Partial cDNA sequence of rabbit BMP15 was cloned by RT-PCR from rabbit ovaries, showing a similarity of 83%-90% with the BMP15 nucleotide sequences in humans, mice, ovine, sheep, cows and pigs. The expression of BMP15...Partial cDNA sequence of rabbit BMP15 was cloned by RT-PCR from rabbit ovaries, showing a similarity of 83%-90% with the BMP15 nucleotide sequences in humans, mice, ovine, sheep, cows and pigs. The expression of BMP15 in rabbit cumulus-oocyte complexs during oocytes in vitro maturation （IVM） was measured by fluorescent quantitative RT-PCR method. BMP 15 was expressed at low levels in immature oocytes and increased to the highest level at 16h of IVM, which coincides with the time of cumulus cell expansion, then declined slowly under IVM cultivation. The expression pattern of BMP 15 suggested that it might be important in cumulus expansion in rabbits.展开更多
The gene encoding bone morphogenetic protein-7(BMP7) is expressed in the developing kidney in embryos and also in the mature organ in adults. During kidney development, expression of BMP7 is essential to determine the...The gene encoding bone morphogenetic protein-7(BMP7) is expressed in the developing kidney in embryos and also in the mature organ in adults. During kidney development, expression of BMP7 is essential to determine the final number of nephrons in and proper size of the organ. The secreted BMP7 acts on the nephron progenitor cells to exert its dual functions: To maintain and expand the progenitor population and to provide them with competence to respond to differentiation cues, each relying on distinct signaling pathways. Intriguingly, in the adult organ, BMP7 has been implicated in protection against and regeneration from injury. Exogenous administration of recombinant BMP7 to animal models of kidney diseases has shown promising effects in counteracting inflammation, apoptosis and fibrosis evoked upon injury. Although the expression pattern of BMP7 has been well described, the mechanisms by which it is regulated have remained elusive and the processes by which the secretion sites of BMP7 impinge upon its functions in kidney development and diseases have not yet been assessed. Understanding the regulatory mechanisms will pave the way towards gaining better insight into the roles of BMP7, and to achieving desired control of the gene expression as a therapeutic strategy for kidney diseases.展开更多
On the basis of the ovine bone morphogenetic protein 15(BMP15)gene,two pairs of primers(PI and P2)were designed to amplify exons 1 and 2 of the BMP15 gene in five randomly selected does of both Angora and Jining Grey ...On the basis of the ovine bone morphogenetic protein 15(BMP15)gene,two pairs of primers(PI and P2)were designed to amplify exons 1 and 2 of the BMP15 gene in five randomly selected does of both Angora and Jining Grey goats.The sequences of BMP15 exon 1(P1 amplification)of Angora and Jining Grey goats were identical.There was a 3-nucleotide(CTT)insertion in positions 268 to 270 of goat