间刺。
具体实施方式
下面结合实例对本发明作进一步的详细说明。以下实例旨在说明本发明，并不限制本发明的范围。
下述实施例中所使用的实验方法如无特殊说明均为常规方法。所用材料、试剂、方法和仪器，未经特殊说明，均为本领域常规材料、试剂、方法和仪器，本领域技术人员均可通过商业渠道获得。
具体实施方式一：本实施方式无肌间刺鲫品系培育方法：
针对bmp6基因在鲫基因组中的2个拷贝bmp6a和bmp6b分别设计敲除靶位点，然后通过2轮基因敲除和筛选，获得F2代bmp6a和bmp6b双基因突变纯合系，然后利用F2代bmp6a和bmp6b双基因突变纯合系进行扩繁形成无肌间刺鲫新品系；
其中，用bmp6a或bmp6b或bmp6a和bmp6b上对应的sgRNA与Cas9蛋白混合后显微注射鲫单细胞期胚胎，进行第一轮基因敲除，构建F0代群体，然后F0代群体养殖3～5个月进行PIT标记和DNA提取，再进行测序确定鲫个体体细胞突变的等位基因及突变率，选取体细胞突变系且体细胞突变率为95％以上的F0代个体作为亲本制备0代受精卵；
用bmp6a或bmp6b或bmp6a和bmp6b上对应的sgRNA与Cas9蛋白混合后显微注射0代受精卵，进行第二轮基因敲除，构建F1代群体，然后F1代群体养殖3～5个月进行PIT标记和DNA提取，再进行测序确定鲫个体体细胞突变的等位基因及突变率，选取体细胞bmp6a和bmp6b双基因突变系且体细胞突变率为95％以上的F1代作为亲本进行繁殖，构建F2代，再从F2代中选取bmp6a和bmp6b双基因突变纯合系。
其中，所述的DNA提取和测序方法为：在0.3～0.5cm  2鳍条样品中加入100μL裂解液(裂解液成分为蛋白酶K0.5mg/mL、Tris(pH8.0)10mM、KCl 50mM、0.3％Tween 20、0.3％NP40)；裂解方法为：PCR仪设置55℃6h，98℃10min进行裂解；裂解后混匀，1000～2000rpm离心2min，取上清液作为PCR扩增模板。
PCR扩增：采用2对引物进行PCR扩增，其中1对引物为目标位点引物(如表2所示用于扩增目标片段的引物序列和5’端M13通用引物序列)，另1对引物为索引引物(M13通用引物序列和5’端用于标识样本的索引碱基序列)。采用2步法进行PCR扩增，第1步PCR扩增体系为10μL，包括基因组DNA上清液1μL、1μM上下游目标引物各0.5μL、2×Dream Taq Master Mix(Thermo Fisher,CA,USA)5μL，无酶水补充至10μL；PCR程序为95℃3min；95℃30s、60℃30s、72℃30s，10个循环；72℃2min；第2步PCR扩增在第1步PCR扩增产物中加入5μM上、下游索引引物各0.5μL，PCR程序设置为95℃2min；95℃30s、58℃30s、72℃30s，6个循环；95℃30s，72℃30s，15个循环；72℃2min。
PCR扩增后将PCR产物等量混合，用TrueSeq建库试剂盒构建DNA测序文库，采用Illumina MiSeq测序平台进行300bp Pair-End测序。高通量测序获得的数据首先采用Tong等 [14]描述的方法对扩增位点和样本进行识别，之后采用CRISPResso2 [15]程序分析每个样本在每个PCR产物中的读长数据，确定体细胞突变的等位基因及突变率。
其中，突变体检测采用Sanger测序：采用表2所示的引物对样本进行PCR扩增，体系设置为25μL：基因组DNA上清液2μL、10μM上下游目标引物各1μL、2×DreamTaq Master Mix(Thermo Fisher,CA,USA)12.5μL、无酶水补充至25μL；PCR反应程序为95℃3min；95℃30s，60℃30s，72℃30s，35个循环；72℃5min。PCR产物用1.5％琼脂糖凝胶电泳检测。检测后纯化回收，进行TA克隆，菌落PCR扩增后取2μL PCR产物，用8％的聚丙烯检测，挑选与对照条带大小有差异的菌落进行Sanger测序。
