An Amorpha fruticosa cDNA encoding 4 coumarate:CoA ligase (4CL), a key enzyme of phenylpropanoid metabolism related to lignin forming, was cloned by degenerating oligo primed polymerase chain reaction (PCR) and ...An Amorpha fruticosa cDNA encoding 4 coumarate:CoA ligase (4CL), a key enzyme of phenylpropanoid metabolism related to lignin forming, was cloned by degenerating oligo primed polymerase chain reaction (PCR) and rapid amplification of cDNA end (RACE) PCR. We designed 5′RACE primers based on 4CLA1 fragment which obtained from degenerate PCR. Inverse PCR and nested PCR enabled cloning of the remainder fragments of the gene included 5′ and 3′ end sequence. The ORF encodes a polypeptide of 540 amino acids. The predicted amino acid sequence exhibits significant homology with those of other cloned 4CL genes, contain domains typical of predicted 4CL proteins, in particular a postulated AMP binding site, catalytic domain, and conserved Cys residues.展开更多
A method based on degenerate Oligo primed polymerase chain reaction (PCR) and random amplification of cDNA end (RACE) PCR for cloning a full length cDNA is described. An Amorpha fruticosa cDNA clone encoding UDP gluco...A method based on degenerate Oligo primed polymerase chain reaction (PCR) and random amplification of cDNA end (RACE) PCR for cloning a full length cDNA is described. An Amorpha fruticosa cDNA clone encoding UDP glucose pyrophosphorylase (UGP), a key enzyme producing UDP glucose in the synthesis of sucrose and cellulose, is cloned by using this method. We design 5’ RACE primers based on UGPA1 fragment, which obtains from degenerate PCR. Inverse PCR and nested PCR enable cloning of the remainder 5’ and 3’ end fragments of the gene. The deduced amino acid sequence exhibits significant homology with the other UGP genes cloned. This method is more simple and inexpensive than screening cDNA library, and can be easily adapted to clone other genes.展开更多
The differential hybridization technique hasbeen widely used to identify genes that are dif-ferentially expressed.However,this approachhas several drawbacks.First,the screeningprocedures are rather labor-intensive and...The differential hybridization technique hasbeen widely used to identify genes that are dif-ferentially expressed.However,this approachhas several drawbacks.First,the screeningprocedures are rather labor-intensive and time-consuming.Second,the amount of phageDNAs transferred onto the two filters may notbe equivalent,which leads to an inaccurate se-lection of a positive clone.Third,isolation ofphage DNA is slow and cumbersome.Here,aPCR based differential screening method that展开更多
ANXA2（AnnexinA2）, a calcium-dependent phospholipid bind- ing protein, is involved in various Ca2＋-related biological activities. In the present study, full-length cDNA of ANXA2 was isolated from the velvet antler t...ANXA2（AnnexinA2）, a calcium-dependent phospholipid bind- ing protein, is involved in various Ca2＋-related biological activities. In the present study, full-length cDNA of ANXA2 was isolated from the velvet antler tip tissue of sika deer （Cervus nippon hortulomm）; the amino acid sequence and gene expression was analyzed by using bioinformatics and real-time reverse transcdptase polymerase chain reaction （RT-PCR） techniques. Nucleotide sequence analysis reveals that the full-length cDNA of the ANXA2 gene was 1372 bp, of which 1020 bp was in the opan-reading frame （OR.F） encoding 339 amino acids; its relative mo- lecular weight was 38.3 kDa; and isoelectrie point was 6.72. Sequence analysis indicates that the protein includes four conserved tan- dem-duplication ANX domains. The gene-aceession nucleotide sequence number in GenBank is JX315571. Expression analysis by RT-PCR re- veals that ANXA2 gene expression has a significant positive correlation with the antler-tissue mineralization process, indicating that this gene may play an important role in the regulation of antler-tissue mineraliza- tion.展开更多
