CN111560401A

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Info

Publication number: CN111560401A
Application number: CN202010451893.7A
Authority: CN
Inventors: 鲍宝龙; 车金远
Original assignee: Shanghai Ocean University
Current assignee: Shanghai Ocean University
Priority date: 2020-05-26
Filing date: 2020-05-26
Publication date: 2020-08-21

Abstract

Translated fromChinese

本发明公开了一种翘嘴红鲌和团头鲂肌间刺变粗的分子育种方法，属于水产生物育种技术领域。本发明的分子育种方法是基于基因编辑的技术手段，获得靶向突变翘嘴红鲌和团头鲂mstn基因的F0代，通过传代获得mstn缺失且肌间刺骨化面积增加的突变体个体。在本发明中，首次提出了利用基因编辑技术来获得肌间刺骨化面积增加的翘嘴红鲌和团头鲂的亲本方法。该方法有助于生产上大规模培育出肌间刺粗大而且可遗传的野生翘嘴红鲌和团头鲂，区别于转基因方法，可应用于人工养殖，克服生产上因肌间刺细小难以加工的难点，不用担心转基因食品对人们的影响，且方便人们食用翘嘴红鲌和团头鲂时更容易发现肌间刺，易于生产上推广。The invention discloses a molecular breeding method for thickening the intermuscular spines of red bream and bream, belonging to the technical field of aquatic species breeding. The molecular breeding method of the present invention is based on the technical means of gene editing, obtains the F0 generation of targeted mutation of the red bream and bream mstn gene, and obtains mutant individuals with mstn deletion and increased intermuscular ossification area through passage. In the present invention, it is the first time to propose a method of using gene editing technology to obtain the parental method of the red tuna and the head bream with increased intermuscular ossification area. The method is helpful for the large-scale cultivation of wild tuna and bream with thick and heritable intermuscular spines. It is different from the transgenic method and can be applied to artificial breeding, which overcomes the difficulty of processing due to the small size of the intermuscular spines in production. There is no need to worry about the impact of genetically modified food on people, and it is convenient for people to find intermuscular spines when they eat red bream and bream, and it is easy to promote in production.

Description

Translated fromChinese

一种翘嘴红鲌和团头鲂肌间刺变粗的分子育种方法A molecular breeding method for thickening of the intermuscular spines of the red-billed bream and the head bream

技术领域technical field

本发明属于水产生物育种技术领域，具体涉及一种翘嘴红鲌和团头鲂肌间刺变粗的分子育种方法。The invention belongs to the technical field of aquatic species breeding, and particularly relates to a molecular breeding method for thickening intermuscular spines of red-billed bream and bream.

背景技术Background technique

翘嘴红鲌、团头鲂，属于四大家鱼，是我国主要的淡水养殖鱼类，其肉质细嫩，肉味鲜美，深受广大消费者的喜爱,在我国淡水鱼类养殖中占据有主导地位。利用基因编辑是鱼类性状选育的重要技术手段，通过基因编辑技术可以靶向编辑特定基因从而获得具有优良性状的新品种，对水产业培育出养殖性能优良的品种有着重要的意义。目前，国内外很多研究者开展了通过基因编辑技术研究养殖鱼类的体色、抗病、性腺发育等分子育种工作，这对培育优良品种具有积极作用。Red-billed tuna and ball-headed bream belong to the four major fish species and are the main freshwater aquaculture fish in my country. Their meat is tender and delicious, and they are deeply loved by consumers and occupy a dominant position in freshwater fish farming in my country. . The use of gene editing is an important technical means for the breeding of fish traits. Through gene editing technology, specific genes can be edited to obtain new varieties with excellent traits, which is of great significance for the aquaculture industry to cultivate varieties with excellent aquaculture performance. At present, many researchers at home and abroad have carried out molecular breeding work on the body color, disease resistance, and gonadal development of farmed fish through gene editing technology, which has a positive effect on the cultivation of excellent varieties.

鲤科鱼类都具有一定数量的肌间刺，肌间刺(intermuscular bone,IB)即为肌间骨，位于脊椎骨两侧肌间隔中。肌间刺不同于脊椎骨，分散在肌间隔中，对鱼肉的深加工及食用造成较大困难。目前，已有国家发明专利“一种分离脆肉翘嘴红鲌鱼背肉和鱼脊骨肉的方法“(专利号：CN201110206703.6)”利用解剖将翘嘴红鲌肌间刺与鱼背肉分离，但也会损失很多鱼肉；国家发明专利“异育银鲫新品系优化选育育苗方法”(专利号：ZL201010140103.X)，利用兴国红鲤精子作为异源精子刺激银鲫卵子雌核发育生殖产生的全雌性后代，获得的异育银鲫新品系肌间刺减少；国家发明专利“一种生长快及少肌间刺的杂交鲂鲌的构建方法”(专利号CN201710788633)利用三角鲂为母本与翘嘴鲌父本进行远缘杂交，获得的杂种表现出生长快、形态优、肌间刺少且形态简单等优良性状。但是通过传统育种方法获得的品种对减少肌间刺数量效果一般。肌肉生长抑制素mstn(myostatin)是一类在骨骼肌中广泛表达的糖蛋白，是肌肉生长的负调控因子。目前国内外尚未有文献报道mstn基因会影响鱼类肌间刺的骨化面积。Cyprinids all have a certain number of intermuscular spines. The intermuscular bone (IB) is the intermuscular bone, which is located in the intermuscular space on both sides of the vertebra. The intermuscular spines are different from the vertebrae and are scattered in the intermuscular space, causing great difficulties for the deep processing and consumption of fish meat. At present, there is a national invention patent "a method for separating the back meat and the fish spine meat of crispy red tuna" (patent number: CN201110206703.6)" using dissection to separate the intermuscular spines and the back meat of the red tuna However, it will also lose a lot of fish meat; the national invention patent "Optimized Breeding and Breeding Method of New Lines of Heterophytic Silver Carp" (Patent No.: ZL201010140103.X), uses Xingguo red carp sperm as heterologous sperm to stimulate the gynogenesis of silver carp eggs The all-female offspring produced by reproduction have reduced intermuscular spines in a new line of heterotrophic silver crucian carp; the national invention patent "a construction method of a hybrid bream with fast growth and few intermuscular spines" (patent number CN201710788633) uses the triangular bream as The female parent was crossed with the male parent of A. chinensis, and the obtained hybrid showed excellent characters such as fast growth, excellent shape, few intermuscular spines and simple shape. However, the varieties obtained by traditional breeding methods have an effect on reducing the number of intermuscular spines. General. Myostatin mstn (myostatin) is a kind of glycoprotein that is widely expressed in skeletal muscle and is a negative regulator of muscle growth. At present, there is no literature report that mstn gene can affect the ossification of fish intermuscular spines area.

我国翘嘴红鲌和团头鲂的养殖规模不断壮大，产量稳步持续增长，是我国重要的淡水养殖良种。但是翘嘴红鲌、团头鲂肌肉中存在大量肌间刺，一定程度上影响食品加工和人们的食用。从基因层面改良肌间刺的形状，培育出肌间刺骨化面积增加的翘嘴红鲌和团头鲂新品种，使人们在食用或加工翘嘴红鲌和团头鲂时，更容易剔除肌间刺。降低被肌间刺伤害的风险，从而更方便人们食用翘嘴红鲌和团头鲂，进而有望提高市场上翘嘴红鲌和团头鲂的消费量。The aquaculture scale of red tuna and tulip bream in my country has been expanding, and the output has been increasing steadily. They are important freshwater aquaculture varieties in my country. However, there are a large number of intermuscular spines in the muscles of red-billed red tuna and ball-headed bream, which affect food processing and people's consumption to a certain extent. From the genetic level, the shape of the intermuscular spines was improved, and new varieties of red-billed red tuna and ball-headed bream with increased ossification area of the intermuscular spines were cultivated, which made it easier for people to remove the muscle when eating or processing red-billed red tuna and ball-headed bream. between thorns. The risk of being injured by intermuscular thorns is reduced, so that it is more convenient for people to eat red tuna and head bream, which is expected to increase the consumption of red tuna and head bream in the market.