具体实施方式二：本实施方式与具体实施方式一的不同点在于：bmp6基因的敲除靶位点如表1所示，
表1 bmp6基因的敲除靶位点
Figure PCTCN2022070608-appb-000003
Figure PCTCN2022070608-appb-000004
其它与具体实施方式一相同。
表2 PCR扩增用引物
Figure PCTCN2022070608-appb-000005
具体实施方式三：本实施方式与具体实施方式一或二的不同点在于：采用如表1所示的sgRNA上游引物和序列为5’-GATCCGCACCGACTCGGTGCCACTTTTTCAAGTTGATAACGGACTAGCCTTATTTTAACTTGCTATTTCTAGCTCTAAAAC-3’的sgRNA下游引物，进行sgRNA体外合成；sgRNA体外合成体系为：10μM sgRNA上游和下游引物各2.5μL、2×Dream Taq Master mix(ThermoFisher,CA,USA)25μL、无酶水补充至50μL；共扩增2管共100μL；sgRNA体外合成程序为95℃变性3min，之后30个循环设置为95℃30s、58℃30s、72℃30s；72℃延伸5min；然后对扩增产物进行纯化回收，之后进行体外转录，每个靶位点建立30μL反应体系：sgRNAPCR回收产物1μg、NTP Buffer Mix 10μL、T7 RNA Polymerase Mix 2μL，用无酶水补足30μL；37℃转录4h， 反应结束加入20μL无酶的水，混匀后加入2μL DNase I，在37℃消化15min，去除未反应的DNA。其它与具体实施方式一或二相同。
sgRNA体外PCR产物用1.5％琼脂糖凝胶电泳检测，条带为120bp。检测后用PCR产物纯化回收试剂盒(Exygen)将PCR产物纯化回收，采用Qubit 3试剂盒(Thermo Fisher，CA，USA)测定浓度待用。sgRNA PCR产物回收浓度在100～160ng/μL之间。
将体外转录的sgRNA用RNA纯化试剂盒(Qiagen)纯化回收。采用Qubit 3试剂盒(Thermo Fisher，CA，USA)测定回收产物浓度，于-80℃冰箱中保存待用。体外转录sgRNA回收浓度在800～3000ng/μL之间。
具体实施方式四：本实施方式与具体实施方式一或二或三的不同点在于：显微注射的方法是：体外合成的sgRNA和Cas9蛋白(NEB M0646,MA,USA)按3:1的摩尔浓度比混合后室温孵育10min，再加入25％的酚红，注射到鲫单细胞期的胚胎中；其中，每个外显子上的靶位点sgRNA等量混合后进行注射，每个sgRNA终浓度均不低于50ng/μL，对照组注射25％的酚红，每粒受精卵注射量1nL±0.02nL。其它与具体实施方式一或二或三相同。
实施例1
无肌间刺鲫品系培育方法：
针对bmp6基因在鲫基因组中的2个拷贝bmp6a和bmp6b分别设计敲除靶位点，然后通过2轮基因敲除和筛选，获得F2代bmp6a和bmp6b双基因突变纯合系，然后利用F2代bmp6a和bmp6b双基因突变纯合系进行扩繁形成无肌间刺鲫新品系；
其中，用(bmp6a)或(bmp6b)或(bmp6a和bmp6b)上对应的sgRNA与Cas9蛋白混合后显微注射鲫单细胞期胚胎，进行第一轮基因敲除，构建F0代群体，然后F0代群体养殖3～5个月进行PIT标记和DNA提取，再进行测序确定鲫个体体细胞突变的等位基因及突变率，选取体细胞突变系且体细胞突变率为95％以上的F0代个体作为亲本制备0代受精卵；