[目的 ]扩增和克隆猪带绦虫囊尾蚴 Ag B基因的 c DNA编码区。 [方法 ]提取猪囊尾蚴总 RNA中 ,利用 RT- PCR技术扩增出 Ag B基因 c DNA编码区 ,然后将其克隆到载体 p UC118中进行序列分析。 [结果 ]PCR反应产物为单一条带 ,大小为 2 .6 k...[目的 ]扩增和克隆猪带绦虫囊尾蚴 Ag B基因的 c DNA编码区。 [方法 ]提取猪囊尾蚴总 RNA中 ,利用 RT- PCR技术扩增出 Ag B基因 c DNA编码区 ,然后将其克隆到载体 p UC118中进行序列分析。 [结果 ]PCR反应产物为单一条带 ,大小为 2 .6 kb。测序结果与澳大利亚株猪囊尾蚴 Ag B序列有 99.8%的同源性 ,二者的氨基酸序列有 99.3%的同源性。 [结论 ]成功地克隆了猪囊尾蚴 Ag B基因 c DNA编码区。展开更多
利用RT-PCR和cDNA末端快速扩增技术(rapid amplification of cDNA ends,RACE)克隆了黄颡鱼DMRT1基因cDNA全长序列,并利用实时荧光定量RT-PCR技术对该基因在黄颡鱼成体不同组织及不同发育阶段的表达情况进行研究。结果表明,黄颡鱼DMRT1基...利用RT-PCR和cDNA末端快速扩增技术(rapid amplification of cDNA ends,RACE)克隆了黄颡鱼DMRT1基因cDNA全长序列,并利用实时荧光定量RT-PCR技术对该基因在黄颡鱼成体不同组织及不同发育阶段的表达情况进行研究。结果表明,黄颡鱼DMRT1基因cDNA序列全长1 381bp,其中5′端非翻译区30bp,3′端非翻译区454bp[不包括poly(A)],开放阅读框885bp,编码295个氨基酸。氨基酸序列同源性分析表明,黄颡鱼DMRT1基因与革胡子鲶同源性最高(为81%),与黑鲷、虹鳟、斑马鱼、青鳉的同源性分别为60%、59%、64%和52%,与小鼠、人的同源性较低,分别为42%和44%。实时荧光定量RT-PCR分析表明:DMRT1基因在黄颡鱼胚胎发育阶段及胚后发育的1～51d仔鱼均有表达,且在胚后发育的第31天表达量最高;在成体,只在雄性精巢中特异性表达,其他组织均无表达,且性腺发育阶段的Ⅳ期精巢表达量最高,表明该基因可能在黄颡鱼雄性性腺的形成或功能维持上具有重要作用。展开更多
Acetylcholinesterase (AChE) is the target of organophosphate and carbamate pesticides. Organophosphate resistance is worldspread in the cotton bollworm[Helicoverpa armigera (Hübner)]. With the degenerate primers ...Acetylcholinesterase (AChE) is the target of organophosphate and carbamate pesticides. Organophosphate resistance is worldspread in the cotton bollworm[Helicoverpa armigera (Hübner)]. With the degenerate primers we amplified a 281 bp cDNA fragment of acetylcholinesterase (AChE) gene in H. armigera by reverse transcription-polymerase chain reaction (RT-PCR) method using total RNA extracted from 4th larva as the template. The cDNA fragment was inserted into pGEMT vector and then cloned. The deduced amino acid sequence of AChE consisted of 94 residues. The sequence analysis indicated that the deduced amino acid sequence of the cDNA fragment shares high identity with AChE gene from other published insects and animals. The acquired sequence had 84%,79%,74%,70%,70%,72%,68%,61%,55% and 57% of amino acid residues identical to those of Leptinotarsa decemlineata(L.d.), Nephotettix cincticeps(N.c.),Anopheles stephensi(A.s.), Aedes aegypti(A.a.), Lucilia cuprina(L.c.), Drosophila melanogaster(D.m.), Musca domestica(M.d.), Meloidogyne incognita(M.i.), Torpedo californica(T.c.) and Gallus gallus(G.g.), respectively. All these results firmly established that the amplified cDNA fragment was the partial sequence of AChE gene in H. armigera. This is the first report of partial cDNA sequence of AChE in H. armigera.展开更多
文摘An Amorpha fruticosa cDNA encoding 4 coumarate:CoA ligase (4CL), a key enzyme of phenylpropanoid metabolism related to lignin forming, was cloned by degenerating oligo primed polymerase chain reaction (PCR) and rapid amplification of cDNA end (RACE) PCR. We designed 5′RACE primers based on 4CLA1 fragment which obtained from degenerate PCR. Inverse PCR and nested PCR enabled cloning of the remainder fragments of the gene included 5′ and 3′ end sequence. The ORF encodes a polypeptide of 540 amino acids. The predicted amino acid sequence exhibits significant homology with those of other cloned 4CL genes, contain domains typical of predicted 4CL proteins, in particular a postulated AMP binding site, catalytic domain, and conserved Cys residues.
