陆川猪MYL9基因克隆及其表达差异分析
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摘要
【目的】克隆陆川猪肌球蛋白调节轻链9基因(MYL9),并进行生物信息学分析及检测其在不同猪种中的表达差异,为明确陆川猪骨骼肌生长规律及研究MYL9的生理功能打下基础。【方法】根据NCBI上的家猪MYL9基因序列设计特异性引物,采用RT-PCR克隆陆川猪MYL9基因,利用在线软件对其进行生物信息学分析,并以实时荧光定量PCR检测陆川猪和杜洛克猪背最长肌中MYL9基因的表达情况。【结果】陆川猪MYL9基因编码区(CDS)序列长519bp,共编码172个氨基酸;与NCBI上已公布的家猪MYL9基因(NM_001244472.1)CDS序列比对,陆川猪MYL9基因存在2处碱基突变,分别是195 bp处C→A和498 bp处T→C,均为同义突变;陆川猪MYL9基因推导氨基酸序列与家猪MYL9基因推导氨基酸序列的同源性最高,为99.6%,与鸡的同源性最低,为88.4%。陆川猪MYL9蛋白存在3个N糖基化位点和15个磷酸化位点,不存在跨膜结构域和信号肽,符合一般EFh-PEF超家族的结构特征。陆川猪MYL9蛋白二级结构中α-螺旋占54.65%,无规则卷曲占35.47%,β-转角占8.14%,延伸链占1.74%。MYL9基因在杜洛克猪背最长肌中的相对表达量极显著高于陆川猪背最长肌(P<0.01)。【结论】MYL9基因序列在不同物种中具有较高的保守性,其在10周龄陆川猪背最长肌中的相对表达量极显著低于杜洛克猪,与其在不同猪种背最长肌中的磷酸化程度有关,也说明MYL9基因对猪肌肉生长速度有一定影响。
【Objective】In this study,myosin regulatory light chain 9 gene(MYL9)in the longissimus dorsi muscle of Luchuan pig was cloned,its bioinformatics analysis was carried out and its expressions in different pig breeds were determined in order to lay a foundation for further studying the mechanism of skeletal muscle growth of Luchuan pig and functions of MYL9.【Method】According to MYL9 gene sequence of pig on NCBI,the specific primers were designed in order to clone MYL9 gene from Luchuan pig by RT-PCR. Bioinformatics analysis on it was conducted by online software. Expression of MYL9 gene in longissimus dorsi muscle of Luchuan pig and Duroc pig was detected by fluorescence quantitative PCR.【Result】The results showed that the length of Luchuan pig MYL9 gene coding region sequence(CDS)was 519 bp and it encoded 172 amino acids. Compared with CDS sequence of pig MYL9 gene published in NCBI(NM_001244472.1),there were two synonymous mutations on 195 bp C→A and 498 bp T→C,and both were synonymous mutations. The homology between amino acid sequence of MYL9 gene in Luchuan pig and that of the published gene MYL9 in pig from NCBI were the highest,reaching 99.6%. Luchuan pig shared the lowest(88.4%)homology with amino acid sequences of Gallus. There were three N glycosylation sites,fifteen phosphorylation sites,no transmembrane domain,no signal peptide and structural features of the general EFh-PEF superfamily in MYL9 protein of Luchuan pig. The ratios of α-helix,random coil,β-turn and extending strand in the secondary structure were 54.65%,35.47%,8.14% and 1.74%,respectively.The expression of MYL9 gene in the longissimus dorsi muscle of Duroc pig was extremely higher than that of Luchuan pig(P<0.01).【Conclusion】The MYL9 gene sequence is highly conserved among different species,and its relative expression in the longissimus dorsi muscle of 10-week old Luchuan pig is extremely lower than that of Duroc pig,which is related to the degree of phosphorylation in the longissimus dorsi of different pig breeds. It also shows that the MYL9 gene has a certain effect on the growth rate of muscles in pigs.