BMP 15 exon1 compared with that of sheep(GenBank accession number AF236078),which caused a leucine insertion in the 12th position of amino acid sequence.Sequence length of goat BMP 15 exon 2(P2 amplification)was identical with that of sheep(AF236079),but there were seven nucleotide and four amino acid changes between goat and sheep.The nucleotide in the 963rd position of BMP15 exon 2 was A for Angora goat and sheep,and G for Jining Grey goat.Based on this A963G mutation,primer pair P3 was designed to detect single nucleotide polymorphism of BMP15 exon 2 in breeds of high prolificacy(Jining Grey),moderate prolificacy(Boer)and low prolificacy(Angora and Inner Mongolia Cashmere)by polymerase chain reactionsingle strand conformation polymorphism(PCR-SSCP).Three genotypes(AA,AG and GG)were detected in Jining Grey goats,two genotypes(AG and GG)in Boer,and only the AA genotype in Angora and Inner Mongolia Cashmere goats.Sequencing revealed one mutation(A963G)in genotype GG compared with genotype AA,and this mutation resulted in an amino acid change of serine→glycine(S300G).In Jining Grey goats,frequencies of AA,AG and GG genotypes were 0.008,0.059 and 0.933,respectively.Genotypic distributions of the BMP 15 gene were significantly different(P<0.05 or P<0.001)between Jining Grey and Boer,Angora,and Inner Mongolia Cashmere goats.In Jining Grey goats,the does with the GG genotype had 0.71(P<0.05)or 1.57(P<0.05)additional kids than did those with AG or AA genotypes,and does with the AG genotype had 0.86(P<0.05)more kids than did those with the AA genotype.These results tentatively indicate that the BMP15 gene is either a major gene that affects prolificacy in Jining Grey goats,or may be a molecular marker in close linkage with such a gene.展开更多
To express die mature peptide of human bone morphogenetic protein-2 in Escherichia coil. Methods: TheDNA fragment encoding the mature peptide of human bone morphogenetic protein-2 (hBMP-2m) was inserted into expressio...To express die mature peptide of human bone morphogenetic protein-2 in Escherichia coil. Methods: TheDNA fragment encoding the mature peptide of human bone morphogenetic protein-2 (hBMP-2m) was inserted into expression vectorpDH in which foreign gene was controlled by PRPL promoters. E. coli DH5a transformed with recombinant plasmid pDHB2m wasinduced at 42℃to express the target protein. The expressed product was partially purified and refolded, and then implanted intorat thigh muscles to assay its bone inductive activity. Results: After induction, a protein band on SDS-PAGE gel with an apparentmol. wt. of 13kD was observed to anticipate in the strain carrying pDHB2m, but not in the control. The expressed hBMP-2m accounted for 45%-60% of the total bacterial protein. The expressed product existed in a form of inclusion body. After partially purified and refolded, rhBMP-2m could induce the formation of cartilage and bone tissue heterotopically. Conclusion: The maturepeptide of human bone morphogenetic protein-2 has ben successfully expressed in E. coli and the product has ectopic bone inductive activity.展开更多