用bmp6a或bmp6b或bmp6a和bmp6b上对应的sgRNA与Cas9蛋白混合后显微注射0代受精卵，进行第二轮基因敲除，构建F1代群体，然后F1代群体养殖3～5个月进行PIT标记和DNA提取，再进行测序确定 鲫个体体细胞突变的等位基因及突变率，选取体细胞bmp6a和bmp6b双基因突变系且体细胞突变率为95％以上的F1代作为亲本进行繁殖，构建F2代，再从F2代中选取bmp6a和bmp6b双基因突变纯合系。
其中，所述的DNA提取和测序方法为：在0.3～0.5cm 2鳍条样品中加入100μL裂解液(裂解液成分为蛋白酶K 0.5mg/mL、Tris(pH8.0)10mM、KCl 50mM、0.3％Tween20、0.3％NP40)；裂解方法为：PCR仪设置55℃6h，98℃10min进行裂解；裂解后混匀，1000～2000rpm离心2min，取上清液作为PCR扩增模板。PCR扩增：采用2对引物进行PCR扩增，其中1对引物为目标位点引物(如表2所示用于扩增目标片段的引物序列和5’端M13通用引物序列)，另1对引物为索引引物(M13通用引物序列和5’端用于标识样本的索引碱基序列)。采用2步法进行PCR扩增，第1步PCR扩增体系为10μL，包括基因组DNA上清液1μL、1μM上下游目标引物各0.5μL、2×Dream Taq Master Mix(Thermo Fisher,CA,USA)5μL，无酶水补充至10μL；PCR程序为95℃3min；95℃30s、60℃30s、72℃30s，10个循环；72℃2min；第2步PCR扩增在第1步PCR扩增产物中加入5μM上、下游索引引物各0.5μL，PCR程序设置为95℃2min；95℃30s、58℃30s、72℃30s，6个循环；95℃30s，72℃30s，15个循环；72℃2min。PCR扩增后将PCR产物等量混合，用TrueSeq建库试剂盒构建DNA测序文库，采用Illumina MiSeq测序平台进行300bp Pair-End测序。高通量测序获得的数据首先采用Tong等(Tong et al.2018)描述的方法对扩增位点和样本进行识别，之后采用CRISPResso2(Fiume et al.2019)程序分析每个样本在每个PCR产物中的读长数据，确定体细胞突变的等位基因及突变率。
其中，突变体检测采用Sanger测序：采用表2所示的引物对样本进行PCR扩增，体系设置为25μL：基因组DNA上清液2μL、10μM上下游目标引物各1μL、2×DreamTaq Master Mix(Thermo Fisher,CA,USA)12.5μL、无酶水补充至25μL；PCR反应程序为95℃3min；95℃30s，60℃30s，72℃30s，35个循环；72℃5min。PCR产物用1.5％琼脂糖凝胶电泳检测。检测后纯化回收，进行TA克隆，菌落PCR扩增后取2μL PCR产物，用8％的聚丙烯检测，挑选与对照条带大小有差异的菌落进行 Sanger测序。
其中，bmp6基因的敲除靶位点如表1所示。
采用如表1所示的sgRNA上游引物和序列为5’-GATCCGCACCGACTCGGTGCCACTTTTTCAAGTTGATAACGGACTAGCCTTATTTTAACTTGCTATTTCTAGCTCTAAAAC-3’的sgRNA下游引物，进行sgRNA体外合成；sgRNA体外合成体系为：10μM sgRNA上游和下游引物各2.5μL、2×DreamTaq Master mix(Thermo Fisher,CA,USA)25μL、无酶水补充至50μL；共扩增2管共100μL；sgRNA体外合成程序为95℃变性3min，之后30个循环设置为95℃30s、58℃30s、72℃30s；72℃延伸5min；然后对扩增产物进行纯化回收，之后进行体外转录，每个靶位点建立30μL反应体系：sgRNA PCR回收产物1μg、NTP Buffer Mix 10μL、T7 RNA Polymerase Mix 2μL，用无酶水补足30μL；37℃转录4h，反应结束加入20μL无酶的水，混匀后加入2μL DNase I，在37℃消化15min，去除未反应的DNA。
