WO2020207002A1 - 一种肌肉生长抑制素mstn的突变体及其应用 - Google Patents

一种肌肉生长抑制素mstn的突变体及其应用 Download PDF

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WO2020207002A1
WO2020207002A1 PCT/CN2019/115749 CN2019115749W WO2020207002A1 WO 2020207002 A1 WO2020207002 A1 WO 2020207002A1 CN 2019115749 W CN2019115749 W CN 2019115749W WO 2020207002 A1 WO2020207002 A1 WO 2020207002A1
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mstn
gene
mutant
animal
muscle
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连正兴
李岩
邓守龙
刘国世
连玲
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中国农业大学
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers

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  • the invention relates to the fields of genetic engineering and genetic breeding, in particular to a mutant of myostatin (MSTN) and its application.
  • MSTN myostatin
  • Myostatin also known as GDF-8, belongs to the TGF ⁇ (transforming growth factor beta) superfamily. McPherron et al. cloned the cDNA sequence of myostatin gene from different species, and found that the amino acid sequence homology of the C-terminal region of the myostatin protein of mouse, rat, human, pig, chicken and turkey was as high as 100%. In 1999, Stratil and others cloned the pig myostatin gene. The pig Myostatin gene contains 3 exons and 2 introns.
  • Myostatin protein contains 376 amino acid residues, mainly including three parts, the N-terminal signal peptide sequence, a protease hydrolysis site containing 4 amino acid residues (RSRR), and the C-terminal mature region containing 109 amino acid residues sequence. Myostatin has a significant negative regulatory effect on the growth and development of skeletal muscle.
  • the MSTN gene of Pyrmont cattle has a missense mutation of a base in the third exon, which causes the cysteine in the C-terminal mature peptide region of the Myostatin protein to be replaced by tyrosine, which cannot form a normal biological activity.
  • the C-terminal mature peptide protein of Myostatin causes abnormally developed skeletal muscles in the hips, thighs, and upper arms of the double-muscle cattle.
  • the content of skeletal muscle is 20%-25% higher than that of ordinary cattle.
  • the skeletal muscle content of Texel sheep was significantly increased.
  • the MSTN gene has a frameshift mutation.
  • the translation of Myostatin protein is terminated prematurely, and the Myostatin protein with normal biological functions cannot be formed. It has also been found in humans that the base mutation of the first intron of the MSTN gene leads to errors in the splicing of the Myostatin mRNA sequence, which makes the Myostatin protein lose its normal biological activity.
  • the purpose of the present invention is to provide a mutant of myostatin (MSTN) and its application.
  • the activity of myostatin MSTN has a complicated relationship with the regulation of animal skeletal muscle growth and development.
  • MSTN plays a negative role in skeletal muscle growth and development, the loss of MSTN gene or the loss of MSTN activity can promote myogenesis and development. It's not obvious.
  • Different forms of low-activity mutants of muscle MSTN gene have great differences in the effects of skeletal muscle growth and muscle content.
  • the present invention screens and obtains a sheep MSTN mutant by randomly performing mutations on the MSTN gene of sheep, and the mutant is the 73rd half of the mutant lacking the wild-type MSTN mature peptide (shown in SEQ ID NO.
  • Cystine is the 339th cysteine of the full-length MSTN protein (Protein ID: NP_001009428.1) containing the signal peptide; the coding gene of this MSTN mutant is the deletion of the wild-type MSTN mature peptide gene (such as SEQ ID No. 4) of the third exon 269-271, that is, the MSTN full-length gene coding region (Gene ID: 443449) containing the signal peptide 1016-1018, the MSTN mutant can significantly improve The muscle fiber density of the animal, the animal carrying the MSTN mutant has obvious double gluteal traits.
  • the present invention provides a myostatin MSTN protein mutant having any one of the following amino acid sequences:
  • the MSTN protein mutant as shown in SEQ ID NO.1 is missing the 73rd cysteine of the mature peptide of the sheep wild-type MSTN protein as shown in SEQ ID NO.3, that is, it contains the signal peptide.
  • MSTN protein of other animals obtained by homology comparison with position 73 of the MSTN protein shown in SEQ ID NO.3
  • MSTN protein mutants of other animals with the same function obtained after the amino acid corresponding to cysteine are deleted are also within the protection scope of the present invention.