Objective:To clone the full-length human bane morphogenetic protein-7 (BMP-7 ) gene and analyse its sequence, to aid in investigation of its function and structure. Methods : Total RNA was isolated from Chinese fetal ...Objective:To clone the full-length human bane morphogenetic protein-7 (BMP-7 ) gene and analyse its sequence, to aid in investigation of its function and structure. Methods : Total RNA was isolated from Chinese fetal kidney by the acid gmnidinium thiocyanate phenol-chloroform method. Two overlapping segments of human BMP- 1 cDNA were obtained by reverse transcription (RT)-PCR. Following application, the two segments were ligated to each other and subcloned into POEM-T easy vector to form PEGM-T easy/hBMP-7 recombinant plasmid. Sanger dideoxy chain-termination method was used to sequence the cDNA. Results. There was 750 bp fragment obtained RT-PCR using #2 primer from 5' end of BMP-7 gene (PCR by using # 2 and # 1) ,and 540 bp fragment from 3' end was generated by KT-PCR using # 4 primer (PCR using # 3 and # 4). Full-length cDNA encoding BMP-7 was obtained by religation of two segments. When compared with hBMP-7 sequence in Gene bank (XM30619) ,our full-length BMP-7 cDNA has a G instead of a T at nucleotide 862. This change results in valine substituting for phenylalanine in the protein. Conclusion. This is the first time that BMP-7 cDNA was successfully cloned from Chinese fetal kidney. BMP-7 cDNA plays an important role in healing injuries of the osteo-articular system. This makes BMP-7 is an attractive target far various clinical applications.展开更多
以控制R om ney Inverdale绵羊和R om ney H anna绵羊高繁殖力的骨形态发生蛋白15(BM P 15)基因为候选基因,采用PCR-RFLP方法检测BM P 15基因在高繁殖力山羊品种(济宁青山羊)以及低繁殖力山羊品种(内蒙古绒山羊、安哥拉山羊、波尔山羊)...以控制R om ney Inverdale绵羊和R om ney H anna绵羊高繁殖力的骨形态发生蛋白15(BM P 15)基因为候选基因,采用PCR-RFLP方法检测BM P 15基因在高繁殖力山羊品种(济宁青山羊)以及低繁殖力山羊品种(内蒙古绒山羊、安哥拉山羊、波尔山羊)中的多态性,同时研究该基因对济宁青山羊高繁殖力的影响。结果表明:BM P 15基因在济宁青山羊、内蒙古绒山羊、安哥拉山羊和波尔山羊中既未发生与Inverdale绵羊相同的V 31D突变,也未发生与H anna绵羊相同的Q 23T er突变。这表明BM P 15基因这2个突变位点对济宁青山羊的高繁殖力没有显著影响。展开更多
根据绵羊骨形态发生蛋白15(bone morphogenetic protein 15,BMP15)基因序列设计2对引物(P1和P2),分别扩增随机选取的安哥拉山羊和济宁青山羊各5个个体的BMP15基因外显子1、2并克隆测序。测序结果与绵羊BMP15基因外显子1(AF236078)相比,...根据绵羊骨形态发生蛋白15(bone morphogenetic protein 15,BMP15)基因序列设计2对引物(P1和P2),分别扩增随机选取的安哥拉山羊和济宁青山羊各5个个体的BMP15基因外显子1、2并克隆测序。测序结果与绵羊BMP15基因外显子1(AF236078)相比,山羊BMP15基因外显子1在第268-270位插入3个核苷酸CTT(268insCTT),导致编码的氨基酸第12位插入亮氨酸(12insL);山羊BMP15基因外显子2序列长度与绵羊(AF236079)一致,但存在7处核苷酸不同,引起4个氨基酸变化,济宁青山羊与安哥拉山羊相比在963位存在核苷酸变化(A963G)。依据A963G变异设计引物P3,利用PCR-SSCP方法检测BMP15基因外显子2在高繁殖力品种(济宁青山羊)、中等繁殖力品种(波尔山羊)和低繁殖力品种(安哥拉山羊和内蒙古绒山羊)中的单核苷酸多态性,分析该基因对济宁青山羊多羔性的影响。结果在济宁青山羊中检测到AA、AG和GG基因型,在波尔山羊中检测到AG和GG基因型,在安哥拉山羊和内蒙古绒山羊中仅检测到AA基因型;测序分析发现GG与AA基因型相比存在1个突变(A963G),导致第300位的丝氨酸变为甘氨酸(S300G);济宁青山羊AA、AG和GG基因型频率分别为0.008、0.059和0.933;高繁殖力山羊品种与中、低繁殖力山羊品种之间BMP15基因型分布存在显著(P<0.05)和极显著(P<0.001)差异;GG基因型济宁青山羊产羔数最小二乘均值比AG和AA基因型的分别多0.71只(P<0.05)和1.57只(P<0.05),AG基因型比AA基因型的多0.86只(P<0.05)。本研究结果初步显示BMP15基因可能是影响济宁青山羊多羔性的一个主效基因或是与之紧密连锁的一个标记。展开更多
