牦牛Lkb1基因编码区克隆及其在骨骼肌的表达分析
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摘要
研究表明肝激酶b1(liver kinase b1, Lkb1)在小鼠(Mus musculus)骨骼肌发育与机体能量代谢调控中发挥重要作用。牦牛(Bos grunniens)作为青藏高原及毗邻地区的特有家畜,其骨骼肌的生长发育是影响产肉性能的主要因素之一。为研究Lkb1在牦牛骨骼肌生长发育及能量代谢中的潜在生物学功能,本研究以金川牦牛背最长肌cDNA为模板,采用PCR技术克隆Lkb1基因的CDS序列,并通过生物信息学方法,预测牦牛Lkb1氨基酸序列的理化性质、分析其在物种间的保守性;最后利用qRT-PCR技术检测Lkb1基因在成年金川牦牛不同组织中的表达模式,并且分析Lkb1基因在同龄不同品种牦牛骨骼肌的表达差异。研究结果显示,牦牛Lkb1基因的CDS序列全长为1 317 bp (GenBank登录号:MH412713),编码438个氨基酸。预测Lkb1蛋白为具有核质穿梭、发生磷酸化/去磷酸化的亲水性蛋白,可能参与调节机体能量和脂肪酸代谢。物种的保守性分析发现牦牛Lkb1基因与绵羊(Ovis aries)和山羊(Capra hircus)的同源性最高,经核苷酸和氨基酸序列比对同源性均高达97.50%和99.54%;而与小鼠核苷酸和氨基酸序列的同源性仅为45.48%和44.19%。基因表达分析显示,Lkb1基因在成年金川牦牛的骨骼肌中具有较高的表达量,在脂肪和肝脏组织中的表达量相对较低。在不同品种中,金川牦牛半腱肌Lkb1表达量显著高于麦洼牦牛(P<0.05),由此推测,Lkb1基因可能与牦牛骨骼肌的生长和能量代谢相关。该研究结果为进一步揭示Lkb1的功能和调控机制提供参考资料。
Research shows that Liver kinase b1(Lkb1) plays key roles in mouse(Mus musculus) skeletal muscle development and energetic metabolism processes. Yak(Bos grunniens) is regarded as the specific livestock in Qinghai Tibet Plateau and its adjacent regions, whose growth and development of skeletal muscle is one of most important factors effecting on meat performance. To explore the underlying functions of Yak Lkb1 regulation on skeletal muscle development, growth and energy metabolism, its CDS region was firstlycloned using cDNA as template from longissimus dorsi muscle(L-muscle) by PCR. Next, the physic-chemical properties of Lkb1 amino acid sequence and evolutionary conservation among species were predicted by bioinformatic methods. Lastly, the tissues expression pattern in Jinchuan Yak was detected by quantified PCR and subsequently the expression difference between Jinchuan and Maiwa Yak in skeletal muscle was analyzed. The result showed that the CDS of Yak Lkb1 gene was 1 317 bp(GenBank No. MH412713),encoding 438 amino acids. ProtScal and TMpred software predicted that Lkb1 was a hydrophilic protein with characters of nuclear/cytoplasm translocation, existed transmembrane domain in the region of 231～253(from inside to outside) and 238～255(from outside to inside) amino acids. Phosphorylation/dephosphorylation sites were analyzed by NetPhos2.0 Server showed that Lkb1 had 6 phosphorylation/dephosphorylation sites in the N-terminal of this protein, including Ser13, Ser18, Thr7, Tyr8, Tyr19 and Tyr23. Further, functional prediction by Protfun2.2 Server showed Lkb1 might regulating energy and fatty acid metabolism. In addition, the secondary structure of Lkb1 was mainly α-helices and random coil, α-helices with 33.79% and random coil with 44.75%, respectively. Moreover, the conservation analysis showed that Yak Lkb1 gene has highest homology comparison of sheep(Ovis aries) and goat(Capra hircus), both with 97.5% comparability of nucleotide sequence and 99.54% of amino acids sequence. The amino acid homology of Lkb1 comparing to chicken(Gallus gallus), pig(Sus scrofa), dog(Canis lupus familiaris), zebrafish(Danio rerio), panda(Giant panda), gorilla(Pan troglodytes), human(Homo sapiens) and chimpanzee(Rattus norvegicus) were 79.73%,95.89%, 84.05%, 83.31%, 92.71%, 81.51%, 99.54% and 87.47%. Therefore, the Lkb1 protein had high homology among species. However, the comparability of Lkb1 between Yak and mouse just reached at45.48% and 44.19% of nucleotide and protein sequence, respectively. Interestingly, m RNA level of Lkb1 gene was enriched in skeletal muscle, compared to that of in liver and fat tissues. Furthermore, the differential expression detection in L-muscle and semitendinosus muscle(S-muscle) from Jin chuan and Maiwa Yak revealed that Jinchuan Yak had significantly higher m RNA level of Lkb1 gene in S-muscle than that of Maiwa Yak, but its mRNA level was indistinguishable in L-muscle between these 2 strains(P<0.05). Thus, it's speculated that Lkb1 might be involved in skeletal muscle growth and energy metabolism. Collectively, these data will provide reference to elucidate the function and mechanism in Yak skeletal muscle biology in the further study.
