合浦珠母贝matrilin-1基因的克隆和表达分析
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摘要
以合浦珠母贝(Pinctada fucata)转录组中的matrilin-1基因片段为基础,开展了matrilin-1的c DNA全长(Pfmatrilin-1)克隆和定量表达分析。Pfmatrilin-1基因c DNA全长2 036 bp,开放阅读框(open reading frame,ORF)1 194 bp,编码397个氨基酸,包括1个信号肽序列和2个血管性血友病因子A(von Willebrand factor A,VWA)样结构域。系统进化分析显示无脊椎动物的matrilins与脊椎动物的matrilins进化关系较远。荧光定量PCR表达分析结果显示,Pfmatrilin-1 mRNA在合浦珠母贝各个组织中均有表达,其中在血液中表达量最高(P<0.05);Pfmatrilin-1从担轮幼虫期至变态期均有表达,眼点期表达量最高(P<0.05),而贝类幼虫发育到眼点期就会开始附着,眼点期幼虫原壳生长停滞,次生壳形成,因此推测其可能与次生壳的形成有关。这些结果表明Pfmatrilin-1在合浦珠母贝生物矿化中发挥重要作用。
Matrilin-1 gene was cloned from the Pinctada fucata based on its transcriptome sequences and named as Pfmatrilin-1. The whole length of Pfmatrilin-1 was 2 036 bp,including a 1 194 bp ORF encoding 397 amino acids,in which there were a signal peptide and two VWA( von Willebrand factor A) motifs. Phylogeny analysis proves that the matrilins in vertebrates and invertebrates were of distant relationship. The qRT-PCR tissue expression analysis indicates that Pfmatrilin-1 was expressed in all the tested tissues with significantly high expression level in blood cells( P < 0. 05). The Pf-matrilin-1 was also continuously expressed from trochophore to metamorphosis stages,reaching the peak at pediveliger stage( P < 0. 05). The prodissoconch Ⅱ formed during pediveliger stage,so the Pfmatrilin-1 may be involved in regulating the formation of prodissoconchⅡ. Thus,it is proved that the Pfmatrilin-1 plays an important role of biomineralization in P. fucata.
Matrilin-1 gene was cloned from the Pinctada fucata based on its transcriptome sequences and named as Pfmatrilin-1. The whole length of Pfmatrilin-1 was 2 036 bp,including a 1 194 bp ORF encoding 397 amino acids,in which there were a signal peptide and two VWA( von Willebrand factor A) motifs. Phylogeny analysis proves that the matrilins in vertebrates and invertebrates were of distant relationship. The qRT-PCR tissue expression analysis indicates that Pfmatrilin-1 was expressed in all the tested tissues with significantly high expression level in blood cells( P < 0. 05). The Pf-matrilin-1 was also continuously expressed from trochophore to metamorphosis stages,reaching the peak at pediveliger stage( P < 0. 05). The prodissoconch Ⅱ formed during pediveliger stage,so the Pfmatrilin-1 may be involved in regulating the formation of prodissoconchⅡ. Thus,it is proved that the Pfmatrilin-1 plays an important role of biomineralization in P. fucata.
引文
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[23]毕晓敏,黄桂菊,范嗣刚,等.合浦珠母贝矿化基因Pearlin重组蛋白的表达条件优化[J].南方水产科学,2015,11(6):100-106.
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[29]KLATT A R,PAULSSON M,WAGENER R.Expression of matrilins during maturation of mouse skeletal tissues[J].Matrix Biol,2002,21(3):289-296.
[30]HUANG X,BIRK D E,GOETINCK P F.Mice lacking matrilin-1(cartilage matrix protein)have alterations in type II collagen fibrillogenesis and fibril organization[J].Dev Dyn,1999,216(4/5):434-441.
[31]张岑.合浦珠母贝贝壳形成相关蛋白及基因的研究[D].北京:清华大学,2006:61-75.
[32]闫芳.马氏珠母贝珍珠层形成相关基因的克隆与功能研究[D].湛江:广东海洋大学,2014:9-28.
[33]薛桂英,郭奕惠,黄桂菊,等.合浦珠母贝不同壳基质蛋白基因的表达水平比较[J].广东农业科学,2013,40(17):140-142,145.
[2]PAULSSON M,KLATT A R,KOBBE B,et al.The matrilins:a novel family of extracellular adaptor proteins[M]∥HASCALL V C,KUETTNER K E.The many faces of osteoarthritis.Basel:Birkhuser,2002:151-158.
[3]LUO J M,WANG Y C,YUAN X,et al.Matrilins family and cartilage diseases[J].Precision Med,2016,2:e1233.
[4]FRESQUET M,JOWITT T A,STEPHEN L A,et al.Structural and functional investigations of Matrilin-1 A-domains reveal insights into their role in cartilage ECM assembly[J].J Biol Chem,2010,285(44):34048-34061.
[5]PAULSSON M,HEINEGRD D.Matrix proteins bound to associatively prepared proteoglycans from bovine cartilage[J].Biochem J,1979,183(3):539-545.
[6]WIBERG C,KLATT A R,WAGENER R,et al.Complexes of matrilin-1 and biglycan or decorin connect collagen VI microfibrils to both collagen II and aggrecan[J].J Biol Chem,2003,278(39):37698-37704.
