山麻鸭OPN基因的克隆、表达及生物信息学分析
|
摘要
应用RT-PCR、3′/5′RACE PCR扩增,克隆了全长为893 bp的山麻鸭Osteopontin(OPN)基因序列,GenBank登录号为XM_013102822。运用生物信息学工具进行分析显示:OPN蛋白一级结构中丝氨酸、半胱氨酸、精氨酸、异亮氨酸、谷氨酸所占比例较高,预测分子式为C_(883)H_(1383)N_(253)O_(267)S_(12)。二级结构预测有8.33%的α螺旋,35.56%的β折叠,56.11%的无规则卷曲。基于编码的氨基酸序列所构建的分子进化树表明,与绿头鸭的OPN基因属于同一分支,亲缘关系最近。在mRNA水平上,OPN基因在山麻鸭心、肝、脾、肺、肾、胸肌、胃、下丘脑、垂体、卵巢、漏斗部、膨大部、卵壳腺中均有不同程度的表达,其中在肾和卵壳腺中的表达量极显著高于其他组织(P<0.01),在肝脏、卵巢、漏斗部和膨大部中的表达量也相对较高,在垂体、胃、脾和下丘脑中的表达量较低。
The Osteopontin cDNA in size of 893 bp was cloned by RT-PCR and 3′/5′RACE PCR amplification.Bioinformatics analysis showed that Ser, Cys, Arg, Ile, Glu had the highest proportion in OPN primary structure, and the predicted molecular formula was C_(883)H_(1383)N_(253)O_(267) S_(12). The secondary structure was predicted to have 8.33% alpha helix, 35.56% beta fold, and 56.11% random coil.The phylogenetic tree was constructed based on the protein sequence showed that the relationship between Shanma duck and Anas platyrhynchos was the closest and had the same evolutionary status.OPN mRNA was expressed in many tissues of Shanma duck, including heart, liver, spleen, lung, kidney, pectoralis,stomach, hypothalamus, pituitary, ovary, infundibulum, ampulla, glandula conchae. Among them, the expression in the kidney and egg shell gland volume was significantly higher than that in other tissues(P<0.01), and relatively high in the liver, ovary, funnel, bulking, but lower in pituitary, stomach, spleen and hypothalamus.
The Osteopontin cDNA in size of 893 bp was cloned by RT-PCR and 3′/5′RACE PCR amplification.Bioinformatics analysis showed that Ser, Cys, Arg, Ile, Glu had the highest proportion in OPN primary structure, and the predicted molecular formula was C_(883)H_(1383)N_(253)O_(267) S_(12). The secondary structure was predicted to have 8.33% alpha helix, 35.56% beta fold, and 56.11% random coil.The phylogenetic tree was constructed based on the protein sequence showed that the relationship between Shanma duck and Anas platyrhynchos was the closest and had the same evolutionary status.OPN mRNA was expressed in many tissues of Shanma duck, including heart, liver, spleen, lung, kidney, pectoralis,stomach, hypothalamus, pituitary, ovary, infundibulum, ampulla, glandula conchae. Among them, the expression in the kidney and egg shell gland volume was significantly higher than that in other tissues(P<0.01), and relatively high in the liver, ovary, funnel, bulking, but lower in pituitary, stomach, spleen and hypothalamus.
引文
[1]林东文,李益明,陈小春,等.龙岩山麻鸭产业状况调查与发展建议[J].福建畜牧兽医,2016,38(1):26-28.
[2]周世业.不同羽色蛋鸭繁殖性能比较分析[J].福建畜牧兽医,2018,40(4):5-6.
[3]章世元,周光玉,徐春燕,等.禽类蛋壳基质蛋白的研究进展[J].中国家禽,2009,31(11):37-40.
[4]赵焱.种植体龈沟液中骨桥蛋白(OPN)水平的研究[D].太原:山西医科大学,2013.
[5]BROWN L F,BERSE B,WATER L V D,et al.Expression and distribution of osteopontin in human tissues:Widespread association with luminal epithelial surfaces[J].Molecular Biology of the Cell,1992,3(10):1169-1180.
[6]SODEK J,GANSS B,MCKEE M D.Osteopontin[J].Crit Rev Oral Biol Med,2000,11(3):279-303.
[7]魏本娟,陈轶君,陆群,等.OLP患者唾液和血清中骨桥蛋白等细胞因子的表达[J].临床口腔医学杂志,2010,26(10):594-596.