  • the present invention provides a gene encoding the MSTN protein mutant.
  • the gene encoding the MSTN protein mutant has any one of the following nucleotide sequences:
  • nucleotide sequence of the same functional protein obtained by replacing, deleting or inserting one or more nucleotides in the nucleotide sequence shown in SEQ ID NO. 2.
  • the coding gene of the MSTN protein mutant shown in SEQ ID NO. 2 is the deletion of positions 269-271 of the third exon of the sheep wild-type MSTN mature peptide coding gene shown in SEQ ID NO. 4 , That is, positions 1016-1018 of the coding region of the full-length MSTN gene containing the signal peptide.
  • the present invention provides biological materials containing genes encoding the MSTN protein mutants.
  • the biological material includes recombinant DNA, vectors, host cells, and engineered bacteria.
  • the vector of the present invention can be a vector used for cloning, expression and other gene editing, as long as it contains the gene of the present invention or the vector of the present invention, it is within the protection scope of the present invention. within.
  • the present invention provides the application of the gene encoding the MSTN protein mutant or the biological material containing the gene in any of the following aspects:
  • the application is to mutate the MSTN encoding gene of the animal into the gene encoding the MSTN protein mutant of the present invention.
  • the animal can be various domestic animals, poultry and other large or small mammals.
  • the animals described in the present invention are pigs, cattle, sheep, horses, chickens, dogs, mice, rats and the like.
  • the present invention provides a genetic breeding method for a double-muscular gluteal animal, which is to mutate the MSTN encoding gene of the animal into the gene encoding the MSTN protein mutant of the present invention.
  • the present invention provides a gene-edited animal that contains the MSTN mutant or is prepared by the genetic breeding method of the double muscular gluteal animal.
  • the present invention also provides a sheep, which carries the encoding gene of the MSTN mutant shown in SEQ ID NO.2.
  • the beneficial effects of the present invention are: the present invention obtains a MSTN protein mutant through random mutation and screening, and the mutant can obviously promote the growth and development of animal skeletal muscle.
  • the muscle fiber density of the animal carrying the MSTN mutant of the present invention is significantly increased (the muscle fiber density of the sheep carrying the MSTN mutant is increased by more than 50%) and the proportion of slow-twitch fibers is significantly increased.
  • the animal has an obvious double-muscle gluteal phenotype, thereby producing meat The amount has also increased significantly.
  • the MSTN protein mutant and its encoding gene provided by the present invention can be used in practice to construct a double gluteal phenotype animal model or for genetic breeding of double gluteal muscle trait animals.
  • Figure 1 is a schematic structural diagram of the MSTN gene targeting site in Example 1 of the present invention.
  • the sequence in the figure includes the corresponding targeting site of the genome and the PAM segment of the site.
  • Figure 2 is an alignment of the mutant sequence of MSTN gene and protein in Example 3 of the present invention, where A is the alignment of the wild-type sheep MSTN gene sequence and the mutant MSTN gene sequence, WT represents the wild-type sequence, #3005 represents gene editing For sheep, -3 represents the deletion of 3 bases; B is the amino acid sequence alignment of wild-type MSTN protein and MSTN protein mutant, #3005 represents gene-edited sheep, and aa represents amino acid.
  • Figure 3 is a comparison diagram of the body condition of the MSTN mutant sheep (marked with dots on the tail or dots on the chest) and half-sib gene-edited negative sheep (without dots) in Example 3 of the present invention, where A is sheep Rear view of the body, B is a partial enlarged view of the buttocks, C is the front view of the sheep, and D is the side view of the MSTN mutant sheep.
  • Figure 4 is a muscle section diagram of the hind gluteal muscle and longissimus dorsi of the MSTN mutant sheep (MSTN-/-) and half-sib gene-edited negative sheep (MSTN+/+) in Example 3 of the present invention, where A is the MSTN mutant sheep (MSTN-/-) posterior gluteal muscle section; B: half-sib gene-edited negative sheep (MSTN+/+) posterior gluteal muscle section; C: MSTN mutant sheep (MSTN-/-) with the longest back Muscle section; D is the longissimus dorsi muscle section of half-sib gene-edited negative sheep (MSTN+/+).