[7]FORADORI M J,CHEN Q,FERNANDEZ C A,et al.Matrilin-1is an inhibitor of neovascularization[J].J Biol Chem,2014,289(20):14301-14309.
[8]NEACSU C D,KO Y P,TAGARIELLO A,et al.Matrilin-1 is essential for zebrafish development by facilitating collagen II secretion[J].J Biol Chem,2014,289(3):1505-1518.
[9]JENKINS R N,OSBORNE-LAWRENCE S L,SINCLAIR A K,et al.Structure and chromosomal location of the human gene encoding cartilage matrix protein[J].J Biol Chem,1990,265(32):19624-19631.
[10]KO Y P,KOBBE B,PAULSSON M,et al.Zebrafish(Danio rerio)matrilins:shared and divergent characteristics with their mammalian counterparts[J].Biochem J,2005,386(Pt 2):367-379.
[11]PEDERSEN M E,TAKLE H,YTTEBORG E,et al.Matrilin-1expression is increased in the vertebral column of Atlantic salmon(Salmo salar L.)individuals displaying spinal fusions[J].Fish Physiol Biochem,2011,37(4):821-831.
[12]李鑫.Matrilin-1在小鼠胫骨闭合骨折模型愈合过程中的作用研究[D].长春:吉林大学,2011:4-7.
[13]MONTANARO L,PARISINI P,GREGGI T,et al.Evidence of a linkage between matrilin-1 gene(MATN1)and idiopathic scoliosis[J].Scoliosis,2006,1(1):21.
[14]ZHANG H,ZHAO S,ZHAO Z,et al.The association of rs1149048 polymorphism in matrilin-1(MATN1)gene with adolescent idiopathic scoliosis susceptibility:a meta-analysis[J].Mol Biol Rep,2014,41(4):2543-2549.
[15]BAE J W,CHO C H,MIN W K,et al.Associations between matrilin-1 gene polymorphisms and adolescent idiopathic scoliosis curve patterns in a Korean population[J].Mol Biol Rep,2012,39(5):5561-5567.
[16]刘晓敏,陈银河,申才良.Matrilin-1基因rs1149048单核苷酸多态性与青少年特发性脊柱侧凸易感性的Meta分析[J].安徽医药,2015,19(10):1933-1936.
[17]ZHANG G,FANG X,GUO X,et al.The oyster genome reveals stress adaptation and complexity of shell formation[J].Nature,2012,490(7418):49-54.
[18]BOUCHUT A,ROGER E,COUSTAU C,et al.Compatibility in the Biomphalaria glabrata/Echinostoma caproni model:potential involvement of adhesion genes[J].Int J Parasitol,2006,36(2):175-184.
[19]XU W,FAISAL M.Matrilin-like molecules produced by circulating hemocytes of the zebra mussel(Dreissena polymorpha)upon stimulation[J].Dev Comp Immunol,2007,31(12):1205-1210.
[20]龙敏明,黄桂菊,邹记兴,等.育珠对合浦珠母贝N19和Prismalin-14基因表达水平的影响[J].南方水产科学,2013,9(5):58-63.
[21]刘晓军,李家乐.养殖珍珠贝贝壳基质蛋白研究进展[J].上海海洋大学学报,2013,22(2):200-205.
[22]王小玉,喻达辉,黄桂菊,等.合浦珠母贝3个家系的AFLP标记分离与遗传多样性研究[J].南方水产,2007,3(5):54-60.
[23]毕晓敏,黄桂菊,范嗣刚,等.合浦珠母贝矿化基因Pearlin重组蛋白的表达条件优化[J].南方水产科学,2015,11(6):100-106.
[24]MARIN F,LUQUET G,MARIE B,et al.Molluscan shell proteins:primary structure,origin,and evolution[J].Curr Top Dev Biol,2008,80(3):209-276.
[25]罗会.合浦珠母贝经济性状的遗传分析[D].上海:上海海洋大学,2013:1-52.
[26]王琦,何毛贤.合浦珠母贝实时定量PCR内参基因的稳定性比较[J].南方水产科学,2013,9(6):33-40.
[27]LIVAK K J,SCHMITTGEN T D.Analysis of relative gene expression data using real-time quantitative PCR and the 2-ΔΔCTmethod[J].Methods,2001,25(4):402-408.
[28]ZHANG Y,CHEN Q.Changes of matrilin forms during endochondral ossification.Molecular basis of oligomeric assembly[J].J Biol Chem,2000,275(42):32628-32634.
[29]KLATT A R,PAULSSON M,WAGENER R.Expression of matrilins during maturation of mouse skeletal tissues[J].Matrix Biol,2002,21(3):289-296.
[30]HUANG X,BIRK D E,GOETINCK P F.Mice lacking matrilin-1(cartilage matrix protein)have alterations in type II collagen fibrillogenesis and fibril organization[J].Dev Dyn,1999,216(4/5):434-441.
[31]张岑.合浦珠母贝贝壳形成相关蛋白及基因的研究[D].北京:清华大学,2006:61-75.
[32]闫芳.马氏珠母贝珍珠层形成相关基因的克隆与功能研究[D].湛江:广东海洋大学,2014:9-28.
[33]薛桂英,郭奕惠,黄桂菊,等.合浦珠母贝不同壳基质蛋白基因的表达水平比较[J].广东农业科学,2013,40(17):140-142,145.