sgRNA体外PCR产物用1.5％琼脂糖凝胶电泳检测，条带为120bp。检测后用PCR产物纯化回收试剂盒(Exygen)将PCR产物纯化回收，采用Qubit 3试剂盒(Thermo Fisher，CA，USA)测定浓度待用。sgRNA PCR产物回收浓度在100～160ng/μL之间。
将体外转录的sgRNA用RNA纯化试剂盒(Qiagen)纯化回收。采用Qubit 3试剂盒(Thermo Fisher，CA，USA)测定回收产物浓度，于-80℃冰箱中保存待用。体外转录sgRNA回收浓度在800～3000ng/μL之间。
其中显微注射的方法是：体外合成的sgRNA和Cas9蛋白(NEB M0646,MA,USA)按3:1的摩尔浓度比混合后室温孵育10min，再加入25％的酚红，注射到鲫单细胞期的胚胎中；其中，每个外显子上的靶位点sgRNA等量混合后进行注射，每个sgRNA终浓度均不低于50ng/μL，对照组注射25％的酚红，每粒受精卵注射量1nL±0.02nL。
本实施例中所采用的二倍体鲫来自于中国水产科学研究院黑龙江水产研究所呼兰试验场。亲鱼于700m 2鱼池中饲养，繁殖时注射催产素(促黄体素释放激素类似物A24μg/kg；马来酸地欧酮1mg/k，促性激素绒毛激素HCG，100单位/kg)，人工授精后受精卵于24cm×20cm×24cm的孵 化缸中孵化，孵化水温22℃～23℃。孵化后待仔鱼卵黄吸收完毕，投喂丰年虫，4次/d。水花在孵化缸中养殖到2cm～3cm大小的乌仔时，移至室外500m 2鱼池中饲养，在鱼种养殖阶段投喂人工饲料，2次/d。
对本实施例形成的无肌间刺鲫新品系进行突变体骨骼染色观察，发现5尾样本肌间刺完全缺失，4尾样本中肌间刺数量仅4～20枚(如图1～4所示，图9，图10)。对上述这些样本在bmp6a的1号外显子和bmp6b的1号外显子进行检测发现1号外显子的3个靶位点均有突变(如图5和图6所示)，基因敲除造成了蛋白翻译提前终止(如图7和图8所示)。
尽管上述实施例对本发明做出了详尽的描述，但它仅仅是本发明一部分实施例，而不是全部实施例，人们还可以根据本实施例在不经创造性前提下获得其它实施例，这些实施例都属于本发明保护范围。

Claims (11)

  1. 一种无肌间刺鲫品系培育方法，其特征在于，按以下步骤进行无肌间刺鲫品系培育：
    针对bmp6基因在鲫基因组中的2个拷贝bmp6a和bmp6b分别设计敲除靶位点，然后通过2轮基因敲除和筛选，获得F2代bmp6a和bmp6b双基因突变纯合系，然后利用F2代bmp6a和bmp6b双基因突变纯合系进行扩繁形成无肌间刺鲫新品系；
    其中，用bmp6a或bmp6b或bmp6a和bmp6b上对应的sgRNA与Cas9蛋白混合后显微注射鲫单细胞期胚胎，进行第一轮基因敲除，构建F0代群体，然后F0代群体养殖3～5个月进行PIT标记和DNA提取，再进行测序确定鲫个体体细胞突变的等位基因及突变率，选取体细胞突变系且体细胞突变率为95％以上的F0代个体作为亲本制备0代受精卵；
    用bmp6a或bmp6b或bmp6a和bmp6b上对应的sgRNA与Cas9蛋白混合后显微注射0代受精卵，进行第二轮基因敲除，构建F1代群体，然后F1代群体养殖3～5个月进行PIT标记和DNA提取，再进行测序确定鲫个体体细胞突变的等位基因及突变率，选取体细胞bmp6a和bmp6b双基因突变系且体细胞突变率为95％以上的F1代作为亲本进行繁殖，构建F2代，再从F2代中选取bmp6a和bmp6b双基因突变纯合系。