  • A is the MSTN mutant sheep (MSTN-/-) posterior gluteal muscle section
  • B half-sib gene-edited negative sheep (MSTN+/+) posterior gluteal muscle section
  • C MSTN mutant sheep (MSTN-/-) with the longest back Muscle section
  • D is the longissimus do
  • Figure 5 is a statistical diagram of muscle fiber density of the Gluteus muscles and Longissimus dorsi of MSTN mutant sheep (MSTN-KO) and half-sib gene-edited negative sheep (WT) in Example 3 of the present invention ( A), and the distribution map of the posterior gluteal muscle fiber area (B).
  • Example 1 Design of CRISPR/Cas9 gene editing sgRNA of sheep MSTN gene
  • a targeting site was designed for the MSTN gene of sheep.
  • the target site is located on the third exon of the MSTN gene. Its position and structure in the sheep genome are shown in Figure 1.
  • the sgRNA sequence used for targeting is shown in SEQ. ID NO.5 shown.
  • a total of 134 fertilized eggs were obtained from 14 donor ewes, and 128 fertilized eggs were transplanted into Cas9 mRNA and MSTN sgRNA (the molar concentration ratio of Cas9 mRNA and MSTN sgRNA is 1:10) using prokaryotic microinjection.
  • 31 recipient ewes 30 days after embryo transfer, after B-ultrasound examination, 9 recipients became pregnant and 6 lambs were born.
  • Lamb blood was taken to extract genomic DNA, and MSTN detection primers were used for PCR amplification.
  • the PCR products were sent to the company for sequencing. The sequencing results showed that a total of 1 lamb had mutations and were homozygous. The positive rate of gene editing was 16.7%.
  • the MSTN detection primer sequence is as follows:
  • SEQ ID NO. 6 F: 5’-AAGTCAAGGTAACAGACACACC-3’;
  • SEQ ID NO. 7 R: 5'-TGTGTTTTAGGAAGCTATGAAC-3'.
  • MSTN protein The sequence of the mutant is shown in SEQ ID NO. 1; the sequence of the MSTN gene mutant is shown in SEQ ID NO. 2; the wild-type MSTN protein mature peptide sequence is shown in SEQ ID NO. 3; the wild-type MSTN mature peptide code The sequence of the gene is shown in SEQ ID NO.4.
  • MSTN mutant lambs and half-sib gene-edited negative lambs were fed and phenotyped at the same time.
  • the table of mutant sheep on the 180th day (6 months of age) and the same-bred half-sib gene-edited negative sheep The results of the type comparison are shown in Figure 3. The results show that compared with half-sib gene-edited negative sheep, MSTN mutant sheep (with dots on the tail) have abnormally developed and plump buttocks muscle groups, and a clear double-muscle gluteal phenotype appears. .
  • MSTN mutant sheep MSTN-/-, Figure 4, A
  • half-sib gene editing negative sheep MSTN+/+, Figure 4, B
  • the muscle fiber density and muscle fiber area distribution of the posterior gluteal muscle and longissimus dorsi of MSTN mutant sheep and half-sib gene-edited negative sheep were quantitatively detected. The results are shown in Figure 5. Compared with half-sib gene-edited negative sheep, MSTN mutation Sheep’s muscle fiber density has increased by more than 50% (shown in Figure 5, A), and the proportion of slow muscle fibers has increased significantly ( Figure 5, B). Due to the developed capillaries of the slow muscle fibers and more mitochondria, the MSTN mutant sheep have stronger exercise endurance. .
  • the MSTN mutant sheep carrying the cysteine 73 deletion of the MSTN protein mature peptide provided by the present invention is stored in the breeding farm under the Tianjin Institute of Animal Husbandry and Veterinary Medicine.
  • the present invention provides a mutant of myostatin MSTN and its application.
  • the MSTN protein mutant provided by the present invention has an amino acid sequence as shown in SEQ ID NO.1.
  • the mutant can obviously promote the growth and development of the animal's skeletal muscle, the muscle fiber density of the animal carrying the MSTN mutant of the present invention is significantly increased, the animal exhibits obvious double-muscle gluteal traits, and the meat yield can also increase accordingly.
  • the MSTN protein mutant and its encoding gene provided by the present invention can be used in practice to prepare a double gluteal phenotype animal model or for genetic breeding of double gluteal muscle trait animals, and has good economic value and application prospects.