Research shows that Liver kinase b1(Lkb1) plays key roles in mouse(Mus musculus) skeletal muscle development and energetic metabolism processes. Yak(Bos grunniens) is regarded as the specific livestock in Qinghai Tibet Plateau and its adjacent regions, whose growth and development of skeletal muscle is one of most important factors effecting on meat performance. To explore the underlying functions of Yak Lkb1 regulation on skeletal muscle development, growth and energy metabolism, its CDS region was firstlycloned using cDNA as template from longissimus dorsi muscle(L-muscle) by PCR. Next, the physic-chemical properties of Lkb1 amino acid sequence and evolutionary conservation among species were predicted by bioinformatic methods. Lastly, the tissues expression pattern in Jinchuan Yak was detected by quantified PCR and subsequently the expression difference between Jinchuan and Maiwa Yak in skeletal muscle was analyzed. The result showed that the CDS of Yak Lkb1 gene was 1 317 bp(GenBank No. MH412713),encoding 438 amino acids. ProtScal and TMpred software predicted that Lkb1 was a hydrophilic protein with characters of nuclear/cytoplasm translocation, existed transmembrane domain in the region of 231～253(from inside to outside) and 238～255(from outside to inside) amino acids. Phosphorylation/dephosphorylation sites were analyzed by NetPhos2.0 Server showed that Lkb1 had 6 phosphorylation/dephosphorylation sites in the N-terminal of this protein, including Ser13, Ser18, Thr7, Tyr8, Tyr19 and Tyr23. Further, functional prediction by Protfun2.2 Server showed Lkb1 might regulating energy and fatty acid metabolism. In addition, the secondary structure of Lkb1 was mainly α-helices and random coil, α-helices with 33.79% and random coil with 44.75%, respectively. Moreover, the conservation analysis showed that Yak Lkb1 gene has highest homology comparison of sheep(Ovis aries) and goat(Capra hircus), both with 97.5% comparability of nucleotide sequence and 99.54% of amino acids sequence. The amino acid homology of Lkb1 comparing to chicken(Gallus gallus), pig(Sus scrofa), dog(Canis lupus familiaris), zebrafish(Danio rerio), panda(Giant panda), gorilla(Pan troglodytes), human(Homo sapiens) and chimpanzee(Rattus norvegicus) were 79.73%,95.89%, 84.05%, 83.31%, 92.71%, 81.51%, 99.54% and 87.47%. Therefore, the Lkb1 protein had high homology among species. However, the comparability of Lkb1 between Yak and mouse just reached at45.48% and 44.19% of nucleotide and protein sequence, respectively. Interestingly, m RNA level of Lkb1 gene was enriched in skeletal muscle, compared to that of in liver and fat tissues. Furthermore, the differential expression detection in L-muscle and semitendinosus muscle(S-muscle) from Jin chuan and Maiwa Yak revealed that Jinchuan Yak had significantly higher m RNA level of Lkb1 gene in S-muscle than that of Maiwa Yak, but its mRNA level was indistinguishable in L-muscle between these 2 strains(P<0.05). Thus, it's speculated that Lkb1 might be involved in skeletal muscle growth and energy metabolism. Collectively, these data will provide reference to elucidate the function and mechanism in Yak skeletal muscle biology in the further study.
引文
艾鹥,文勇立,傅昌秀,等.2013.金川多胸椎牦牛宰后肌肉矿物质、脂肪酸及肉色分析[J].食品科学,34(16):251-256.(Ai Y,Wen Y L,Fu C X et al.2013.Analysis of muscle trace elements,fatty acid composition and color parameters of Jinchuan multi-vertebrate Yak[J].Food Science.34(16):251-256.)
李强,傅昌秀,文勇立,等.2012.金川多肋牦牛体尺和屠宰性状测定及其相关性分析[J].中国草食动物科学,32(02):18-20.(Li Q,Fu C X,Wen Y L et al.2012.Determination of body size and slaughter character and its correlation analysis of Jinchuan Multi-Ribs Yak[J].China Herbivore Science.32(02):18-20.)