【Objective】In this study,myosin regulatory light chain 9 gene(MYL9)in the longissimus dorsi muscle of Luchuan pig was cloned,its bioinformatics analysis was carried out and its expressions in different pig breeds were determined in order to lay a foundation for further studying the mechanism of skeletal muscle growth of Luchuan pig and functions of MYL9.【Method】According to MYL9 gene sequence of pig on NCBI,the specific primers were designed in order to clone MYL9 gene from Luchuan pig by RT-PCR. Bioinformatics analysis on it was conducted by online software. Expression of MYL9 gene in longissimus dorsi muscle of Luchuan pig and Duroc pig was detected by fluorescence quantitative PCR.【Result】The results showed that the length of Luchuan pig MYL9 gene coding region sequence(CDS)was 519 bp and it encoded 172 amino acids. Compared with CDS sequence of pig MYL9 gene published in NCBI(NM_001244472.1),there were two synonymous mutations on 195 bp C→A and 498 bp T→C,and both were synonymous mutations. The homology between amino acid sequence of MYL9 gene in Luchuan pig and that of the published gene MYL9 in pig from NCBI were the highest,reaching 99.6%. Luchuan pig shared the lowest(88.4%)homology with amino acid sequences of Gallus. There were three N glycosylation sites,fifteen phosphorylation sites,no transmembrane domain,no signal peptide and structural features of the general EFh-PEF superfamily in MYL9 protein of Luchuan pig. The ratios of α-helix,random coil,β-turn and extending strand in the secondary structure were 54.65%,35.47%,8.14% and 1.74%,respectively.The expression of MYL9 gene in the longissimus dorsi muscle of Duroc pig was extremely higher than that of Luchuan pig(P<0.01).【Conclusion】The MYL9 gene sequence is highly conserved among different species,and its relative expression in the longissimus dorsi muscle of 10-week old Luchuan pig is extremely lower than that of Duroc pig,which is related to the degree of phosphorylation in the longissimus dorsi of different pig breeds. It also shows that the MYL9 gene has a certain effect on the growth rate of muscles in pigs.
引文
何若钢,刘桂武,林德源,覃小荣,黄伟杰,刘丁健,张家富,罗丽萍.2011.杜洛克与陆川猪杂交后代的肉质研究[J].黑龙江畜牧兽医,(12):37-39.[He R G,Liu G W,Lin D Y,Qin X R,Huang W J,Liu D J,Zhang J F,Luo L P.2011.The meat quality of offspring of Duroc pig with Luchuan pig[J].Heilongjiang Animal Science and Veterinary Medicine,(12):37-39.]
黄亚强.2015.基于蛋白质组学的前列腺癌间质标记物筛选及MYL9表达的临床研究[D].广州:南方医科大学.[Huang Y Q.2015.The proteomics-based screening of stromal marker in prostate cancer and the clinical study of MYL9 expression[D].Guangzhou:Southern Medical University.]
黄艳娜,兰干球,蒋钦杨.2014a.广西陆川猪和巴马香猪的分子生物学研究进展[J].中国畜牧兽医,41(11):79-84.[Huang Y N,Lan G Q,Jiang Q Y.2014a.Research progress on molecular biology of Guangxi Luchuan pig and Bama miniature pig[J].China Animal Husbandry&Veterinary Medicine,41(11):79-84.]
黄艳娜,李莉,韦玲静,郭晓萍,梁明振,兰干球,蒋钦杨.2014b.陆川猪肌肉生长抑制素基因的克隆与序列分析[J].南方农业学报,45(4):659-663.[Huang Y N,Li L,Wei L J,Guo X P,Liang M Z,Lan G Q,Jiang Q Y.2014b.Gene cloning and sequence analysis of myostatin in Luchuan pig[J].Journal of Southern Agriculture,45(4):659-663.]
刘明君,覃兆鲜,陈宝剑,王崇洲,温斌华,吴永绍,梁旦,蓝晞,张冰,潘天彪,谢炳坤.2018.陆川猪与杜洛克杂交猪的屠宰性能和肉质性状分析[J].江苏农业科学,46(17):186-188.[Liu M J,Qin Z X,Chen B J,Wang C Z,Wen B H,Wu Y S,Liang D,Lan X,Zhang B,Pan T B,Xie B K.2018.Analysis of slaughter performance and meat quality traits of Luchuan pig and Duroc hybrid pigs[J].Jiangsu Agricultural Sciences,46(17):186-188.]
王丛阳,王辉,沈传陆.2010.肌球蛋白轻链的调节及其对肿瘤细胞增殖、转移的影响[J].医学综述,16(21):3251-3254.[Wang C Y,Wang H,Shen C L.2010.Regulation of myosin light chain and its effect on tumor cell proliferation and metastasis[J].Medical Recapitulate,16(21):3251-3254.]
王永勇,谭翔,戴磊,阳诺,欧进,崔亚威,陈铭伍.2017.MYL9在非小细胞肺癌淋巴结和外周血中的表达及其临床意义[J].广西医科大学学报,34(9):1303-1306.[Wang Y Y,Tan X,Dai L,Yang N,Ou J,Cui Y W,Chen M W.2017.Expression and clinical significance of MYL9 in lymph nodes and peripheral blood of patients with nonsmall cell lung cancer[J].Journal of Guangxi Medical University,34(9):1303-1306.]