本研究旨在阐明骨形态发生蛋白15(Bone morphogenetic protein 15,BMP15)基因多态性与邵伯鸡母系产蛋性状之间的关系,为鸡繁殖性状的标记辅助选择提供科学依据。采用PCR-RFLP技术检测261只邵伯鸡母系BMP15的基因多态性,用最小二乘法分...本研究旨在阐明骨形态发生蛋白15(Bone morphogenetic protein 15,BMP15)基因多态性与邵伯鸡母系产蛋性状之间的关系,为鸡繁殖性状的标记辅助选择提供科学依据。采用PCR-RFLP技术检测261只邵伯鸡母系BMP15的基因多态性,用最小二乘法分析该基因多态性与邵伯鸡母系产蛋性状的关系。发现BMP15基因外显子1序列中存在3个多态位点C397T、A474G和C594T,其中C397T位点C→T的突变使亮氨酸变为苯丙氨酸,经RFLP检测,3个多态位点均发现3种基因型。χ2检验表明,邵伯鸡母系在这3个位点均处于Hardy-Weinberg平衡。用最小二乘法分析这3个位点的多态性与邵伯鸡母系产蛋性状之间的关系,结果发现,C397T位点TT基因型个体的开产日龄显著早于CT型个体(P<0.05),TT型个体的300日龄产蛋数显著高于CT型个体(P<0.05);A474G位点AA、AG和GG型个体间的各性状差异均不显著(P>0.05);C594T位点CC型个体的开产日龄显著早于CT与TT型个体。3个位点的合并基因型TTAATT对开产日龄、开产体质量、开产蛋质量、300日龄平均蛋质量、300日龄产蛋数均有显著影响(P<0.05)。对于邵伯鸡母系而言,TTAATT是最有利基因型,本研究结果初步表明,BMP15基因合并基因型TTAATT可以作为邵伯鸡母系产蛋性状潜在的DNA分子标记。展开更多
文摘Partial cDNA sequence of rabbit BMP15 was cloned by RT-PCR from rabbit ovaries, showing a similarity of 83%-90% with the BMP15 nucleotide sequences in humans, mice, ovine, sheep, cows and pigs. The expression of BMP15 in rabbit cumulus-oocyte complexs during oocytes in vitro maturation （IVM） was measured by fluorescent quantitative RT-PCR method. BMP 15 was expressed at low levels in immature oocytes and increased to the highest level at 16h of IVM, which coincides with the time of cumulus cell expansion, then declined slowly under IVM cultivation. The expression pattern of BMP 15 suggested that it might be important in cumulus expansion in rabbits.
基金Supported by Grants-in-Aid for Young Scientists(B)(No.15K18454 to Tsujimura T)Scientific Research(B)(No.15H03001 to Hishikawa K)Scientific Research(C)(Nos.25461208 to Takase O,15K09244 to Yoshikawa M and 26462400 to Idei M)from the Japan Society for the Promotion of Science
文摘The gene encoding bone morphogenetic protein-7(BMP7) is expressed in the developing kidney in embryos and also in the mature organ in adults. During kidney development, expression of BMP7 is essential to determine the final number of nephrons in and proper size of the organ. The secreted BMP7 acts on the nephron progenitor cells to exert its dual functions: To maintain and expand the progenitor population and to provide them with competence to respond to differentiation cues, each relying on distinct signaling pathways. Intriguingly, in the adult organ, BMP7 has been implicated in protection against and regeneration from injury. Exogenous administration of recombinant BMP7 to animal models of kidney diseases has shown promising effects in counteracting inflammation, apoptosis and fibrosis evoked upon injury. Although the expression pattern of BMP7 has been well described, the mechanisms by which it is regulated have remained elusive and the processes by which the secretion sites of BMP7 impinge upon its functions in kidney development and diseases have not yet been assessed. Understanding the regulatory mechanisms will pave the way towards gaining better insight into the roles of BMP7, and to achieving desired control of the gene expression as a therapeutic strategy for kidney diseases.