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Abstract

本发明涉及基因工程及遗传育种技术领域，具体涉及一种肌肉生长抑制素MSTN的突变体及其应用。本发明提供的MSTN蛋白突变体，具有如SEQ ID NO.1所示的氨基酸序列。所述突变体能够明显促进动物的骨骼肌的生长发育，携带本发明所述的MSTN突变体的动物的肌纤维密度显著提高，动物表现明显的双肌臀性状，产肉量也可随之增加。本发明提供的MSTN蛋白突变体及其编码基因可在实践中用于制备双肌臀表型动物模型或用于双臀肌性状动物的遗传育种。

Description

一种肌肉生长抑制素MSTN的突变体及其应用
交叉引用
本申请要求2019年4月8日提交的专利名称为“一种肌肉生长抑制素MSTN的突变体及其应用”的第201910277191.9号中国专利申请的优先权，其全部公开内容通过引用整体并入本文。
技术领域
本发明涉及基因工程及遗传育种领域，具体涉及一种肌肉生长抑制素(MSTN)的突变体及其应用。
背景技术
肌肉生长抑制素(myostatin，MSTN)，又称GDF-8，属于TGFβ(transforming growth factor beta)超家族的成员。McPherron等克隆了不同物种的myostatin基因的cDNA序列,经分析发现，小鼠、大鼠、人、猪、鸡和火鸡的myostatin蛋白的C-末端区域的氨基酸序列的同源性高达100％。1999年，Stratil等克隆出了猪的myostatin基因，猪Myostatin基因含有3个外显子和2个内含子。Myostatin蛋白含有376个氨基酸残基，主要包括三个部分，N末端的信号肽序列，一个含有4个氨基酸残基(RSRR)的蛋白酶水解位点，C末端的含有109个氨基酸残基的成熟区序列。Myostatin对骨骼肌的生长发育具有明显的负调控作用。
目前，已在不同物种中发现MSTN基因序列的不同突变类型导致了Myostatin蛋白活性的降低，进而导致骨骼肌肥大的现象。皮尔蒙特牛的MSTN基因在第三外显子上有一个碱基的错义突变,导致Myostatin蛋白的C端成熟肽区域的半胱氨酸被酪氨酸替换，不能形成具有正常生物学活性的Myostatin的C端成熟肽蛋白，该突变造成双肌牛的臀部、大腿和上臂等部位的骨骼肌异常发达，其骨骼肌的含量比普通牛高出20％-25％。比利时兰牛的MSTN基因的第三外显子上有11个碱基的缺失，导致移码突变，翻译过程提前终止，使得Myostatin蛋白的C端成熟肽部分仅仅只 有7个氨基酸，剩下的102个氨基酸(274～375)全部缺失，不能形成具有正常生物学活性的C端成熟肽蛋白。特赛尔绵羊的MSTN基因的3’UTR区域存在一个G到A的碱基突变，这部分序列恰好是mir1和mir206的靶向序列，因此导致mir1和mir206的活性受损，Myostatin蛋白的活性受到了明显的抑制，特赛尔绵羊的骨骼肌含量显著提高。惠比特犬的MSTN基因的第三外显子上存在两个碱基对的缺失，MSTN基因出现了移码突变，Myostatin蛋白的翻译提前终止，无法形成具有正常生物学功能Myostatin蛋白。在人类也发现MSTN基因的第一内含子的碱基突变导致Myostatin的mRNA序列的剪切方式出现错误，使得Myostatin蛋白失去了正常的生物学活性。
发明内容
为了解决现有技术中存在的问题，本发明的目的是提供一种肌肉生长抑制素(MSTN)的突变体及其应用。
