Cloning and Sequence Analysis of Myostatin(MSTN) Gene from Fujian White Rabbit
-
摘要: 为掌握福建白兔负向调控骨骼肌生长发育的肌肉生长抑制素（Myostatin，MSTN）基因分子基础，应用RT-PCR和RACE技术从肌肉组织总RNA中克隆出福建白兔MSTN基因的cDNA序列并进行分析。结果表明，该基因序列全长1 551 bp（GenBank登录号：KX084386），其中5'非翻译区136 bp，3'非翻译区287 bp，1 128 bp的开放阅读框，编码375个氨基酸，MSTN蛋白在第97~768核苷酸处编码的氨基酸存在1个TGF-β前肽超家族结构域，在第841~1 125核苷酸处编码的氨基酸存在1个TGF-β家族结构域。序列分析表明：福建白兔与穴兔、人、猪、家鼠、马、牛、绵羊、鹿、狒狒、猩猩、猴的MSTN核苷酸序列同源性分别为99.9%、92.8%、94.4%、86.9%、95.2%、89.1%、91.0%、90.3%、95.9%、96.4%、95.9%。研究结果为进一步开展福建白兔MSTN基因的结构、表达以及分子育种等相关研究提供了基础数据。Abstract: In order to master the molecular basis of Myostatin(MSTN) gene that negatively regulates skeletal muscle growth and development in Fujian white rabbit, Fujian white rabbit MSTN gene cDNA was amplified and cloned from total RNA of muscle by RT-PCR and RACE(rapid amplification of cDNA ends). Sequence analysis indicated that the full cDNA (1 551 bp, Gen Bank accession number:KX084386) consisted of a 5'-terminal untranslated region (UTR) (136 bp), a 3'-terminal UTR (287 bp) and an ORF(1 128 bp). The open reading frame encoded a deduced protein with 375 amino acids residues. There were one TGF-beta propeptide superfamily domains at amino acids encoded by the 97 to 768 nucleotides and one TGF-beta family domains at amino acids encoded by the 841 to 1 125 nucleotides. Comparison of Fujian white rabbit MSTN with those of other rabbit, human, pig, mouse, horse, cattle, sheep, deer, baboons, orangutan and monkey showed that the amino acids horno1ogy were 99.9%, 92.8%, 94.4%, 86.9%, 95.2%, 89.1%, 91.0%, 90.3%, 95.9%, 96.4%, 95.9%, respectively. It provides the basic data for further research on structure function, expression regulation and molecular breeding of MSTN gene in Fujian white rabbit.
-
Key words:
- Fujian white rabbit /
- MSTN /
- clone /
- sequence analysis
-
图 1 产物电泳
注：A为总RNA(M：DL2000 DNA Marker，1：总RNA)；B为MSTN的同源序列(M：DL2000 DNA Marker，1:PCR产物; 2:阴性对照)；C为MSTN的3′RACE(M：DL2000 DNA Marker，1:PCR产物，2:阴性对照)；D为MSTN的5′RACE(M：DL2000 DNA Marker，1：PCR产物)；E为RACE验证(M：DL2000 DNA Marker，1：阴性对照，2：PCR产物)。
Figure 1. Products were detected by gel electrophoresis
图 2 福建白兔MSTN cDNA序列及其预测的氨基酸序列
Figure 2. Sequence of MSTN from Fujian white rabbit and its amino acid sequence
表 1 设计的引物
Table 1. Primer sequence
名称 序列(5′-3′) 长度/(mers) F1(已知序列) GTTTATGCTGATCGTGGCTG 20 R1(已知序列) CACCCACAGCGGTCTACTAC 20 F2(已知序列) CTGTGTAATGCATGCACTTG 20 F3(3′RACE) AGCAGGTCCTTGCTGTACTC 20 F4(3′RACE) CCAATTACTGCTCTGGAGAC 20 F5(3′RACE) GAGCACTCAACAGAATCACG 20 R4(5′RACE) CTTCCAAAGAGCCATCACTG 20 R6(5′RACE) CTGAACGTCGTACTGATC 18 YZF1(序列验证) CTCAAGCTGTTCATGCAT 18 YZR1(序列验证) CCAACCATTGCATGTATTC 19 
下载: 导出CSV
-
[1] MCPHERRON A C, LAWLER A M, LEE S J. Regulation of skeletal muscle mass by a new TGF-β superfamily member[J]. Nature, 1997, 387(6628):83-90. doi: 10.1038/387083a0 [2] KAMBADUR R, SHARMA M, SMITH TP, et al. Mutations in myostatin (GDF8) in double-muscled Belgian Blue and Piedmontese cattle[J]. Genome Res, 1997, 7(9):910-916. doi: 10.1101/gr.7.9.910 [3] SCHUELKE M, WAGNER K R, STOLX L E, et al. Myostatin mutation associated with gross muscle hypertrophy in a child[J]. N Engl J Med, 2004, 350(26):2682-2688. doi: 10.1056/NEJMoa040933 [4] MCPHERRON A C, LEE S J.Suppression of body fat accumulation in myostatin deficient mice[J]. J Clin Invest, 2002, 109:595-601. doi: 10.1172/JCI0213562 [5] ALEXANDRA C, McPHERRON, SE-JIN LEE. Double muscling in cattle due to mutations in the myostatin gene[J]. USA: Proc Natl Acad Sci, 1997, 94: 12457-12461. [6] MARK THOMAS, BRETT LANGLEY. Myostatin, a negative regulator of muscle growth, function of inhibiting myoblast proliferation[J]. J Biol Chem, 2000, 275(51):40235-40243. doi: 