文摘A method based on degenerate Oligo primed polymerase chain reaction (PCR) and random amplification of cDNA end (RACE) PCR for cloning a full length cDNA is described. An Amorpha fruticosa cDNA clone encoding UDP glucose pyrophosphorylase (UGP), a key enzyme producing UDP glucose in the synthesis of sucrose and cellulose, is cloned by using this method. We design 5’ RACE primers based on UGPA1 fragment, which obtains from degenerate PCR. Inverse PCR and nested PCR enable cloning of the remainder 5’ and 3’ end fragments of the gene. The deduced amino acid sequence exhibits significant homology with the other UGP genes cloned. This method is more simple and inexpensive than screening cDNA library, and can be easily adapted to clone other genes.
文摘The differential hybridization technique hasbeen widely used to identify genes that are dif-ferentially expressed.However,this approachhas several drawbacks.First,the screeningprocedures are rather labor-intensive and time-consuming.Second,the amount of phageDNAs transferred onto the two filters may notbe equivalent,which leads to an inaccurate se-lection of a positive clone.Third,isolation ofphage DNA is slow and cumbersome.Here,aPCR based differential screening method that
基金funded by the General Program of the National Natural Science Foundation of China(31271324)the Fundamental Research Funds for the Central Universities(2572014EA05-03)the Fundamental Research Funds for the Central Universities(DL10BA08)
文摘ANXA2（AnnexinA2）, a calcium-dependent phospholipid bind- ing protein, is involved in various Ca2＋-related biological activities. In the present study, full-length cDNA of ANXA2 was isolated from the velvet antler tip tissue of sika deer （Cervus nippon hortulomm）; the amino acid sequence and gene expression was analyzed by using bioinformatics and real-time reverse transcdptase polymerase chain reaction （RT-PCR） techniques. Nucleotide sequence analysis reveals that the full-length cDNA of the ANXA2 gene was 1372 bp, of which 1020 bp was in the opan-reading frame （OR.F） encoding 339 amino acids; its relative mo- lecular weight was 38.3 kDa; and isoelectrie point was 6.72. Sequence analysis indicates that the protein includes four conserved tan- dem-duplication ANX domains. The gene-aceession nucleotide sequence number in GenBank is JX315571. Expression analysis by RT-PCR re- veals that ANXA2 gene expression has a significant positive correlation with the antler-tissue mineralization process, indicating that this gene may play an important role in the regulation of antler-tissue mineraliza- tion.
文摘[目的 ]扩增和克隆猪带绦虫囊尾蚴 Ag B基因的 c DNA编码区。 [方法 ]提取猪囊尾蚴总 RNA中 ,利用 RT- PCR技术扩增出 Ag B基因 c DNA编码区 ,然后将其克隆到载体 p UC118中进行序列分析。 [结果 ]PCR反应产物为单一条带 ,大小为 2 .6 kb。测序结果与澳大利亚株猪囊尾蚴 Ag B序列有 99.8%的同源性 ,二者的氨基酸序列有 99.3%的同源性。 [结论 ]成功地克隆了猪囊尾蚴 Ag B基因 c DNA编码区。
文摘Acetylcholinesterase (AChE) is the target of organophosphate and carbamate pesticides. Organophosphate resistance is worldspread in the cotton bollworm[Helicoverpa armigera (Hübner)]. With the degenerate primers we amplified a 281 bp cDNA fragment of acetylcholinesterase (AChE) gene in H. armigera by reverse transcription-polymerase chain reaction (RT-PCR) method using total RNA extracted from 4th larva as the template. The cDNA fragment was inserted into pGEMT vector and then cloned. The deduced amino acid sequence of AChE consisted of 94 residues. The sequence analysis indicated that the deduced amino acid sequence of the cDNA fragment shares high identity with AChE gene from other published insects and animals. The acquired sequence had 84%,79%,74%,70%,70%,72%,68%,61%,55% and 57% of amino acid residues identical to those of Leptinotarsa decemlineata(L.d.), Nephotettix cincticeps(N.c.),Anopheles stephensi(A.s.), Aedes aegypti(A.a.), Lucilia cuprina(L.c.), Drosophila melanogaster(D.m.), Musca domestica(M.d.), Meloidogyne incognita(M.i.), Torpedo californica(T.c.) and Gallus gallus(G.g.), respectively. All these results firmly established that the amplified cDNA fragment was the partial sequence of AChE gene in H. armigera. This is the first report of partial cDNA sequence of AChE in H. armigera.