肌肉生长抑制素MSTN的活性与动物骨骼肌生长发育的调控作用关系复杂，尽管MSTN对于骨骼肌生长发育起负调控的作用，但是，MSTN基因的缺失或MSTN活性的丧失对于肌肉生成发育的促进作用并不明显。肌肉MSTN基因的不同形式的低活性突变体，对于骨骼肌生长发育以及肌肉的含量水平的影响存在较大差异。本发明通过对绵羊的MSTN基因进行随机的基因突变，筛选得到一种绵羊MSTN的突变体，该突变体为缺失了野生型MSTN成熟肽(如SEQ ID NO.3所示)的第73位半胱氨酸，即含有信号肽的MSTN全长蛋白(Protein ID:NP_001009428.1)的第339位半胱氨酸；该MSTN突变体的编码基因为缺失了野生型MSTN成熟肽基因(如SEQ ID NO.4所示)的第3外显子的269-271位，即含有信号肽的MSTN全长基因编码区(Gene ID:443449)的第1016-1018位，所述MSTN突变体能够明显提高动物的肌纤维密度，携带MSTN突变体的动物具有明显的双肌臀性状。
第一方面，本发明提供一种肌肉生长抑制素MSTN蛋白突变体，具 有如下任意一种氨基酸序列：
(1)如SEQ ID NO.1所示的氨基酸序列；
(2)如SEQ ID NO.1所示的氨基酸序列经一个或多个氨基酸替换、缺失或插入得到的具有相同功能蛋白的氨基酸序列。
本发明中，如SEQ ID NO.1所示的MSTN蛋白突变体为缺失了如SEQ ID NO.3所示的绵羊野生型MSTN蛋白成熟肽的第73位半胱氨酸，即含有信号肽的MSTN蛋白全长的第339位半胱氨酸。
本领域技术人员应该理解，根据不同动物MSTN蛋白的保守性和同源性，将其它动物的MSTN蛋白中通过同源比对获得的与如SEQ ID NO.3所示的MSTN蛋白的第73位半胱氨酸相对应的氨基酸缺失后获得的具有相同功能的其它动物的MSTN蛋白突变体也在本发明保护范围内。
第二方面，本发明提供编码所述MSTN蛋白突变体的基因。
具体地，所述编码MSTN蛋白突变体的基因具有如下任意一种核苷酸序列：
(1)如SEQ ID NO.2所示的核苷酸序列；
(2)如SEQ ID NO.2所示的核苷酸序列经一个或多个核苷酸替换、缺失或插入得到的具有相同功能蛋白的核苷酸序列。
本发明中，如SEQ ID NO.2所示的MSTN蛋白突变体的编码基因为缺失了如SEQ ID NO.4所示的绵羊野生型MSTN成熟肽编码基因第三外显子的269-271位，即含有信号肽的MSTN全长基因编码区的第1016-1018位。
第三方面，本发明提供包含编码所述MSTN蛋白突变体的基因的生物材料。
作为优选，所述生物材料包括重组DNA、载体、宿主细胞、工程菌。
本领域技术人员应当理解，本发明所述的载体可以为用于克隆、表达等各种基因编辑的载体，只要含有本发明所述基因或本发明所述的载体，均在本发明的保护范围之内。
第四方面，本发明提供编码所述MSTN蛋白突变体的基因或包含所述基因的生物材料在如下任意一方面的应用：
(1)在提高动物肌肉含量或促进动物肌肉发育中的应用；
(2)提高动物产肉量中的应用；
(3)在制备基因编辑动物或在双肌臀动物遗传育种中的应用。
本发明中，所述应用为将所述动物的MSTN编码基因突变为本发明所述的编码MSTN蛋白突变体的基因。
本发明中，所述动物可以为各种家畜、家禽以及其它大型或小型哺乳动物。
作为优选实施方式，本发明所述的动物为猪、牛、羊、马、鸡、狗、小鼠、大鼠等。
第五方面，本发明提供一种双肌臀动物的遗传育种方法，为将所述动物的MSTN编码基因突变为本发明所述的编码MSTN蛋白突变体的基因。
第六方面，本发明提供含有所述MSTN突变体或利用所述双肌臀动物的遗传育种方法制备得到的基因编辑动物。
第七方面，本发明还提供一种绵羊，其携带如SEQ ID NO.2所示的MSTN突变体的编码基因。
本发明的有益效果在于：本发明通过随机突变和筛选得到了一种MSTN蛋白突变体，该突变体能够明显促进动物骨骼肌的生长发育。携带本发明所述的MSTN突变体的动物的肌纤维密度显著提高(携带MSTN突变体的绵羊的肌纤维密度提高50％以上)且慢肌纤维比例显著提高，动物出现明显的双肌臀表型，进而产肉量也显著增加。本发明提供的MSTN蛋白突变体及其编码基因可在实践中用于构建双肌臀表型动物模型或用于双臀肌性状动物的遗传育种。
附图说明