10.1074/jbc.M004356200 [7] RIOS R, CAMEIRO I. Myostatin is an inhibitor of myogenic differentiation[J]. Am J Physiol Cell Physiol, 2002, 282(5):993-999. doi: 10.1152/ajpcell.00372.2001 [8] ALEXANDRA C, MCPHERRON A C. Suppression of body fat accumulation in myostatin-deficient mice[J]. J Clin Invest, 2002, 109(5):595-601 doi: 10.1172/JCI0213562 [9] LIN J, ARNOLD H B, DELLA-FERA M A. Myostatin knockout in mice increases myogenesis and decreases adipogenesis[J]. Biochem Biophys Res Commun, 2002, 291(3):701-706. doi: 10.1006/bbrc.2002.6500 [10] KOLLIAS H D, McDERMOTT C.Transforming growth factor-βand myostatin signaling in skeletal muscle[J]. J Appl Physiol, 2008, 104:579-587. doi: 10.1152/japplphysiol.01091.2007 [11] KRIK S, OLKHAM J. Myostatin regulation during skeletal muscle regeneration[J]. J Cell Physiol, 2000, 184(3):356-363. doi: 10.1002/(ISSN)1097-4652 [12] 李帅, 杨舒黎, 苟潇.肌肉生成抑制素(MSTN)基因研究进展[J].中国畜牧兽医, 2011, 38(2):60-63. http://d.old.wanfangdata.com.cn/Periodical/zgxmsy201102013 [13] HWANG W Y, FU Y, REYON D, et al. Efficialent genome editing in zebrafish using a CRISPR-CAS system[J]. Nat Biotechnol, 2013, 31(3):227-229. http://mbe.oxfordjournals.org/external-ref?access_num=23360964&link_type=MED [14] 王伟, 连林生, 李继中.猪MSTN基因生物信息学分析[J].安徽农业科学2012, 40(10):5943-5945. doi: 10.3969/j.issn.0517-6611.2012.10.083 [15] 吴清洪, 岳敏, 田雨光, 等.西藏小型猪MSTN基因分子克隆及序列分析[J].中国比较医学杂志, 2015, 25(3):18-22. doi: 10.3969/j.issn.1671-7856.2015.03.005 [16] 王娇娇, 张世伟, 张丽君, 等.辽育白牛MSTN基因分子克隆及序列分析[J].现代畜牧兽医, 2016(10):21-27. doi: 10.3969/j.issn.1672-9692.2016.10.004 [17] 张丽, 刘丽霞, 李强子, 等.天祝白牦牛MSTN基因编码区克隆及生物信息学分析[J].浙江农业学报, 2017, 29(4):618-624. doi: 10.3969/j.issn.1004-1524.2017.04.15 [18] 梁春年, 丁学智, 包鹏甲, 等.牦牛MSTN基因分子克隆及序列分析[J].华北农学报, 2013, 28(1):76-81. doi: 10.3969/j.issn.1000-7091.2013.01.015 [19] 金鑫燕.山羊肌生成抑制素(MSTN)基因的克隆及生物信息学分析[J].中国畜牧兽医, 2011, 38(9):11-114. http://d.old.wanfangdata.com.cn/Periodical/zgxmsy201109026 [20] JEANPLONG F, FALCONER S J, OLDHAM J M. et al.Discovery of a mammalian splice variant of myostatin that stimulates myogenesis[J]. PloS ONE, 2013, 8:e81713. doi: 10.1371/journal.pone.0081713 [21] JAIN H, SINGH S, KADAM M, et al. Knockdown of the myostatin gene by RNA interference in caprine fibroblast cells[J]. J Biotechnol, 2010, 145(2):99-102. doi: 10.1016/j.jbiotec.2009.10.017 [22] TRIPAT A K, RAMANI U V, PATEL A K, et al.short hairpin RNA-induced myostatin gene silencing in caprine myoblast cells in vitro[J]. Appl Biochem Biotechnol, 2013, 169:688-694. doi: 10.1007/s12010-012-0021-1 [23] TANG D, ZHU H, WU J, et al.Silencing myostatin gene by RNAi in sheep embryos[J]. J Biotechnol, 2012, 158:69-74. doi: 10.1016/j.jbiotec.2012.01.008 [24] TRIPATHI A K, APARNATHI M K, VYAVAHARE S S, et al.Myostatin gene silencing by RNA interference in chicken embryo fibroblast cells[J]. J Biotechnol, 2012, 160:140-145. doi: 10.1016/j.jbiotec.2012.03.001 [25] HU S, NI W, SAI W, et al.Knockdown of myostatin expression by RNAi enhances muscle growth in transgenic sheep[J]. PloS ONE, 2013, 8(3):e58521. doi: 10.1371/journal.pone.0058521 [26] ZHON Z R, ZHONG B S, JIA R X, et al.Production of myostatin-targeted goat by nuclear transfer from cultured adult somatic cells[J]. Theriogenology, 2013, 79(2):225-233. doi: 10.1016/j.theriogenology.2012.08.006 [27] LUO J, SONG Z, YU S, et al. Efficient generation of myostatin (MSTN) biallelic mutations in cattle using zinc finger nucleases[J]. PloS ONE, 2014, 9:e95225. doi: 10.1371/journal.pone.0095225 [28] 高丽, 谌颜, 廷茂, 等.肌肉生长抑制素基因在哺乳动物中的最新研究进展[J].生物技术进展, 2014, 4(6):381-388. doi: 10.3969/j.issn.2095-2341.2014.06.01 -
点击查看大图
图(6) / 表(1)
计量
- 文章访问数: 1205
- HTML全文浏览量: 333
- PDF下载量: 9
- 被引次数: 0