[8]张冬杰,刘娣,汪晓鸿,等.大白猪骨桥蛋白基因的克隆、表达及功能分析[J].畜牧兽医学报,2009,40(7):965-970.
[9]符策广,刘洁.骨桥蛋白的生物学特性及在骨改建中的作用[J].临床口腔医学杂志,2012,28(8):506-508.
[10]TAKAHASHI K,TAKAHASHI F,TANABE K K,et al.The carboxyl-terminal fragment of osteopontin suppresses arginine-glycine-asparatic acid-dependent cell adhesion[J].Biochem Mol Biol Int,2010,46(6):1081-1092.
[11]周帅,杨春,庄逢源.骨桥蛋白与力学信号传导研究进展[J].航天医学与医学工程,2015,28(3):223-228.
[12]刘莹莹.骨桥蛋白促进肝干细胞/肝前体细胞增殖和迁移的研究[D].上海:第二军医大学,2013.
[13]范哲,李鸿雁,龙毅,等.骨桥蛋白的研究进展[J].中国实验诊断学,2005,9(2):307-309.
[14]赖淑静,陈黎,傅衍,等.蛋鸭骨桥蛋白基因OPN的多态性与蛋品质性状的相关性研究[J].农业生物技术学报,2015,23(3):352-358.
[15]李梦寻,黄涛,马力鹏,等.猪lncRNA-ENSSSCT00000018610的克隆及其在猪卵泡中的表达[J].畜牧兽医学报,2018,49(9):1830-1839.
[16]叶幼荣.蛋鸡子宫内膜细胞钙转运及蛋壳钙化早期钙沉积机制的研究[D].石河子:石河子大学,2015.
[17]陈杰,尹玉港.基质蛋白及其与蛋壳形成的关系研究[J].家禽科学,2009(11):40-43.
[18]FERNANDEZ M S,PASSALACQUA K,ARIAS J I,et al.Partial biomimetic reconstitution of avian eggshell formation[J].Journal of Structural Biology,2004,148(1):1-10.
[19]HERN NDEZ-HERN NDEZ A,VIDAL M L,G MEZ-MORALES J,et al.Influence of eggshell matrix proteins on the precipitation of calcium carbonate(CaCO 3)[J].Journal of Crystal Growth,2008,310(7):1754-1759.
[20]CHIEN Y C,HINCKE M T,VALI H,et al.Ultrastructural matrix-mineral relationships in avian eggshell,and effects of osteopontin on calcite growth in vitro[J].Journal of Structural Biology,2008,163(1):84-99.
[21]FERNANDEZ M S,ESCOBAR C,LAVELIN I,et al.Localization of osteopontin in oviduct tissue and eggshell during different stages of the avian egg laying cycle[J].Journal of Structural Biology,2003,143(3):171-180.
[22]LAVELIN I,YARDEN N,BEN-BASSAT S,et al.Regulation of Osteopontin Gene Expression During Egg Shell Formation in the Laying Hen by Mechanical Strain[J].Matrix Biology Journal of the International Society for Matrix Biology,1998,17(8-9):615.
[23]PINES M,KNOPOV V,BAR A.Involvement of osteopontin in egg shell formation in the laying chicken[J].Matrix Biology,1994,14(9):765-771.
[24]CHIEN Y C,HINCKE M T,MCKEE M D.Avian eggshell structure and osteopontin[J].Cells Tissues Organs,2009,189(1-4):38-43.
[25]SHET D,GHOSH J,AJITH S,et al.Efficacy of dietary phytase supplementation on laying performance and expression of osteopontin and calbindin genes in eggshell gland[J].Anim Nutr,2018,4(1):52-8.
[26]李诗燕.磷酸化Osteopontin抑制磷酸钙和草酸钙结晶的动力学研究[D].武汉:华中农业大学,2015.
[2]周世业.不同羽色蛋鸭繁殖性能比较分析[J].福建畜牧兽医,2018,40(4):5-6.
[3]章世元,周光玉,徐春燕,等.禽类蛋壳基质蛋白的研究进展[J].中国家禽,2009,31(11):37-40.
[4]赵焱.种植体龈沟液中骨桥蛋白(OPN)水平的研究[D].太原:山西医科大学,2013.