图1为本发明实施例1中MSTN基因打靶位点的结构示意图，图中 序列包含基因组相应的打靶位点以及该位点的PAM区段。
图2为本发明实施例3中MSTN基因和蛋白的突变序列比对，其中，A为野生型绵羊MSTN基因的序列和突变体MSTN基因序列比对，WT代表野生型序列，#3005代表基因编辑绵羊，-3代表3个碱基的缺失；B为野生型MSTN蛋白和MSTN蛋白突变体的氨基酸序列比对，#3005代表基因编辑绵羊，aa代表氨基酸。
图3为本发明实施例3中MSTN突变绵羊(尾部标记有圆点或胸部标记有圆点)与半同胞基因编辑阴性绵羊个体(无圆点标记)的体况对比图，其中，A为绵羊身体后视图，B为臀部局部放大图，C为绵羊前视图，D为MSTN突变绵羊侧视图。
图4为本发明实施例3中MSTN突变绵羊(MSTN-/-)与半同胞基因编辑阴性绵羊(MSTN+/+)的后臀肌和背最长肌的肌肉切片图，其中A为MSTN突变绵羊(MSTN-/-)的后臀肌肌肉切片图；B为半同胞基因编辑阴性绵羊(MSTN+/+)的后臀肌肌肉切片图；C为MSTN突变绵羊(MSTN-/-)的背最长肌肌肉切片图；D为半同胞基因编辑阴性绵羊(MSTN+/+)的背最长肌肌肉切片图。
图5为本发明实施例3中MSTN突变绵羊(MSTN-KO)与半同胞基因编辑阴性绵羊(WT)的后臀肌(Gluteus muscles)和背最长肌(Longissimus dorsi)的肌纤维密度统计图(A)，以及后臀肌肌纤维面积分布图(B)。
具体实施方式
下面将结合实施例对本发明的优选实施方式进行详细说明。需要理解的是以下实施例的给出仅是为了起到说明的目的，并不是用于对本发明的范围进行限制。本领域的技术人员在不背离本发明的宗旨和精神的情况下，可以对本发明进行各种修改和替换。
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。
下述实施例中所用的材料、试剂等，如无特殊说明，均可从商业途径 得到。
实施例1：绵羊MSTN基因CRISPR/Cas9基因编辑sgRNA的设计
针对绵羊的MSTN基因的设计了1个打靶位点，该靶点位于MSTN基因第三外显子上，其在绵羊基因组中的位置和结构如图1所示，用于打靶的sgRNA序列如SEQ ID NO.5所示。
实施例2：绵羊的MSTN基因编辑及基因编辑绵羊的筛选鉴定
从14只供体母羊共获取了134枚受精卵，将采用原核显微注射方法导入Cas9 mRNA和MSTN sgRNA(Cas9 mRNA和MSTN sgRNA的摩尔浓度比例为1：10)的128枚受精卵移植至31只受体母羊，胚胎移植30天后，经过B超检查，9只受体怀孕，共产羔6只。取羔羊血液提取基因组DNA，采用MSTN检测引物进行PCR扩增，将PCR产物送至公司测序，测序结果显示，共有1只羔羊产生了突变，且为纯合子，基因编辑的阳性率为16.7％。MSTN检测引物序列如下：
SEQ ID NO.6：F：5’-AAGTCAAGGTAACAGACACACC-3’；
SEQ ID NO.7：R：5’-TGTGTTTTAGGAAGCTATGAAC-3’。
表1 绵羊的MSTN基因突变效率
Figure PCTCN2019115749-appb-000001
实施例3 MSTN基因的突变体检测和表型分析
将实施例2得到的PCR扩增产物连接T载体后挑取了49个单克隆进行测序，测序结果经与NCBI收录的参考绵羊MSTN基因(登录号NC_019459.2)的核苷酸序列进行比对，发现49个克隆均为MSTN基因第3外显子的269-271位(即含信号肽的MSTN全长基因编码区的第1016-1018位)出现了3个碱基的缺失(如图2的A所示)，对应MSTN蛋白成熟肽的第73位(即含信号肽的MSTN全长蛋白的第339位氨基酸)半胱氨酸发生了缺失(如图2的B所示)；MSTN蛋白突变体的序列如SEQ  ID NO.1所述；MSTN基因突变体的序列如SEQ ID NO.2所述；野生型MSTN蛋白成熟肽序列如SEQ ID NO.3所示；野生型MSTN成熟肽编码基因的序列如SEQ ID NO.4所示。