发明内容SUMMARY OF THE INVENTION

针对现有技术中存在的问题，本发明的目的在于采用CRISPR/Cas9的基因编辑方法突变翘嘴红鲌和团头鲂的mstn基因，从而改变肌间刺大小，获得肌间刺骨化面积变大的翘嘴红鲌和团头鲂的方法。In view of the problems existing in the prior art, the purpose of the present invention is to use the gene editing method of CRISPR/Cas9 to mutate the mstn gene of the red-billed red bream and the head bream, so as to change the size of the intermuscular spine and obtain a larger area of ossification of the intermuscular spine The method of the raised-billed red bream and ball-headed bream.

为了达到上述目的，本发明采用如下技术方案：In order to achieve the above object, the present invention adopts the following technical solutions:

一种翘嘴红鲌和团头鲂肌间刺变粗的分子育种方法，步骤如下：A molecular breeding method for thickening intermuscular thorns of red-billed red bream and head bream, the steps are as follows:

(1)克隆翘嘴红鲌和团头鲂mstn基因核苷酸序列；(1) Cloning the nucleotide sequences of the mstn genes of the red tuna and the head bream;

(2)采用基因敲除方法构建翘嘴红鲌和团头鲂mstn基因的缺失突变体品系；(2) The gene knockout method was used to construct the deletion mutant lines of the red tuna and the head bream mstn gene;

(3)筛选肌间刺粗大个体：选取经过基因敲除后比同龄野生种肌肉肥大的翘嘴红鲌和团头鲂分别进行杂交，收集受精卵，孵化培育后获得稳定遗传的纯合突变体，检测肌间刺变化并筛选肌间刺粗大个体。(3) Screening of individuals with thick intermuscular spines: After gene knockout, the red-billed red bream and the head bream with muscle hypertrophy of the same age were selected for hybridization, fertilized eggs were collected, and homozygous mutants with stable inheritance were obtained after incubation and cultivation. , to detect the changes of intermuscular spines and to screen out individuals with thick intermuscular spines.

在上述方案的基础上，所述步骤1)中克隆的翘嘴红鲌mstn基因核苷酸序列如SEQID NO:1所示，克隆的团头鲂mstn基因核苷酸序列如SEQ ID NO:2所示。On the basis of the above-mentioned scheme, the nucleotide sequence of the cloned red bream mstn gene in the step 1) is shown in SEQ ID NO: 1, and the nucleotide sequence of the cloned bream mstn gene is shown in SEQ ID NO: 2 shown.

SEQ ID NO:1翘嘴红鲌mstn基因核苷酸序列nucleotide sequence

SEQ ID NO:2团头鲂mstn基因核苷酸序列SEQ ID NO:2 Nucleotide sequence of bream mstn gene

在上述方案的基础上，所述步骤1)中克隆翘嘴红鲌mstn基因核苷酸序列的引物对为：On the basis of the above-mentioned scheme, the primer pair of cloning the nucleotide sequence of the red trout mstn gene in the step 1) is:

F:5’-CATGTGGTCCAGTGGGTAATG-3’(SEQ ID NO:3)；F: 5'-CATGTGGTCCAGTGGGTAATG-3' (SEQ ID NO: 3);

R:5’-GTTGATGGGAGACATCTTGGTG-3’(SEQ ID NO:4)；R: 5'-GTTGATGGGAGACATCTTGGTG-3' (SEQ ID NO: 4);

所述步骤1)中克隆团头鲂mstn基因核苷酸序列的引物对为：In the described step 1), the primer pair of the nucleotide sequence of the cloning group head bream mstn gene is:

F:5’-GCAAACATCCTCTAGCACGC-3’(SEQ ID NO:5)；F: 5'-GCAAACATCCTCTAGCACGC-3' (SEQ ID NO: 5);

R:5’-CCACAGCGGTCTACTACCA-3’(SEQ ID NO:6)。R: 5'-CCACAGCGGTCTACTACCA-3' (SEQ ID NO: 6).

在上述方案的基础上，步骤(2)所述的基因敲除方法为TALEN法或crispr/cas9法。On the basis of the above scheme, the gene knockout method described in step (2) is the TALEN method or the CRISPR/cas9 method.

在上述方案的基础上，采用CRISPR/Cas9基因敲除方法构建翘嘴红鲌和团头鲂mstn基因的缺失突变体品系的方法，具体为：On the basis of the above scheme, the method of constructing the deletion mutant strains of the mstn gene of red bream and bream bream by using the CRISPR/Cas9 gene knockout method is as follows:

①在翘嘴红鲌和团头鲂mstn基因序列的外显子区域设计靶点，将设计的靶点体外转录合成sgRNA，并与Cas9 mRNA按比例混合，配成用于基因编辑注射的药物；①Design targets in the exon region of the mstn gene sequence of the red tuna and bream, and transcribe the designed target in vitro to synthesize sgRNA, and mix it with Cas9 mRNA in proportion to prepare a drug for gene editing injection;

②取性腺发育成熟的翘嘴红鲌和团头鲂，注射绒促性素HCG，间隔1～3天后进行人工授精，待胚胎吸水膨胀后开始注射①中的基因编辑注射药物，注射时将药物准确注射到一细胞期的细胞内，每个胚胎注射体积约2nL；2. Take the red tuna and bream with mature gonads, inject chorionic gonadotropin HCG, and perform artificial insemination after an interval of 1 to 3 days. After the embryo absorbs water and swells, start to inject the gene editing injection drug in ①. Accurately inject into cells at the one-cell stage, and the injection volume per embryo is about 2nL;

③注射完成后随机抽取三组注射12h后的翘嘴红鲌和团头鲂胚胎，每组三颗，用剪刀剪碎胚胎，提取基因组DNA，用荧光毛细管电泳法检测敲除效率；③After the injection, randomly select three groups of 12h post-injection red tuna and bream embryos, three in each group, cut the embryos with scissors, extract the genomic DNA, and detect the knockout efficiency by fluorescent capillary electrophoresis;

④将检测有效的胚胎饲养至1个月，剪取个体尾鳍，提取基因组DNA，进行荧光STR检测，再通过分子克隆确定突变基因型，将编辑数目非3n的有效突变F0代留下，将有效突变的翘嘴红鲌和团头鲂饲养至成年。(4) Raise the effective embryos for 1 month, cut individual tail fins, extract genomic DNA, perform fluorescent STR detection, and then determine the mutant genotype through molecular cloning, and leave the effective mutant F0 generation with an editing number other than 3n, which will be effective. Mutated red-billed bream and ball-headed bream are raised to adulthood.

在上述方案的基础上，所述步骤①中基因编辑注射的药物含Cas9 mRNA终浓度300ng/μL，sgRNA终浓度50ng/μL。On the basis of the above scheme, the drug injected in gene editing in step ① contains a final concentration of Cas9 mRNA of 300 ng/μL and a final concentration of sgRNA of 50 ng/μL.