张家富,何若钢,江永强,覃小荣,黄伟杰,刘丁健,刘桂武,罗丽萍.2010.陆川猪胴体品质及肉质的研究[J].养猪,(6):33-36.[Zhang J F,He R G,Jiang Y Q,Qin X R,Huang W J,Liu D J,Liu G W,Luo L P.2010.Carcass quality and meat quality of Luchuan pig[J].Swine Production,(6):33-36.]
Conti M A,Even-Ram S,Liu C,Yamada K M,Adelstein R S.2004.Defects in cell adhesion and the visceral endoderm following ablation of nonmuscle myosin heavy chain II-Ain mice[J].The Journal of Biological Chemistry,279(40):41263-41266.
Gilles L,Bluteau D,Boukour S,Chang Y,Zhang Y,Robert T,Dessen P,Debili N,Bernard O A,Vainchenker W,Raslova H.2009.MAL/SRF complex is involved in platelet formation and megakaryocyte migration by regulating MYL9(MLC2)and MMP9[J].Blood,114(19):4221-4232.
Hong F,Haldeman B D,Jackson D,Carter M,Baker J E,Cremo C R.2011.Biochemistry of smooth muscle myosin light chain kinase[J].Archives of Biochemistry and Biophysics,510(2):135-146.
Jalagadugula G,Mao G,Kaur G,Goldfinger L E,Dhanasekaran D N,Rao A K.2010.Regulation of platelet myosin light chain(MYL9)by RUNX1:Implications for thrombocytopenia and platelet dysfunction in RUNX1 haplodeficiency[J].Blood,116(26):6037-6045.
Krendel M,Mooseker M S.2005.Myosins:Tails(and heads)of functional diversity[J].Physiology,20:239-251.
Licht A H,Nübel T,Feldner A,Jurisch-Yaksi N,Marcello M,Demicheva E,Hu J H,Hartenstein B,Augustin H G,Hecker M,Angel P,Korff T,Schorpp-Kistner M.2010.Junb regulates arterial contraction capacity,cellular contractility,and motility via its target My19 in mice[J].The Journal of Clinical Investigation,120(7):2307-2318.
Shehadeh L A,Webster K A,Hare J M,Vazquez-Padron R I.2011.Dynamic regulation of vascular myosin light chain(MYL9)with injury and aging[J].PLoS One,6(10):e25855.
Zeng L,Xu H,Chew T L,Eng E,Sadeghi M M,Adler S,Kanwar Y S,Danesh F R.2005.HMG CoA reductase inhibition modulates VEGF-induced endothelial cell hyperpermeability by preventing RhoA activation and myosin regulatory light chain phosphorylation[J].FASEB Journal,19(13):1845-1847.
Zhang Y S,Liu B,Luo X J,Zhang J J,Li N S,Ma Q L,Jiang J L,Li Y J,Li Q,Peng J.2015.A novel function of nuclear nonmuscle myosin regulatory light chain in promotion of xanthine oxidase transcription after myocardial ischemia/reperfusion[J].Free Radical Biology&Medicine,83:115-128.
黄亚强.2015.基于蛋白质组学的前列腺癌间质标记物筛选及MYL9表达的临床研究[D].广州:南方医科大学.[Huang Y Q.2015.The proteomics-based screening of stromal marker in prostate cancer and the clinical study of MYL9 expression[D].Guangzhou:Southern Medical University.]
黄艳娜,兰干球,蒋钦杨.2014a.广西陆川猪和巴马香猪的分子生物学研究进展[J].中国畜牧兽医,41(11):79-84.[Huang Y N,Lan G Q,Jiang Q Y.2014a.Research progress on molecular biology of Guangxi Luchuan pig and Bama miniature pig[J].China Animal Husbandry&Veterinary Medicine,41(11):79-84.]
黄艳娜,李莉,韦玲静,郭晓萍,梁明振,兰干球,蒋钦杨.2014b.陆川猪肌肉生长抑制素基因的克隆与序列分析[J].南方农业学报,45(4):659-663.[Huang Y N,Li L,Wei L J,Guo X P,Liang M Z,Lan G Q,Jiang Q Y.2014b.Gene cloning and sequence analysis of myostatin in Luchuan pig[J].Journal of Southern Agriculture,45(4):659-663.]