对MSTN突变羔羊和半同胞基因编辑阴性羔羊(MSTN基因型为野生型)同时进行饲养和表型观察，第180天(6月龄)的突变绵羊与同样饲养的半同胞基因编辑阴性绵羊的表型对比结果如图3所示，结果表明，与半同胞基因编辑阴性绵羊相比，MSTN突变绵羊(尾部标有圆点)的臀部肌肉群异常发达和丰满，出现了明显的双肌臀表型。MSTN突变绵羊(MSTN-/-，图4的A)与半同胞基因编辑阴性绵羊(MSTN+/+，图4的B)的后臀肌的肌肉切片图相比可知，MSTN突变绵羊的后臀肌的肌纤维密度显著提高；MSTN突变绵羊(MSTN-/-，图4的C)与半同胞基因编辑阴性绵羊(MSTN+/+，图4的D)的背最长肌的肌肉切片相比可知，MSTN突变绵羊的背最长肌的肌纤维密度显著提高。对MSTN突变绵羊与半同胞基因编辑阴性绵羊的后臀肌和背最长肌的肌纤维密度及肌纤维面积分布进行定量检测，结果如图5所示，与半同胞基因编辑阴性绵羊相比，MSTN突变绵羊的肌纤维密度提高了50％以上(图5的A所示)，慢肌纤维比例显著增加(图5的B)，由于慢肌纤维毛细血管发达，线粒体较多，使得MSTN突变绵羊的运动耐力更强。
本发明提供的携带MSTN蛋白成熟肽的第73位半胱氨酸缺失的MSTN突变体绵羊保存在天津畜牧兽医研究所下属种羊场。
以上所述仅是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明技术原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。
工业实用性
本发明提供一种肌肉生长抑制素MSTN的突变体及其应用。本发明提供的MSTN蛋白突变体，具有如SEQ ID NO.1所示的氨基酸序列。所述突变体能够明显促进动物的骨骼肌的生长发育，携带本发明所述的 MSTN突变体的动物的肌纤维密度显著提高，动物表现明显的双肌臀性状，产肉量也可随之增加。本发明提供的MSTN蛋白突变体及其编码基因可在实践中用于制备双肌臀表型动物模型或用于双臀肌性状动物的遗传育种，具有较好的经济价值和应用前景。

Claims (10)

  1. 一种肌肉生长抑制素MSTN蛋白突变体，其特征在于，具有如下任意一种氨基酸序列：
    (1)如SEQ ID NO.1所示的氨基酸序列；
    (2)如SEQ ID NO.1所示的氨基酸序列经一个或多个氨基酸替换、缺失或插入得到的具有相同功能蛋白的氨基酸序列。
  2. 编码权利要求1所述的MSTN蛋白突变体的基因。
  3. 根据权利要求2所述的基因，其特征在于，具有如下任意一种核苷酸序列：
    (1)如SEQ ID NO.2所示的核苷酸序列；
    (2)如SEQ ID NO.2所示的核苷酸序列经一个或多个核苷酸替换、缺失或插入得到的具有相同功能蛋白的核苷酸序列。
  4. 包含权利要求2或3所述基因的生物材料。
  5. 根据权利要求4所述的生物材料，其特征在于，包括重组DNA、载体、宿主细胞、工程菌。
  6. 权利要求2或3所述基因或权利要求4或5所述生物材料在提高动物肌肉含量或促进动物肌肉发育中的应用。
  7. 权利要求2或3所述基因或权利要求4或5所述生物材料在提高动物产肉量中的应用。
  8. 权利要求2或3所述基因或权利要求4或5所述生物材料在制备基因编辑动物或在双肌臀动物遗传育种中的应用。
  9. 根据权利要求6～8任一项所述的应用，其特征在于，所述应用为将所述动物的MSTN编码基因突变为权利要求2或3所述的基因。
  10. 一种双肌臀动物的遗传育种方法，其特征在于，将所述动物的MSTN编码基因突变为权利要求2或3所述基因。
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