在上述方案的基础上，所述步骤①设计的翘嘴红鲌mstn基因敲除靶点序列为以下序列中的至少一条：On the basis of the above scheme, the target sequence for the knockout of the red trout mstn gene designed in step (1) is at least one of the following sequences:

exon1-target1:5’-GGTTCTGGGGGATGACAGTA-3’(SEQ ID NO:7)；exon1-target1:5'-GGTTCTGGGGGATGACAGTA-3' (SEQ ID NO:7);

exon1-target2:5’-GGGATCAGTACGATGTTCTG-3’(SEQ ID NO:8)；exon1-target2:5'-GGGATCAGTACGATGTTCTG-3' (SEQ ID NO:8);

exon1-target3:5’-GGAGCCTTCCACAGCCACGG-3’(SEQ ID NO:9)；exon1-target3:5'-GGAGCCTTCCACAGCCACGG-3' (SEQ ID NO:9);

exon2-target1:5’-GGGCTAATGCCCGTTACGGA-3’(SEQ ID NO:10)；exon2-target1:5'-GGGCTAATGCCCGTTACGGA-3' (SEQ ID NO: 10);

exon2-target2:5’-GGACAACCGGAGACCAACTG-3’(SEQ ID NO:11)；exon2-target2:5'-GGACAACCGGAGACCAACTG-3' (SEQ ID NO:11);

exon2-target3:5’-GGGTCTTCCTCCGTCCGTAA-3(SEQ ID NO:12)；exon2-target3: 5'-GGGTCTTCCTCCGTCCGTAA-3 (SEQ ID NO: 12);

所述步骤①设计的团头鲂mstn基因敲除靶点序列为以下序列中的至少一条：The target sequence for knockout of the bream mstn gene designed in step (1) is at least one of the following sequences:

exon1-target1:5’-GGTTCTGGGGGATGACAGTA-3’(SEQ ID NO:13)；exon1-target1:5'-GGTTCTGGGGGATGACAGTA-3' (SEQ ID NO: 13);

exon1-target2:5’-GGAGCCTTCCACAGCCACGG-3’(SEQ ID NO:14)；exon1-target2:5'-GGAGCCTTCCACAGCCACGG-3' (SEQ ID NO: 14);

exon1-target3:5’-GGGATCAGTACGACGTTCTG-3’(SEQ ID NO:15)。exon1-target3:5'-GGGATCAGTACGACGTTCTG-3' (SEQ ID NO: 15).

在上述方案的基础上，所述步骤③中翘嘴红鲌检测所用引物为：On the basis of the above scheme, the primers used in the detection of the red tuna in step 3 are:

T1:5’-CATGTGGTCCAGTGGGTAATG-3’(SEQ ID NO:16)；T1: 5'-CATGTGGTCCAGTGGGTAATG-3' (SEQ ID NO: 16);

T2:5’-CGTTTCCCTTCGCGTCATAC-3’(SEQ ID NO:17)；T2: 5'-CGTTTCCCTTCGCGTCATAC-3' (SEQ ID NO: 17);

所述步骤③中团头鲂检测所用引物为：The primers used in the step 3. in the detection of bream in the ball head are:

T1:5’-GCAAACATCCTCTAGCACGC-3’(SEQ ID NO:18)；T1:5'-GCAAACATCCTCTAGCACGC-3' (SEQ ID NO: 18);

T2:5’-GATGGTCTCTGTGGTGGCATG-3’(SEQ ID NO:19)。T2: 5'-GATGGTCTCTGTGGTGGCATG-3' (SEQ ID NO: 19).

在上述方案的基础上，步骤②所述的注射绒促性素HCG的注射用量为：雌鱼1600-2400U/kg，雄鱼800-1200U/kg。On the basis of the above scheme, the injection dosage of chorionic gonadotropin HCG described in step (2) is: 1600-2400U/kg for female fish and 800-1200U/kg for male fish.

在上述方案的基础上，所述步骤(3)中检测肌间刺变化的方法为鱼类硬骨染色和体外分离肌间刺，其中硬骨染色采用茜素红染色法。On the basis of the above scheme, the method for detecting the change of intermuscular spines in the step (3) is fish sclerotinic staining and in vitro separation of intermuscular spines, wherein alizarin red staining method is used for sclerosing staining.

本发明的有益效果是：The beneficial effects of the present invention are:

在本发明中，相较于现有的通过雌核发育、远缘杂交等传统育种方法虽能减少肌间刺数量，但效果不明显。本发明通过基因编辑技术定向编辑翘嘴红鲌和团头鲂mstn基因，获得肌间刺骨化面积增加并且生长速度更快、稳定遗传的突变体翘嘴红鲌和团头鲂，可以解决生产上因肌间刺细小难以发现从而剔除的缺点，通过基因敲除方法，可以获得大量可稳定遗传的突变体翘嘴红鲌和团头鲂，更受大众欢迎。In the present invention, compared with the existing traditional breeding methods such as gynogenesis and distant hybridization, the number of intermuscular spines can be reduced, but the effect is not obvious. The invention uses the gene editing technology to orientately edit the mstn genes of the red tuna and the head bream, to obtain mutants of the red tuna and the head bream with increased intermuscular ossification area, faster growth speed and stable inheritance, which can solve the problem of production problems. Due to the shortcoming that the intermuscular spines are small and difficult to find and thus eliminated, a large number of stably inherited mutants of red bream and bream can be obtained through gene knockout methods, which are more popular among the public.

具体实施方式Detailed ways

在本发明中所使用的术语，除非有另外说明，一般具有本领域普通技术人员通常理解的含义。Terms used in the present invention generally have the meanings commonly understood by those of ordinary skill in the art unless otherwise specified.

下面结合具体实施例，并参照数据进一步详细的描述本发明。以下实施例只是为了举例说明本发明，而非以任何方式限制本发明的范围。The present invention will be described in further detail below with reference to specific embodiments and data. The following examples are only intended to illustrate the present invention and are not intended to limit the scope of the present invention in any way.

实施例1Example 1

(1)翘嘴红鲌mstn基因核苷酸部分序列克隆(1) Cloning of partial nucleotide sequence of mstn gene

在基因数据库NCBI中下载与翘嘴红鲌和团头鲂亲缘关系较近的几种鱼类mstn核苷酸序列，并进行同源比对，选取相对保守区域设计PCR扩增引物，引物序列为：Download the mstn nucleotide sequences of several fishes that are closely related to the red bream and the bream from the gene database NCBI, and perform homology comparison, and select the relatively conserved regions to design PCR amplification primers. The primer sequences are: :

翘嘴红鲌mstn：Red tuna mstn:

以翘嘴红鲌cDNA为模板，进行mstn序列的扩增，获得翘嘴红鲌mstn的编码区部分序列，如SEQ ID NO:1所示。Amplification of the mstn sequence was carried out using the cDNA of the red tuna chinensis as a template to obtain the partial sequence of the coding region of the red tuna mstn, as shown in SEQ ID NO: 1.