刘明君,覃兆鲜,陈宝剑,王崇洲,温斌华,吴永绍,梁旦,蓝晞,张冰,潘天彪,谢炳坤.2018.陆川猪与杜洛克杂交猪的屠宰性能和肉质性状分析[J].江苏农业科学,46(17):186-188.[Liu M J,Qin Z X,Chen B J,Wang C Z,Wen B H,Wu Y S,Liang D,Lan X,Zhang B,Pan T B,Xie B K.2018.Analysis of slaughter performance and meat quality traits of Luchuan pig and Duroc hybrid pigs[J].Jiangsu Agricultural Sciences,46(17):186-188.]
王丛阳,王辉,沈传陆.2010.肌球蛋白轻链的调节及其对肿瘤细胞增殖、转移的影响[J].医学综述,16(21):3251-3254.[Wang C Y,Wang H,Shen C L.2010.Regulation of myosin light chain and its effect on tumor cell proliferation and metastasis[J].Medical Recapitulate,16(21):3251-3254.]
王永勇,谭翔,戴磊,阳诺,欧进,崔亚威,陈铭伍.2017.MYL9在非小细胞肺癌淋巴结和外周血中的表达及其临床意义[J].广西医科大学学报,34(9):1303-1306.[Wang Y Y,Tan X,Dai L,Yang N,Ou J,Cui Y W,Chen M W.2017.Expression and clinical significance of MYL9 in lymph nodes and peripheral blood of patients with nonsmall cell lung cancer[J].Journal of Guangxi Medical University,34(9):1303-1306.]
张家富,何若钢,江永强,覃小荣,黄伟杰,刘丁健,刘桂武,罗丽萍.2010.陆川猪胴体品质及肉质的研究[J].养猪,(6):33-36.[Zhang J F,He R G,Jiang Y Q,Qin X R,Huang W J,Liu D J,Liu G W,Luo L P.2010.Carcass quality and meat quality of Luchuan pig[J].Swine Production,(6):33-36.]
Conti M A,Even-Ram S,Liu C,Yamada K M,Adelstein R S.2004.Defects in cell adhesion and the visceral endoderm following ablation of nonmuscle myosin heavy chain II-Ain mice[J].The Journal of Biological Chemistry,279(40):41263-41266.
Gilles L,Bluteau D,Boukour S,Chang Y,Zhang Y,Robert T,Dessen P,Debili N,Bernard O A,Vainchenker W,Raslova H.2009.MAL/SRF complex is involved in platelet formation and megakaryocyte migration by regulating MYL9(MLC2)and MMP9[J].Blood,114(19):4221-4232.
Hong F,Haldeman B D,Jackson D,Carter M,Baker J E,Cremo C R.2011.Biochemistry of smooth muscle myosin light chain kinase[J].Archives of Biochemistry and Biophysics,510(2):135-146.
Jalagadugula G,Mao G,Kaur G,Goldfinger L E,Dhanasekaran D N,Rao A K.2010.Regulation of platelet myosin light chain(MYL9)by RUNX1:Implications for thrombocytopenia and platelet dysfunction in RUNX1 haplodeficiency[J].Blood,116(26):6037-6045.
Krendel M,Mooseker M S.2005.Myosins:Tails(and heads)of functional diversity[J].Physiology,20:239-251.
Licht A H,Nübel T,Feldner A,Jurisch-Yaksi N,Marcello M,Demicheva E,Hu J H,Hartenstein B,Augustin H G,Hecker M,Angel P,Korff T,Schorpp-Kistner M.2010.Junb regulates arterial contraction capacity,cellular contractility,and motility via its target My19 in mice[J].The Journal of Clinical Investigation,120(7):2307-2318.
Shehadeh L A,Webster K A,Hare J M,Vazquez-Padron R I.2011.Dynamic regulation of vascular myosin light chain(MYL9)with injury and aging[J].PLoS One,6(10):e25855.
Zeng L,Xu H,Chew T L,Eng E,Sadeghi M M,Adler S,Kanwar Y S,Danesh F R.2005.HMG CoA reductase inhibition modulates VEGF-induced endothelial cell hyperpermeability by preventing RhoA activation and myosin regulatory light chain phosphorylation[J].FASEB Journal,19(13):1845-1847.
Zhang Y S,Liu B,Luo X J,Zhang J J,Li N S,Ma Q L,Jiang J L,Li Y J,Li Q,Peng J.2015.A novel function of nuclear nonmuscle myosin regulatory light chain in promotion of xanthine oxidase transcription after myocardial ischemia/reperfusion[J].Free Radical Biology&Medicine,83:115-128.