[5]BROWN L F,BERSE B,WATER L V D,et al.Expression and distribution of osteopontin in human tissues:Widespread association with luminal epithelial surfaces[J].Molecular Biology of the Cell,1992,3(10):1169-1180.
[6]SODEK J,GANSS B,MCKEE M D.Osteopontin[J].Crit Rev Oral Biol Med,2000,11(3):279-303.
[7]魏本娟,陈轶君,陆群,等.OLP患者唾液和血清中骨桥蛋白等细胞因子的表达[J].临床口腔医学杂志,2010,26(10):594-596.
[8]张冬杰,刘娣,汪晓鸿,等.大白猪骨桥蛋白基因的克隆、表达及功能分析[J].畜牧兽医学报,2009,40(7):965-970.
[9]符策广,刘洁.骨桥蛋白的生物学特性及在骨改建中的作用[J].临床口腔医学杂志,2012,28(8):506-508.
[10]TAKAHASHI K,TAKAHASHI F,TANABE K K,et al.The carboxyl-terminal fragment of osteopontin suppresses arginine-glycine-asparatic acid-dependent cell adhesion[J].Biochem Mol Biol Int,2010,46(6):1081-1092.
[11]周帅,杨春,庄逢源.骨桥蛋白与力学信号传导研究进展[J].航天医学与医学工程,2015,28(3):223-228.
[12]刘莹莹.骨桥蛋白促进肝干细胞/肝前体细胞增殖和迁移的研究[D].上海:第二军医大学,2013.
[13]范哲,李鸿雁,龙毅,等.骨桥蛋白的研究进展[J].中国实验诊断学,2005,9(2):307-309.
[14]赖淑静,陈黎,傅衍,等.蛋鸭骨桥蛋白基因OPN的多态性与蛋品质性状的相关性研究[J].农业生物技术学报,2015,23(3):352-358.
[15]李梦寻,黄涛,马力鹏,等.猪lncRNA-ENSSSCT00000018610的克隆及其在猪卵泡中的表达[J].畜牧兽医学报,2018,49(9):1830-1839.
[16]叶幼荣.蛋鸡子宫内膜细胞钙转运及蛋壳钙化早期钙沉积机制的研究[D].石河子:石河子大学,2015.
[17]陈杰,尹玉港.基质蛋白及其与蛋壳形成的关系研究[J].家禽科学,2009(11):40-43.
[18]FERNANDEZ M S,PASSALACQUA K,ARIAS J I,et al.Partial biomimetic reconstitution of avian eggshell formation[J].Journal of Structural Biology,2004,148(1):1-10.
[19]HERN NDEZ-HERN NDEZ A,VIDAL M L,G MEZ-MORALES J,et al.Influence of eggshell matrix proteins on the precipitation of calcium carbonate(CaCO 3)[J].Journal of Crystal Growth,2008,310(7):1754-1759.
[20]CHIEN Y C,HINCKE M T,VALI H,et al.Ultrastructural matrix-mineral relationships in avian eggshell,and effects of osteopontin on calcite growth in vitro[J].Journal of Structural Biology,2008,163(1):84-99.
[21]FERNANDEZ M S,ESCOBAR C,LAVELIN I,et al.Localization of osteopontin in oviduct tissue and eggshell during different stages of the avian egg laying cycle[J].Journal of Structural Biology,2003,143(3):171-180.
[22]LAVELIN I,YARDEN N,BEN-BASSAT S,et al.Regulation of Osteopontin Gene Expression During Egg Shell Formation in the Laying Hen by Mechanical Strain[J].Matrix Biology Journal of the International Society for Matrix Biology,1998,17(8-9):615.
[23]PINES M,KNOPOV V,BAR A.Involvement of osteopontin in egg shell formation in the laying chicken[J].Matrix Biology,1994,14(9):765-771.
[24]CHIEN Y C,HINCKE M T,MCKEE M D.Avian eggshell structure and osteopontin[J].Cells Tissues Organs,2009,189(1-4):38-43.
[25]SHET D,GHOSH J,AJITH S,et al.Efficacy of dietary phytase supplementation on laying performance and expression of osteopontin and calbindin genes in eggshell gland[J].Anim Nutr,2018,4(1):52-8.
[26]李诗燕.磷酸化Osteopontin抑制磷酸钙和草酸钙结晶的动力学研究[D].武汉:华中农业大学,2015.