谭子璇,王琦,徐旭,等.2018.金川牦牛与麦洼牦牛舍饲育肥条件下肉品质的比较研究[J].食品工业科技.1-14.(Tan Z X,Wang Q,Xu X et al.2018.Comparative study on meat quality of Jinchuan Yak and Maiwa Yak under yard-feeding conditions[J].Science and Technology of Food Industry.17:1-14.)
Black B L,Olson E N.1998.Transcriptional control of muscle development by myocyte enhancer factor-2(MEF2)proteins[J].Annual Review of Cell and Developmental Biology,14(1):167-196.
Comai G,Tajbakhsh S.2014.Molecular and cellular regulation of skeletal myogenesis[J].Current Topics in Developmental Biology,110:1-73.
Dusi S,Dellabianca V,Grzeskowiak M,et al.1993.Relationship between phosphorylation and translocation to the plasma-membrane of P47phox and P67phox and activation of the nadph oxidase in normal and Ca2+-depleted human neutrophils[J].Biochemical Journal,290(1):173-178.
Jenne D E,Reimann H,Nezu J,et al.1998.Peutz-Jeghers syndrome is caused by mutations in a novel serine threonine kinase[J].Nature Genetics,18(1):38-44.
Ji H B,Ramsey M R,Hayes D N,et al.2007.LKB1 modulates lung cancer differentiation and metastasis[J].Nature,448(7155):807-U807.
Krock B,Skuli N,Simon M C.2011.The Tumor suppressor LKB1 emerges as a critical factor in hematopoietic stem cell biology[J].Cell Metabolism,13(1):8-10.
Lai D M,Chen Y F,Wang F Y,et al.2012.LKB1 Controls the pluripotent state of human embryonic stem cells[J].Cellular Reprogramming,14(2):164-170.
Mian I,Pierre-Louis W S,Dole N,et al.2012.LKB1 destabilizes microtubules in myoblasts and contributes to myoblast differentiation[J].PLoS One,7(2):e31583.
Parker S J,Svensson R U,Divakaruni A S,et al.2017.LKB1promotes metabolic flexibility in response to energy stress[J].Metabolic Engineering,43(Pt B):208-217.
Ramirez-Espinosa J J,Gonzalez-Davalos L,Shimada A,et al2016.Bovine(Bos taurus)bone marrow mesenchyma cell differentiation to adipogenic and myogenic lineages[J].Cells Tissues Organs,201(1):51-64.
Robelin J,Lacourt A,Bechet D,et al.1991.Muscle differentiation in the bovine fetus:A histological and histochemical approach[J].Growth Development and Aging,55(3):151-160.
Sapkota G P,Boudeau J,Deak M,et al.2002.Identification and characterization of four novel phosphorylation sites(Ser31,Ser325,Thr336 and Thr366)on LKB1/STK11,the protein kinase mutated in Peutz-Jeghers cancer syndrome[J].Biochemical Journal,362(Pt 2):481-490.
Sapkota G P,Kieloch A,Lizcano J M,et al.2001.Phosphorylation of the protein kinase mutated in Peutz-Jeghers cancer syndrome,LKB1/STK11,at Ser431 by p90(RSK)and cAMP-dependent protein kinase,but not its farnesylation at Cys(433),is essential for LKB1 to suppress cell vrowth[J].Journal of Biological Chemistry,276(22):19469-19482.
Shan T,Xiong Y,Zhang P,et al.2016.Lkb1 controls brown adipose tissue growth and thermogenesis by regulating the intracellular localization of CRTC3[J].Nature Communications,7:12205-12215.
Shan T,Xu Z,Liu J,et al.2017.Lkb1 regulation of skeletal muscle development,metabolism and muscle progenitor cell homeostasis[J].Journal of Cellular Physiology,232(10):2653-2656.
Shan T,Zhang P,Liang X,et al.2014a.Lkb1 is indispensable for skeletal muscle development,regeneration,and satellite cell homeostasis[J].Stem Cells,32(11):2893-2907.
Spicer J,Ashworth A.2004.LKB1 kinase:Master and commander of metabolism and polarity[J].Current Biology,14(10):383-385.
Tiainen M,Vaahtomeri K,Ylikorkala A,et al.2002.Growth arrest by the LKB1 tumor suppressor:Induction of p21(WAF1/CIP1)[J].Human Molecular Genetics,11(13):1497-1504.