基金supported by National Key Basic Research and Development Program of China(No.2006CB102105)National High Technology Research and Development Program of China(No.2006AA10Z139)+1 种基金National Natural Science Foundation of China(No.30540052 and No.30871773)Beijing Natural Sciences Foundation of China(No.6062023)
文摘On the basis of the ovine bone morphogenetic protein 15(BMP15)gene,two pairs of primers(PI and P2)were designed to amplify exons 1 and 2 of the BMP15 gene in five randomly selected does of both Angora and Jining Grey goats.The sequences of BMP15 exon 1(P1 amplification)of Angora and Jining Grey goats were identical.There was a 3-nucleotide(CTT)insertion in positions 268 to 270 of goat BMP 15 exon1 compared with that of sheep(GenBank accession number AF236078),which caused a leucine insertion in the 12th position of amino acid sequence.Sequence length of goat BMP 15 exon 2(P2 amplification)was identical with that of sheep(AF236079),but there were seven nucleotide and four amino acid changes between goat and sheep.The nucleotide in the 963rd position of BMP15 exon 2 was A for Angora goat and sheep,and G for Jining Grey goat.Based on this A963G mutation,primer pair P3 was designed to detect single nucleotide polymorphism of BMP15 exon 2 in breeds of high prolificacy(Jining Grey),moderate prolificacy(Boer)and low prolificacy(Angora and Inner Mongolia Cashmere)by polymerase chain reactionsingle strand conformation polymorphism(PCR-SSCP).Three genotypes(AA,AG and GG)were detected in Jining Grey goats,two genotypes(AG and GG)in Boer,and only the AA genotype in Angora and Inner Mongolia Cashmere goats.Sequencing revealed one mutation(A963G)in genotype GG compared with genotype AA,and this mutation resulted in an amino acid change of serine→glycine(S300G).In Jining Grey goats,frequencies of AA,AG and GG genotypes were 0.008,0.059 and 0.933,respectively.Genotypic distributions of the BMP 15 gene were significantly different(P<0.05 or P<0.001)between Jining Grey and Boer,Angora,and Inner Mongolia Cashmere goats.In Jining Grey goats,the does with the GG genotype had 0.71(P<0.05)or 1.57(P<0.05)additional kids than did those with AG or AA genotypes,and does with the AG genotype had 0.86(P<0.05)more kids than did those with the AA genotype.These results tentatively indicate that the BMP15 gene is either a major gene that affects prolificacy in Jining Grey goats,or may be a molecular marker in close linkage with such a gene.
文摘To express die mature peptide of human bone morphogenetic protein-2 in Escherichia coil. Methods: TheDNA fragment encoding the mature peptide of human bone morphogenetic protein-2 (hBMP-2m) was inserted into expression vectorpDH in which foreign gene was controlled by PRPL promoters. E. coli DH5a transformed with recombinant plasmid pDHB2m wasinduced at 42℃to express the target protein. The expressed product was partially purified and refolded, and then implanted intorat thigh muscles to assay its bone inductive activity. Results: After induction, a protein band on SDS-PAGE gel with an apparentmol. wt. of 13kD was observed to anticipate in the strain carrying pDHB2m, but not in the control. The expressed hBMP-2m accounted for 45%-60% of the total bacterial protein. The expressed product existed in a form of inclusion body. After partially purified and refolded, rhBMP-2m could induce the formation of cartilage and bone tissue heterotopically. Conclusion: The maturepeptide of human bone morphogenetic protein-2 has ben successfully expressed in E. coli and the product has ectopic bone inductive activity.
文摘Objective:To clone the full-length human bane morphogenetic protein-7 (BMP-7 ) gene and analyse its sequence, to aid in investigation of its function and structure. Methods : Total RNA was isolated from Chinese fetal kidney by the acid gmnidinium thiocyanate phenol-chloroform method. Two overlapping segments of human BMP- 1 cDNA were obtained by reverse transcription (RT)-PCR. Following application, the two segments were ligated to each other and subcloned into POEM-T easy vector to form PEGM-T easy/hBMP-7 recombinant plasmid. Sanger dideoxy chain-termination method was used to sequence the cDNA. Results. There was 750 bp fragment obtained RT-PCR using #2 primer from 5' end of BMP-7 gene (PCR by using # 2 and # 1) ,and 540 bp fragment from 3' end was generated by KT-PCR using # 4 primer (PCR using # 3 and # 4). Full-length cDNA encoding BMP-7 was obtained by religation of two segments. When compared with hBMP-7 sequence in Gene bank (XM30619) ,our full-length BMP-7 cDNA has a G instead of a T at nucleotide 862. This change results in valine substituting for phenylalanine in the protein. Conclusion. This is the first time that BMP-7 cDNA was successfully cloned from Chinese fetal kidney. BMP-7 cDNA plays an important role in healing injuries of the osteo-articular system. This makes BMP-7 is an attractive target far various clinical applications.