(2)翘嘴红鲌mstn基因的缺失突变体品系的构建(2) Construction of mutant strains of red tuna with deletion of mstn gene

采用基因敲除方法获得翘嘴红鲌mstn基因缺失突变体品系，以crispr/cas9构建缺失品系方法为例，具体步骤如下：The gene knockout method was used to obtain the mutant line of the red tuna mstn gene deletion. Taking the method of constructing a deletion line with CRISPR/cas9 as an example, the specific steps are as follows:

(i)翘嘴红鲌mstn基因敲除靶点选择(i) Target selection for mstn gene knockout

翘嘴红鲌mstn序列有三个外显子：exon1、exon2、exon3。为了更高效的进行基因编辑和长片段的碱基缺失，在第一外显子上设计2个靶点，序列为:There are three exons in the mstn sequence of red tuna: exon1, exon2, and exon3. In order to perform more efficient gene editing and long-range base deletion, two targets were designed on the first exon, and the sequences are:

在第二个外显子in the second exon

(ii)翘嘴红鲌靶基因编辑(ii) Target gene editing of red tuna

a、采用crispr/cas9系统进行编辑：a. Use the CRISPR/cas9 system to edit:

将设计的6个靶点，分别体外转录成sgRNA，分别与Cas9 mRNA按比例共同混合至一管，配成用于基因编辑注射的药物，含：Cas9 mRNA终浓度300The 6 designed targets were transcribed into sgRNA in vitro, respectively mixed with Cas9 mRNA in proportion to a tube, and formulated into a drug for gene editing injection, including: Cas9 mRNA final concentration 300

ng/μl，sgRNA终浓度50ng/μl。ng/μl, the final concentration of sgRNA is 50ng/μl.

b、获取性腺发育成熟的翘嘴红鲌，3冬龄以上、体重1.5kg以上体格健壮无损伤的雌雄鱼，雌雄比2:1，置于帆布缸，注射促性腺激素HCG，注射量为雌鱼1600-2400U/kg，雄鱼800-1200U/kg，中间间隔1-3天，后进行人工授精，为方便显微注射，布卵至一次性塑料培养皿中，待受精卵吸水膨胀后立即于显微镜下进行显微注射，注射时将药物准确注射到一细胞期的细胞内，每个胚胎注射体积约2nl。将注射和未注射的受精卵及时送至于孵化器中，室温孵化(水温24-27℃，约20h出膜)。b. Obtain the red tuna with mature gonads, 3 winter-old or more healthy male and female fish with a body weight of 1.5kg or more, with a male-to-female ratio of 2:1, placed in a canvas tank, and injected with the gonadotropin HCG, the injection amount is female Fish 1600-2400U/kg, male fish 800-1200U/kg, with an interval of 1-3 days, then artificial insemination, for the convenience of microinjection, the eggs are placed in a disposable plastic petri dish, and immediately after the fertilized eggs absorb water and swell The microinjection was carried out under the microscope, and the drug was accurately injected into the cells of one cell stage during the injection, and the injection volume of each embryo was about 2nl. The injected and uninjected fertilized eggs were sent to the incubator in time, and incubated at room temperature (water temperature 24-27°C, about 20h out of the membrane).

(iii)目标基因突变体的筛选及纯合突变体的获得(iii) Screening of target gene mutants and acquisition of homozygous mutants

分别随机抽取三组注射过的翘嘴红鲌胚胎和未注射的野生胚胎(注射12h后)，每组三颗，用剪刀剪碎胚胎，碱裂解法提取基因组DNA。Three groups of injected embryos and uninjected wild embryos (12h after injection) were randomly selected, three in each group, and the embryos were minced with scissors, and the genomic DNA was extracted by alkaline lysis method.

利用荧光毛细管电泳法检测敲除效率。将检测有效的胚胎饲养至1个月，剪取个体尾鳍，提取基因组DNA,进行荧光STR检测，再通过分子克隆确定突变基因型，将编辑数目非3n的有效突变F0代留下，有效突变的翘嘴红鲌至少收集20尾，饲养至成年。Knockout efficiency was detected by fluorescent capillary electrophoresis. The effective embryos were raised for 1 month, the individual tail fins were clipped, genomic DNA was extracted, and fluorescent STR was detected. Then, the mutant genotype was determined by molecular cloning. At least 20 tails were collected and raised to adulthood.

所述检测引物为：The detection primers are:

(iv)肌间刺粗大个体的筛选(iv) Screening of individuals with thick intermuscular spines

由于翘嘴红鲌缺失mstn基因后会导致肌肉肥大，此时我们选取比同龄野生种体型肥大的翘嘴红鲌进行杂交，收集受精卵，发育至孵化期后，随机提取10颗胚胎的基因组DNA，进行STR验证,获得可稳定遗传的纯合突变体F1。将F1代饲养至体长约1cm长左右，随机挑选30条个体分别剪取部分尾巴，提取基因组DNA,进行STR检测和分子克隆验证，直至获得有效突变且稳定遗传的纯合突变体F1，饲养至成年，验证肌间刺变化，采用活体CT扫描，挑选出肌间刺粗大的个体，可用于稳定传代。Since the deletion of the mstn gene in the red-billed red tuna will lead to muscle hypertrophy, at this time, we selected the red-billed red tuna, which is larger than the wild species of the same age, for hybridization, and collected the fertilized eggs. After they developed to the incubation period, the genomic DNA of 10 embryos was randomly extracted. , STR verification was carried out, and the homozygous mutant F1 with stable inheritance was obtained. The F1 generation was raised to a body length of about 1 cm, and 30 individuals were randomly selected to clip part of their tails, extract genomic DNA, carry out STR detection and molecular cloning verification, until the homozygous mutant F1 with effective mutation and stable inheritance was obtained. In adulthood, the changes of intermuscular spines are verified, and in vivo CT scans are used to select individuals with thick intermuscular spines, which can be used for stable passage.

所述检测肌间刺变化的方法为鱼类硬骨染色和体外分离肌间刺，硬骨染色采用茜素红染色法。The method for detecting the change of the intermuscular spine is fish sclerotinic staining and in vitro separation of the intermuscular spine, and the sclerotinic staining adopts the alizarin red staining method.

实施例2Example 2

(1)团头鲂mstn基因核苷酸序列克隆(1) Nucleotide sequence cloning of the mstn gene

通过基因数据库下载团头鲂mstn序列，设计引物，扩增出mstn序列的部分CDS区，引物序列为：Download the mstn sequence from the gene database, design primers, and amplify part of the CDS region of the mstn sequence. The primer sequences are:

团头鲂mstnDory mstn

R:5’-CCACAGCGGTCTACTACCA-3’(SEQ ID NO:6)；R: 5'-CCACAGCGGTCTACTACCA-3' (SEQ ID NO: 6);

以团头鲂cDNA为模板，进行mstn序列的扩增，获得团头鲂mstn的编码区部分序列，如SEQ ID NO:2所示。Amplification of the mstn sequence was carried out using the cDNA of the bream bream as a template to obtain the partial sequence of the coding region of the bream bream mstn, as shown in SEQ ID NO: 2.

(2)团头鲂mstn基因的缺失突变体品系的构建(2) Construction of Mutant Lines Deleting the mstn Gene of Tuantou bream

采用基因敲除方法获得团头鲂mstn基因缺失突变体品系，以crispr/cas9构建缺失品系方法为例，具体步骤如下：The gene knockout method was used to obtain a mutant line of the bream mstn gene deletion. Taking the method of constructing a deletion line with CRISPR/cas9 as an example, the specific steps are as follows:

(i)团头鲂mstn靶基因的选择(i) Selection of mstn target genes

团头鲂mstn序列有三个外显子,为了更高效的进行基因编辑和长片段的碱基缺失，在第一外显子上设计3个靶点，序列为:The bream mstn sequence has three exons. In order to perform more efficient gene editing and long-range base deletion, three targets are designed on the first exon. The sequences are:

(ii)、团头鲂靶基因编辑(ii), target gene editing of Tuantou bream

将设计的3个靶点，分别体外转录成sgRNA，分别与Cas9 mRNA按比例共同混合至一管，配成用于基因编辑注射的药物，含：Cas9 mRNA终浓度300The three designed targets were transcribed into sgRNA in vitro, respectively mixed with Cas9 mRNA in proportion to a tube, and formulated into a drug for gene editing injection, including: Cas9 mRNA final concentration 300

b、获取性腺发育成熟的团头鲂，3-4冬龄以上、体重1.5kg以上体格健壮无损伤的雌雄鱼，雌雄比2:1，置于5m×1.2m的圆形帆布缸中，注射促性腺激素HCG，注射量雌鱼1600-2400U/kg，雄鱼800-1200U/kg。中间间隔1-3天，后进行人工授精，为方便显微注射，布卵至一次性塑料培养皿中，必要时需要脱黏，待受精卵吸水膨胀后立即于显微镜下进行显微注射，注射时将药物准确注射到一细胞期的细胞内，每个胚胎注射体积约2nl。将注射和未注射的受精卵及时送至于孵化器中，室温孵化(水温24-27℃，约20h出膜)b. Obtain the bream with mature gonads, 3-4 winter-old, healthy male and female fish with a body weight of 1.5kg or more, with a male-to-female ratio of 2:1, placed in a 5m×1.2m circular canvas tank, injected Gonadotropin HCG, the injection dose is 1600-2400U/kg for female fish and 800-1200U/kg for male fish. The interval is 1-3 days, and then artificial insemination is carried out. For the convenience of microinjection, the eggs are placed in a disposable plastic petri dish. If necessary, it needs to be debonded. After the fertilized eggs absorb water and swell, microinjection is carried out under the microscope immediately. When the drug is accurately injected into the cells of one cell stage, the injection volume of each embryo is about 2nl. Send the injected and uninjected fertilized eggs to the incubator in time, and incubate at room temperature (water temperature 24-27 ℃, about 20h out of the membrane)

分别随机抽取三组注射过的团头鲂胚胎和未注射的野生胚胎(注射12h后)，每组三颗，用剪刀剪碎胚胎，碱裂解法提取基因组DNA。Three groups of injected bream embryos and uninjected wild embryos (12h after injection) were randomly selected, three in each group. The embryos were cut with scissors, and the genomic DNA was extracted by alkaline lysis method.

利用荧光毛细管电泳法检测敲除效率。将检测有效的胚胎饲养至1个月，剪取个体尾鳍，提取基因组DNA,进行荧光STR检测，再通过分子克隆确定突变基因型，将编辑数目非3n的有效突变F0代留下，有效突变的团头鲂至少收集20尾，饲养至成年。Knockout efficiency was detected by fluorescent capillary electrophoresis. The effective embryos were raised for 1 month, the individual tail fins were clipped, genomic DNA was extracted, and fluorescent STR was detected. Then, the mutant genotype was determined by molecular cloning. At least 20 bream were collected and raised to adulthood.

所述检测引物为：The detection primers are:

T2:5’-GATGGTCTCTGTGGTGGCATG-3’(SEQ ID NO:19)；T2: 5'-GATGGTCTCTGTGGTGGCATG-3' (SEQ ID NO: 19);

由于团头鲂缺失mstn基因后会导致肌肉肥大，此时我们选取比同龄野生种体型肥大的团头鲂进行杂交，收集受精卵，待发育至孵化期后，随机提取10颗胚胎的基因组DNA，进行STR验证,获得可稳定遗传的纯合突变体F1。将F1代饲养至体长约1cm长左右，随机挑选30条个体分别剪取部分尾巴，提取基因组DNA,进行STR检测和分子克隆验证，直至获得有效突变且稳定遗传的纯合突变体F1，饲养至成年，验证肌间刺变化，采用活体CT扫描，挑选出肌间刺粗大的个体，可用于稳定传代。Since the lack of mstn gene in the bream can lead to muscle hypertrophy, we selected the bream that was larger than the wild species of the same age for crossbreeding, and collected fertilized eggs. STR verification was performed to obtain a homozygous mutant F1 that could stably inherit. The F1 generation was raised to a body length of about 1 cm, and 30 individuals were randomly selected to clip part of their tails, extract genomic DNA, carry out STR detection and molecular cloning verification, until the homozygous mutant F1 with effective mutation and stable inheritance was obtained. In adulthood, the changes of intermuscular spines are verified, and in vivo CT scans are used to select individuals with thick intermuscular spines, which can be used for stable passage.

以上所述，仅是本发明的较佳实施例而已，并非是对本发明作其它形式的限制，任何熟悉本专业的技术人员可能利用上述揭示的技术内容加以变更或改型为等同变化的等效实施例。但是凡是未脱离本发明技术方案内容，依据本发明的技术实质对以上实施例所作的任何简单修改、等同变化与改型，仍属于本发明技术方案的保护范。The above are only preferred embodiments of the present invention, and are not intended to limit the present invention in other forms. Any person skilled in the art may use the technical content disclosed above to make changes or modifications to equivalent changes. Example. However, any simple modifications, equivalent changes and modifications made to the above embodiments according to the technical essence of the present invention without departing from the content of the technical solutions of the present invention still belong to the protection scope of the technical solutions of the present invention.

序列表 sequence listing

<110> 上海海洋大学<110> Shanghai Ocean University

<120> 一种翘嘴红鲌和团头鲂肌间刺变粗的分子育种方法<120> A molecular breeding method for thickening the intermuscular spines of red tuna

<160> 19<160> 19

<170> SIPOSequenceListing 1.0<170> SIPOSequenceListing 1.0

<210> 1<210> 1

<211> 2777<211> 2777

<212> DNA<212> DNA

<213> 翘嘴红鲌(Erythroculter ilishaeformis)<213> Erythroculter ilishaeformis

<400> 1<400> 1

catgtggtcc agtgggtaat ggagatataa cggcgcacca gcagccttcc acagccacgg 60catgtggtcc agtgggtaat ggagatataa cggcgcacca gcagccttcc acagccacgg 60

aggaaagcga gcagtgttcc acatgtgagt ttagacaaca cagcaagctg atgagactgc 120aggaaagcga gcagtgttcc acatgtgagt ttagacaaca cagcaagctg atgagactgc 120

atgccatcaa gtcccaaatt cttagcaaac tccgactcaa acaggctcca aacatcagcc 180atgccatcaa gtcccaaatt cttagcaaac tccgactcaa acaggctcca aacatcagcc 180

gggacgtggt caaacagctg ttacccaaag caccgccttt gcaacaactt ctggatcagt 240gggacgtggt caaacagctg ttacccaaag caccgccttt gcaacaactt ctggatcagt 240

acgatgttct gggggatgac agtaaggatg gagctgtgga agaggatgat gaacatgcca 300acgatgttct gggggatgac agtaaggatg gagctgtgga agaggatgat gaacatgcca 300

ccacagagac catcatgacc atggccacag agcgtaagga tcatctattt caaagattca 360ccacagagac catcatgacc atggccacag agcgtaagga tcatctattt caaagattca 360

ttcatgtaaa tgtatcctct taacaatgga caaagaatat gtctacttgg agagaccctt 420ttcatgtaaa tgtatcctct taacaatgga caaagaatat gtctacttgg agagaccctt 420

tacgcagtct ttgtgcccaa gccagttgca cccacaaggc agctactggg cataaaatgc 480tacgcagtct ttgtgcccaa gccagttgca cccacaaggc agctactggg cataaaatgc 480

acagttaaga tgtataaaga cgattcctgt tttttgaggc tttgtgcccc tcttttctca 540acagttaaga tgtataaaga cgattcctgt tttttgaggc tttgtgcccc tcttttctca 540

gccttgctaa agcaatttta cgcacgctga acatggaagc gcggcaaaag cgcaccagcg 600gccttgctaa agcaatttta cgcacgctga acatggaagc gcggcaaaag cgcaccagcg 600

tcggtcagaa gaaaatgttt tcacctttac tacacgtgat aggctataaa ctgtaaattg 660tcggtcagaa gaaaatgttt tcacctttac tacacgtgat aggctataaa ctgtaaattg 660