李强,傅昌秀,文勇立,等.2012.金川多肋牦牛体尺和屠宰性状测定及其相关性分析[J].中国草食动物科学,32(02):18-20.(Li Q,Fu C X,Wen Y L et al.2012.Determination of body size and slaughter character and its correlation analysis of Jinchuan Multi-Ribs Yak[J].China Herbivore Science.32(02):18-20.)
谭子璇,王琦,徐旭,等.2018.金川牦牛与麦洼牦牛舍饲育肥条件下肉品质的比较研究[J].食品工业科技.1-14.(Tan Z X,Wang Q,Xu X et al.2018.Comparative study on meat quality of Jinchuan Yak and Maiwa Yak under yard-feeding conditions[J].Science and Technology of Food Industry.17:1-14.)
Black B L,Olson E N.1998.Transcriptional control of muscle development by myocyte enhancer factor-2(MEF2)proteins[J].Annual Review of Cell and Developmental Biology,14(1):167-196.
Comai G,Tajbakhsh S.2014.Molecular and cellular regulation of skeletal myogenesis[J].Current Topics in Developmental Biology,110:1-73.
Dusi S,Dellabianca V,Grzeskowiak M,et al.1993.Relationship between phosphorylation and translocation to the plasma-membrane of P47phox and P67phox and activation of the nadph oxidase in normal and Ca2+-depleted human neutrophils[J].Biochemical Journal,290(1):173-178.
Jenne D E,Reimann H,Nezu J,et al.1998.Peutz-Jeghers syndrome is caused by mutations in a novel serine threonine kinase[J].Nature Genetics,18(1):38-44.
Ji H B,Ramsey M R,Hayes D N,et al.2007.LKB1 modulates lung cancer differentiation and metastasis[J].Nature,448(7155):807-U807.
Krock B,Skuli N,Simon M C.2011.The Tumor suppressor LKB1 emerges as a critical factor in hematopoietic stem cell biology[J].Cell Metabolism,13(1):8-10.
Lai D M,Chen Y F,Wang F Y,et al.2012.LKB1 Controls the pluripotent state of human embryonic stem cells[J].Cellular Reprogramming,14(2):164-170.
Mian I,Pierre-Louis W S,Dole N,et al.2012.LKB1 destabilizes microtubules in myoblasts and contributes to myoblast differentiation[J].PLoS One,7(2):e31583.
Parker S J,Svensson R U,Divakaruni A S,et al.2017.LKB1promotes metabolic flexibility in response to energy stress[J].Metabolic Engineering,43(Pt B):208-217.
Ramirez-Espinosa J J,Gonzalez-Davalos L,Shimada A,et al2016.Bovine(Bos taurus)bone marrow mesenchyma cell differentiation to adipogenic and myogenic lineages[J].Cells Tissues Organs,201(1):51-64.
Robelin J,Lacourt A,Bechet D,et al.1991.Muscle differentiation in the bovine fetus:A histological and histochemical approach[J].Growth Development and Aging,55(3):151-160.
Sapkota G P,Boudeau J,Deak M,et al.2002.Identification and characterization of four novel phosphorylation sites(Ser31,Ser325,Thr336 and Thr366)on LKB1/STK11,the protein kinase mutated in Peutz-Jeghers cancer syndrome[J].Biochemical Journal,362(Pt 2):481-490.
Sapkota G P,Kieloch A,Lizcano J M,et al.2001.Phosphorylation of the protein kinase mutated in Peutz-Jeghers cancer syndrome,LKB1/STK11,at Ser431 by p90(RSK)and cAMP-dependent protein kinase,but not its farnesylation at Cys(433),is essential for LKB1 to suppress cell vrowth[J].Journal of Biological Chemistry,276(22):19469-19482.
Shan T,Xiong Y,Zhang P,et al.2016.Lkb1 controls brown adipose tissue growth and thermogenesis by regulating the intracellular localization of CRTC3[J].Nature Communications,7:12205-12215.
Shan T,Xu Z,Liu J,et al.2017.Lkb1 regulation of skeletal muscle development,metabolism and muscle progenitor cell homeostasis[J].Journal of Cellular Physiology,232(10):2653-2656.
Shan T,Zhang P,Liang X,et al.2014a.Lkb1 is indispensable for skeletal muscle development,regeneration,and satellite cell homeostasis[J].Stem Cells,32(11):2893-2907.
Spicer J,Ashworth A.2004.LKB1 kinase:Master and commander of metabolism and polarity[J].Current Biology,14(10):383-385.
Tiainen M,Vaahtomeri K,Ylikorkala A,et al.2002.Growth arrest by the LKB1 tumor suppressor:Induction of p21(WAF1/CIP1)[J].Human Molecular Genetics,11(13):1497-1504.