  2. 根据权利要求1所述的无肌间刺鲫品系培育方法，其特征在于，所述bmp6基因的敲除靶位点如表1所示，
    表1
    Figure PCTCN2022070608-appb-100001
    Figure PCTCN2022070608-appb-100002
  3. 根据权利要求2所述的无肌间刺鲫品系培育方法，其特征在于，针对所述bmp6基因的敲除靶位点设计得到的sgRNA上游引物如下表所示，
    Figure PCTCN2022070608-appb-100003
  4. 根据权利要求3所述的无肌间刺鲫品系培育方法，其特征在于，采用所述sgRNA上游引物和序列为 5’-GATCCGCACCGACTCGGTGCCACTTTTTCAAGTTGATAACGGACTAGCCTTATTTTAACTTGCTATTTCTAGCTCTAAAAC-3’的sgRNA下游引物，进行sgRNA体外合成；sgRNA合成扩增体系为：10μM sgRNA上游和下游引物各2.5μL、2×Dream Taq Master mix 25μL、无酶水补充至50μL；sgRNA体外合成程序为95℃变性3min；之后30个循环设置为95℃30s、58℃30s、72℃30s，72℃延伸5min；
    sgRNA体外合成后，将PCR产物纯化回收，然后利用RNA体外转录试剂盒进行sgRNA体外转录，每个靶位点建立30μL的sgRNA体外转录反应体系：sgRNAPCR回收产物1μg、NTP Buffer Mix10μL、T7 RNA Polymerase Mix 2μL，用无酶水补足30μL；37℃转录4h，反应结束加入20μL无酶的水，混匀后加入2μL DNase I，在37℃消化15min，去除未反应的DNA。
  5. 根据权利要求4所述的无肌间刺鲫品系培育方法，其特征在于，所述显微注射的方法是：体外合成的sgRNA和Cas9蛋白按3:1的摩尔浓度比混合后室温孵育10min，再加入25％的酚红，注射到鲫单细胞期的胚胎中；其中，每个外显子上的靶位点sgRNA等量混合后进行注射，每个sgRNA终浓度均不低于50ng/μL，每粒受精卵注射量为1nL±0.02nL。
  6. 根据权利要求4所述的无肌间刺鲫品系培育方法，其特征在于，所述sgRNA的PCR产物回收浓度在100～160ng/μL之间。
  7. 根据权利要求4所述的无肌间刺鲫品系培育方法，其特征在于，所述体外转录的sgRNA回收浓度在800～3000ng/μL之间。
  8. 利用权利要求1～7任一项所述的无肌间刺鲫品系培育方法得到的无肌间刺鲫品系。
  9. 一种敲除鲫基因组中bmp6基因的试剂，所述试剂包括针对表1所示的bmp6基因的敲除靶位点设计得到的sgRNA上游引物和sgRNA下游引物。
  10. 根据权利要求9所述试剂，其特征在于，所述sgRNA上游引物如下表所示，
    Figure PCTCN2022070608-appb-100004
    Figure PCTCN2022070608-appb-100005
  11. 根据权利要求9所述试剂，其特征在于，所述sgRNA下游引物的核苷酸序列为：5’-GATCCGCACCGACTCGGTGCCACTTTTTCAAGTTGATAACGGACTAGCCTTATTTTAACTTGCTATTTCTAGCTCTAAAAC-3’。
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