gttcgtatct tagccaatct gtaggttaaa atgtgtagcc tattatgtgt aacgcctttt 720gttcgtatct tagccaatct gtaggttaaa atgtgtagcc tattatgtgt aacgcctttt 720

atatgtagtg tctaaatata gacactatat ctgtagccga tccattcgtt tttaaaggct 780atatgtagtg tctaaatata gacactatat ctgtagccga tccattcgtt tttaaaggct 780

tctgcaatgc cgtatgtata tgatttgcat gtctttttca ttttttttcc ttttaaataa 840tctgcaatgc cgtatgtata tgatttgcat gtctttttca ttttttttcc ttttaaataa 840

ctacatgtat gcaaactaca aaggctatat tgtcgatcaa aattttacat ataaatgtct 900ctacatgtat gcaaactaca aaggctatat tgtcgatcaa aattttacat ataaatgtct 900

cctttatgtc tgtagtgtat ttgattttgt gggcgtagtc aacgttattt ctttaaaagt 960cctttatgtc tgtagtgtat ttgattttgt gggcgtagtc aacgttattt ctttaaaagt 960

tacattttac attttgtctc ttttcattta tatcagtgcg tcatttcacg gacctcttta 1020tacattttac attttgtctc ttttcattta tatcagtgcg tcatttcacg gacctcttta 1020

ataggctaat aaaactgaaa accagtagat cccgtataat ttactttctc ttctttgttt 1080ataggctaat aaaactgaaa accagtagat cccgtataat ttactttctc ttctttgttt 1080

tcagctgacc ccattgttca agtagatcgg aaaccgaagt gttgtttttt ctccttcagt 1140tcagctgacc ccattgttca agtagatcgg aaaccgaagt gttgttttttt ctccttcagt 1140

ccgaaaatcc aagcgaaccg gatcgtaaga gcgcagctct gggttcatct gagaccggcg 1200ccgaaaatcc aagcgaaccg gatcgtaaga gcgcagctct gggttcatct gagaccggcg 1200

gaagaagcga ccaccgtctt cttacagata tcacggctaa tgcccgttac ggacggagga 1260gaagaagcga ccaccgtctt cttacagata tcacggctaa tgcccgttac ggacggagga 1260

agacacatac gaatacgatc cctgaagatc gacgtgaacg caggagtcac gtcttggcag 1320agacacatac gaatacgatc cctgaagatc gacgtgaacg caggagtcac gtcttggcag 1320

agtatagacg taaagcaggt gctctcggtg tggttaagac aaccggagac caactggggc 1380agtatagacg taaagcaggt gctctcggtg tggttaagac aaccggagac caactggggc 1380

atcgagataa acgcgtatga cgcgaaggga aacgacttgg ccgtcacctc agctgaggct 1440atcgagataa acgcgtatga cgcgaaggga aacgacttgg ccgtcacctc agctgaggct 1440

ggagaggatg gactggtgag ttgagctgtt ttgttaccaa atgtgcgttt tttacacaat 1500ggagaggatg gactggtgag ttgagctgtt ttgttaccaa atgtgcgttt tttacacaat 1500

acaaccgctt ttagacagag ctctgccagc agaaatcgac aatatcaaga aattgtacgg 1560acaaccgctt ttagacagag ctctgccagc agaaatcgac aatatcaaga aattgtacgg 1560

tctagttaaa caacactttg ttttaaatca gtaatctccc aaaaatgtgt gcacattttt 1620tctagttaaa caacactttg ttttaaatca gtaatctccc aaaaatgtgt gcacattttt 1620

attacttgca tgtttcggag cgcgcaacac cacatcagaa tttagagctc gaaacattcg 1680attacttgca tgtttcggag cgcgcaacac cacatcagaa tttagagctc gaaacattcg 1680

ggacccgata cctgaacgaa tgattctttc aatccggttc ttttgagtga atcaaaagcg 1740ggacccgata cctgaacgaa tgattctttc aatccggttc ttttgagtga atcaaaagcg 1740

aacagtgcga ccactgtaat tcggttttag aactaacgat tcttttgacc tagttcttgt 1800aacagtgcga ccactgtaat tcggttttag aactaacgat tcttttgacc tagttcttgt 1800

aatgaatcat cagcattcat caccttagta cagtccgatt cccgaacgaa tgactcttat 1860aatgaatcat cagcattcat caccttagta cagtccgatt cccgaacgaa tgactcttat 1860

tggccggttc tttttcgtga aaaacactta agaatcaatc gaaacggtta cagaattaaa 1920tggccggttc tttttcgtga aaaacactta agaatcaatc gaaacggtta cagaattaaa 1920

ctgactcaaa gaatcgcaag ttactttcgc catatctgac tcgaaacaaa ctgaaaaaaa 1980ctgactcaaa gaatcgcaag ttactttcgc catatctgac tcgaaacaaa ctgaaaaaaa 1980

aatcatgtta aggctcctga aactcaaatc agagtagtta gtggttgctt acatgacaac 2040aatcatgtta aggctcctga aactcaaatc agagtagtta gtggttgctt acatgacaac 2040

ttgtgttgta atcagtgata cattataaaa agaatgtttt agtattttta acataaatat 2100ttgtgttgta atcagtgata cattataaaa agaatgtttt agtattttta acataaatat 2100

aacgcaaagt gtgagaacag tcattaaaaa agcaaaaatt gtattgcctg tatttctgta 2160aacgcaaagt gtgagaacag tcattaaaaa agcaaaaatt gtattgcctg tatttctgta 2160

tattgattct tctgaaaact ataatgatcc catgatttgc tgggggcagt aaatgcaata 2220tattgattct tctgaaaact ataatgatcc catgatttgc tgggggcagt aaatgcaata 2220

tgaattgaat gatttttttt tttttttttt accagaagaa tccttaatgg caccattaag 2280tgaattgaat gatttttttt tttttttttt accagaagaa tccttaatgg caccattaag 2280

gaacaggaat tgttaagtgg aatcggaacc agaatcatta aattaattcc ctttcctaat 2340gaacaggaat tgttaagtgg aatcggaacc agaatcatta aattaattcc ctttcctaat 2340

cgtatctgta ctggctagct gtagcattcg ttaatgcttg gacaaagcaa tctcgacgtc 2400cgtatctgta ctggctagct gtagcattcg ttaatgcttg gacaaagcaa tctcgacgtc 2400

agatgagagt tagccataga tatgaaagta ccaagagtaa actgaagcgc tggttctttg 2460agatgagagt tagccataga tatgaaagta ccaagagtaa actgaagcgc tggttctttg 2460

ggttctttct ctcacagctc ccctttatag aggtgaaaat ctcagagggc ccaaagcgaa 2520ggttctttct ctcacagctc ccctttatag aggtgaaaat ctcagagggc ccaaagcgaa 2520

tccggaggga ctctggactg gactgcgacg agaattcctc agagtctcga tgctgcagat 2580tccggaggga ctctggactg gactgcgacg agaattcctc agagtctcga tgctgcagat 2580

accctctcac tgtggacttc gaggacttcg gctgggactg gattattgct ccgaaacgct 2640accctctcac tgtggacttc gaggacttcg gctgggactg gattattgct ccgaaacgct 2640

ataaggcgaa ttactgttcg ggagaatgcg actacatgca cctgcagaag tatccccaca 2700ataaggcgaa ttactgttcg ggagaatgcg actacatgca cctgcagaag tatccccaca 2700