文摘以控制R om ney Inverdale绵羊和R om ney H anna绵羊高繁殖力的骨形态发生蛋白15(BM P 15)基因为候选基因,采用PCR-RFLP方法检测BM P 15基因在高繁殖力山羊品种(济宁青山羊)以及低繁殖力山羊品种(内蒙古绒山羊、安哥拉山羊、波尔山羊)中的多态性,同时研究该基因对济宁青山羊高繁殖力的影响。结果表明:BM P 15基因在济宁青山羊、内蒙古绒山羊、安哥拉山羊和波尔山羊中既未发生与Inverdale绵羊相同的V 31D突变,也未发生与H anna绵羊相同的Q 23T er突变。这表明BM P 15基因这2个突变位点对济宁青山羊的高繁殖力没有显著影响。
文摘根据绵羊骨形态发生蛋白15(bone morphogenetic protein 15,BMP15)基因序列设计2对引物(P1和P2),分别扩增随机选取的安哥拉山羊和济宁青山羊各5个个体的BMP15基因外显子1、2并克隆测序。测序结果与绵羊BMP15基因外显子1(AF236078)相比,山羊BMP15基因外显子1在第268-270位插入3个核苷酸CTT(268insCTT),导致编码的氨基酸第12位插入亮氨酸(12insL);山羊BMP15基因外显子2序列长度与绵羊(AF236079)一致,但存在7处核苷酸不同,引起4个氨基酸变化,济宁青山羊与安哥拉山羊相比在963位存在核苷酸变化(A963G)。依据A963G变异设计引物P3,利用PCR-SSCP方法检测BMP15基因外显子2在高繁殖力品种(济宁青山羊)、中等繁殖力品种(波尔山羊)和低繁殖力品种(安哥拉山羊和内蒙古绒山羊)中的单核苷酸多态性,分析该基因对济宁青山羊多羔性的影响。结果在济宁青山羊中检测到AA、AG和GG基因型,在波尔山羊中检测到AG和GG基因型,在安哥拉山羊和内蒙古绒山羊中仅检测到AA基因型;测序分析发现GG与AA基因型相比存在1个突变(A963G),导致第300位的丝氨酸变为甘氨酸(S300G);济宁青山羊AA、AG和GG基因型频率分别为0.008、0.059和0.933;高繁殖力山羊品种与中、低繁殖力山羊品种之间BMP15基因型分布存在显著(P<0.05)和极显著(P<0.001)差异;GG基因型济宁青山羊产羔数最小二乘均值比AG和AA基因型的分别多0.71只(P<0.05)和1.57只(P<0.05),AG基因型比AA基因型的多0.86只(P<0.05)。本研究结果初步显示BMP15基因可能是影响济宁青山羊多羔性的一个主效基因或是与之紧密连锁的一个标记。
文摘本研究旨在阐明骨形态发生蛋白15(Bone morphogenetic protein 15,BMP15)基因多态性与邵伯鸡母系产蛋性状之间的关系,为鸡繁殖性状的标记辅助选择提供科学依据。采用PCR-RFLP技术检测261只邵伯鸡母系BMP15的基因多态性,用最小二乘法分析该基因多态性与邵伯鸡母系产蛋性状的关系。发现BMP15基因外显子1序列中存在3个多态位点C397T、A474G和C594T,其中C397T位点C→T的突变使亮氨酸变为苯丙氨酸,经RFLP检测,3个多态位点均发现3种基因型。χ2检验表明,邵伯鸡母系在这3个位点均处于Hardy-Weinberg平衡。用最小二乘法分析这3个位点的多态性与邵伯鸡母系产蛋性状之间的关系,结果发现,C397T位点TT基因型个体的开产日龄显著早于CT型个体(P<0.05),TT型个体的300日龄产蛋数显著高于CT型个体(P<0.05);A474G位点AA、AG和GG型个体间的各性状差异均不显著(P>0.05);C594T位点CC型个体的开产日龄显著早于CT与TT型个体。3个位点的合并基因型TTAATT对开产日龄、开产体质量、开产蛋质量、300日龄平均蛋质量、300日龄产蛋数均有显著影响(P<0.05)。对于邵伯鸡母系而言,TTAATT是最有利基因型,本研究结果初步表明,BMP15基因合并基因型TTAATT可以作为邵伯鸡母系产蛋性状潜在的DNA分子标记。