cccatctggt gaacaaggcc aatccgcgag gcaccgccgg gccctgctgc acccccacca 2760cccatctggt gaacaaggcc aatccgcgag gcaccgccgg gccctgctgc acccccacca 2760

agatgtctcc catcaac 2777agatgtctcc catcaac 2777

<210> 2<210> 2

<211> 1146<211> 1146

<212> DNA<212> DNA

<213> 团头鲂(Megalobrama amblycephala)<213> Megalobrama amblycephala

<400> 2<400> 2

gcaaacatcc tctagcacgc cttggaacat gcattttacg caggttttaa tttctctaag 60gcaaacatcc tctagcacgc cttggaacat gcattttacg caggttttaa tttctctaag 60

tgtattaatt gcatgtggtc cagtgggtaa tggagatata acggcgcacc agcagccttc 120tgtattaatt gcatgtggtc cagtgggtaa tggagatata acggcgcacc agcagccttc 120

cacagccacg gaggaaagcg agcagtgttc cacatgtgag tttagacaac acagcaagct 180cacagccacg gaggaaagcg agcagtgttc cacatgtgag tttagacaac acagcaagct 180

gatgagactg catgccatca agtcccaaat tcttagcaaa ctccgactca aacaggctcc 240gatgagactg catgccatca agtcccaaat tcttagcaaa ctccgactca aacaggctcc 240

aaacatcagc cgggacgtgg tcaaacagct gttacccaaa gcaccgcctt tgcaacaact 300aaacatcagc cgggacgtgg tcaaacagct gttacccaaa gcaccgcctt tgcaacaact 300

tctggatcag tacgacgttc tgggggatga cagtaaggat ggagctgtgg aagaggatga 360tctggatcag tacgacgttc tgggggatga cagtaaggat ggagctgtgg aagaggatga 360

tgaacatgcc accacagaga ccatcatgac catggccaca gagcccgacc ccatcgttca 420tgaacatgcc accacagaga ccatcatgac catggccaca gagcccgacc ccatcgttca 420

agtagatcgg aaaccgaagt gttgtttttt ctccttcagt ccgaaaatcc aagcgaaccg 480agtagatcgg aaaccgaagt gttgtttttt ctccttcagt ccgaaaatcc aagcgaaccg 480

gatcgtaaga gcgcagctct gggttcatct gagaccggcg gaagaagcga ccaccgtctc 540gatcgtaaga gcgcagctct gggttcatct gagaccggcg gaagaagcga ccaccgtctc 540

cttacagata tcacggctga tgcccgttac ggacggagga agacacatac gaatacgatc 600cttacagata tcacggctga tgcccgttac ggacggagga agacacatac gaatacgatc 600

cctgaagatc gatgtgaacg caggagtcac gtcttggcag agtatagacg taaagcaggt 660cctgaagatc gatgtgaacg caggagtcac gtcttggcag agtatagacg taaagcaggt 660

gctctcggtg tggttaagac aaccggagac caactggggc atcgagataa acgcgtatga 720gctctcggtg tggttaagac aaccggagac caactggggc atcgagataa acgcgtatga 720

cgcgaaggga aacgacttgg ccgtcacctc agctgaggct ggagaggatg gactgctccc 780cgcgaaggga aacgacttgg ccgtcacctc agctgaggct ggagaggatg gactgctccc 780

ctttatggag gtgaaaatct cagagggccc aaagcgaatc cggagggact ctggactgga 840ctttatggag gtgaaaatct cagagggccc aaagcgaatc cggagggact ctggactgga 840

ctgcgacgag aattcctcag agtctcgatg ctgcagatac cctctcactg tggacttcga 900ctgcgacgag aattcctcag agtctcgatg ctgcagatac cctctcactg tggacttcga 900

ggacttcggc tgggactgga ttattgctcc gaaacgctat aaggcgaatt actgttcggg 960ggacttcggc tgggactgga ttattgctcc gaaacgctat aaggcgaatt actgttcggg 960

agaatgcgac tacatgcacc tgcagaagta tccccacacc catctggtga acaaggccaa 1020agaatgcgac tacatgcacc tgcagaagta tccccacacc catctggtga acaaggccaa 1020

tccgcgaggc accgccgggc cctgctgcac ccccaccaag atgtctccca tcaacatgct 1080tccgcgaggc accgccgggc cctgctgcac ccccaccaag atgtctccca tcaacatgct 1080

ttacttcaat ggcaaagagc agatcatcta cggcaagatc ccctcaatgg tagtagaccg 1140ttacttcaat ggcaaagagc agatcatcta cggcaagatc ccctcaatgg tagtagaccg 1140

ctgtgg 1146ctgtgg 1146

<210> 3<210> 3

<211> 21<211> 21

<212> DNA<212> DNA

<213> 人工序列(Erythroculter ilishaeformis)<213> Artificial Sequence (Erythroculter ilishaeformis)

<400> 3<400> 3

catgtggtcc agtgggtaat g 21catgtggtcc agtgggtaat g 21

<210> 4<210> 4

<211> 22<211> 22

<212> DNA<212> DNA

<400> 4<400> 4

gttgatggga gacatcttgg tg 22gttgatggga gacatcttgg tg 22

<210> 5<210> 5

<211> 20<211> 20

<212> DNA<212> DNA

<213> 人工序列(Megalobrama amblycephala)<213> Artificial Sequence (Megalobrama amblycephala)

<400> 5<400> 5

gcaaacatcc tctagcacgc 20gcaaacatcc tctagcacgc 20

<210> 6<210> 6

<211> 19<211> 19

<212> DNA<212> DNA

<400> 6<400> 6

ccacagcggt ctactacca 19ccacagcggt ctactacca 19

<210> 7<210> 7

<211> 20<211> 20

<212> DNA<212> DNA

<400> 7<400> 7

ggttctgggg gatgacagta 20ggttctgggg gatgacagta 20

<210> 8<210> 8

<211> 20<211> 20

<212> DNA<212> DNA

<400> 8<400> 8

gggatcagta cgatgttctg 20gggatcagta cgatgttctg 20

<210> 9<210> 9

<211> 20<211> 20

<212> DNA<212> DNA

<400> 9<400> 9

ggagccttcc acagccacgg 20ggagccttcc acagccacgg 20

<210> 10<210> 10

<211> 20<211> 20

<212> DNA<212> DNA

<400> 10<400> 10

gggctaatgc ccgttacgga 20gggctaatgc ccgttacgga 20

<210> 11<210> 11

<211> 20<211> 20

<212> DNA<212> DNA

<400> 11<400> 11

ggacaaccgg agaccaactg 20ggacaaccgg agaccaactg 20

<210> 12<210> 12

<211> 20<211> 20

<212> DNA<212> DNA

<400> 12<400> 12

gggtcttcct ccgtccgtaa 20gggtcttcct ccgtccgtaa 20

<210> 13<210> 13

<211> 20<211> 20

<212> DNA<212> DNA

<400> 13<400> 13

ggttctgggg gatgacagta 20ggttctgggg gatgacagta 20

<210> 14<210> 14

<211> 20<211> 20

<212> DNA<212> DNA

<400> 14<400> 14

ggagccttcc acagccacgg 20ggagccttcc acagccacgg 20

<210> 15<210> 15

<211> 20<211> 20

<212> DNA<212> DNA

<400> 15<400> 15

gggatcagta cgacgttctg 20gggatcagta cgacgttctg 20

<210> 16<210> 16

<211> 21<211> 21

<212> DNA<212> DNA

<400> 16<400> 16

catgtggtcc agtgggtaat g 21catgtggtcc agtgggtaat g 21

<210> 17<210> 17

<211> 20<211> 20

<212> DNA<212> DNA

<400> 17<400> 17

cgtttccctt cgcgtcatac 20cgtttccctt cgcgtcatac 20

<210> 18<210> 18

<211> 20<211> 20

<212> DNA<212> DNA

<400> 18<400> 18

gcaaacatcc tctagcacgc 20gcaaacatcc tctagcacgc 20

<210> 19<210> 19

<211> 21<211> 21

<212> DNA<212> DNA

<400> 19<400> 19

gatggtctct gtggtggcat g 21gatggtctct gtggtggcat g 21

Claims

Translated fromChinese

1.一种翘嘴红鲌和团头鲂肌间刺变粗的分子育种方法，其特征在于，步骤如下：1. a kind of molecular breeding method that the thorns of the red bream and the head bream intermuscular become thick, it is characterized in that, step is as follows:

2.根据权利要求1所述翘嘴红鲌和团头鲂肌间刺变粗的分子育种方法，其特征在于，所述步骤1)中克隆的翘嘴红鲌mstn基因核苷酸序列如SEQ ID NO:1所示，克隆的团头鲂mstn基因核苷酸序列如SEQ ID NO:2所示。2. according to the molecular breeding method that the red trout with cockroaches and the intermuscular spines of the bream are thickened according to claim 1, it is characterized in that, the red bream mstn gene nucleotide sequence of cloned in the described step 1) is such as SEQ ID NO: 1, the cloned bream mstn gene nucleotide sequence is shown in SEQ ID NO: 2.

3.根据权利要求1所述翘嘴红鲌和团头鲂肌间刺变粗的分子育种方法，其特征在于，所述步骤1)中克隆翘嘴红鲌mstn基因核苷酸序列的引物对为：3. according to the molecular breeding method of the described red bream and bream intermuscular thickening of claim 1, it is characterized in that, in described step 1), clone the primer pair of red bream mstn gene nucleotide sequence for:

F:5’-CATGTGGTCCAGTGGGTAATG-3’；F: 5'-CATGTGGTCCAGTGGGTAATG-3';

R:5’-GTTGATGGGAGACATCTTGGTG-3’；R: 5'-GTTGATGGGAGACATCTTGGTG-3';

F:5’-GCAAACATCCTCTAGCACGC-3’；F:5'-GCAAACATCCTCTAGCACGC-3';

R:5’-CCACAGCGGTCTACTACCA-3’。R: 5'-CCACAGCGGTCTACTACCA-3'.

4.根据权利要求1所述翘嘴红鲌和团头鲂肌间刺变粗的分子育种方法，其特征在于，步骤(2)所述的基因敲除方法为TALEN法或crispr/cas9法。4. The molecular breeding method for thickening intermuscular thorns of red bream and bream according to claim 1, is characterized in that, the gene knockout method described in step (2) is TALEN method or CRISPR/cas9 method.

5.根据权利要求4所述翘嘴红鲌和团头鲂肌间刺变粗的分子育种方法，其特征在于，采用CRISPR/Cas9基因敲除方法构建翘嘴红鲌和团头鲂mstn基因的缺失突变体品系的方法，具体为：5. according to the molecular breeding method of the thickened intermuscular spines of the red tuna and the head bream according to claim 4, it is characterized in that, adopt the CRISPR/Cas9 gene knockout method to construct the red bream and the head bream mstn gene. Methods for deletion mutant lines, specifically:

6.根据权利要求5所述翘嘴红鲌和团头鲂肌间刺变粗的分子育种方法，其特征在于，所述步骤①中基因编辑注射的药物含Cas9 mRNA终浓度300ng/μL，sgRNA终浓度50ng/μL。6. The molecular breeding method for thickening the intermuscular thorns of red bream and bream according to claim 5, is characterized in that, the medicine injected by gene editing in step 1. contains Cas9 mRNA final concentration 300ng/μL, sgRNA Final concentration 50ng/μL.

7.根据权利要求5所述翘嘴红鲌和团头鲂肌间刺变粗的分子育种方法，其特征在于，所述步骤①设计的翘嘴红鲌mstn基因敲除靶点序列为以下序列中的至少一条：7. according to the molecular breeding method that the red mussels of cockroaches and the intermuscular thorns of the bream are thickened according to claim 5, it is characterized in that, described step 1. designed red bream mstn gene knockout target sequence is following sequence At least one of:

exon1-target1:5’-GGTTCTGGGGGATGACAGTA-3’；exon1-target1:5'-GGTTCTGGGGGATGACAGTA-3';

exon1-target2:5’-GGGATCAGTACGATGTTCTG-3’；exon1-target2:5'-GGGATCAGTACGATGTTCTG-3';

exon1-target3:5’-GGAGCCTTCCACAGCCACGG-3’；exon1-target3:5'-GGAGCCTTCCACAGCCACGG-3';

exon2-target1:5’-GGGCTAATGCCCGTTACGGA-3’；exon2-target1:5'-GGGCTAATGCCCGTTACGGA-3';

exon2-target2:5’-GGACAACCGGAGACCAACTG-3’；exon2-target2:5'-GGACAACCGGAGACCAACTG-3';

exon2-target3:5’-GGGTCTTCCTCCGTCCGTAA-3；exon2-target3:5'-GGGTCTTCCTCCGTCCGTAA-3;

exon1-target2:5’-GGAGCCTTCCACAGCCACGG-3’；exon1-target2:5'-GGAGCCTTCCACAGCCACGG-3';

exon1-target3:5’-GGGATCAGTACGACGTTCTG-3’。exon1-target3:5'-GGGATCAGTACGACGTTCTG-3'.

8.根据权利要求5所述翘嘴红鲌和团头鲂肌间刺变粗的分子育种方法，其特征在于，所述步骤③中翘嘴红鲌检测所用引物为：8. according to the molecular breeding method that the red tuna with raised mouth and the intermuscular thorns of the head bream thicken according to claim 5, it is characterized in that, described step 3. in the red bream with the raised mouth detects the primers used are:

T1:5’-CATGTGGTCCAGTGGGTAATG-3’；T1:5'-CATGTGGTCCAGTGGGTAATG-3';

T2:5’-CGTTTCCCTTCGCGTCATAC-3’；T2:5'-CGTTTCCCTTCGCGTCATAC-3';

T1:5’-GCAAACATCCTCTAGCACGC-3’；T1:5'-GCAAACATCCTCTAGCACGC-3';

T2:5’-GATGGTCTCTGTGGTGGCATG-3’。T2: 5'-GATGGTTCTCTGTGGTGGCATG-3'.

9.根据权利要求5所述翘嘴红鲌和团头鲂肌间刺变粗的分子育种方法，其特征在于：步骤②所述的注射绒促性素HCG的注射用量为：雌鱼1600-2400U/kg，雄鱼800-1200U/kg。9. according to the molecular breeding method of the described cockroach red bream and the head bream intermuscular thorn thickening of claim 5, it is characterized in that: the injection dosage of step 2. described injection chorionic gonadotropin HCG is: female fish 1600- 2400U/kg, male fish 800-1200U/kg.

10.根据权利要求1～9任一项所述翘嘴红鲌和团头鲂肌间刺变粗的分子育种方法，其特征在于：所述步骤(3)中检测肌间刺变化的方法为鱼类硬骨染色和体外分离肌间刺，其中硬骨染色采用茜素红染色法。10. The molecular breeding method for thickening the intermuscular thorns of the red tuna with cockroaches according to any one of claims 1 to 9, and it is characterized in that: in the step (3), the method for detecting the variation of the intermuscular thorns is: Fish sclerotinic staining and in vitro isolation of intermuscular spines, in which alizarin red staining was used for sclerotinic staining.