Two starch-branching enzyme (SBE) in rice, is known to be a key enzyme in amylopectin biosynthesis. The cDNA of two SBE(starch-branching enzyme) genes Sbe1 and Sbc3 encoding SBE Ⅰ and SBE Ⅲ (two major isoforms in ri...Two starch-branching enzyme (SBE) in rice, is known to be a key enzyme in amylopectin biosynthesis. The cDNA of two SBE(starch-branching enzyme) genes Sbe1 and Sbc3 encoding SBE Ⅰ and SBE Ⅲ (two major isoforms in rice) were cloned by an improved RT-PCR technique, from a template cDNA library derived from the total mRNAs extracted from the immature seeds of a japonica rice Wuyunjing 7. DNA sequence analysis showed that the size of the cloned Sbe1 and Sbe3 cDNAs were 2490 and 2481 bp long, respectively, including their entire coding sequences. Comparison analysis indicated that the nucleotide sequence of Sbe3 was the same as that of sbc3 (Genbank Accession No. D16201) as reported previously. There were only four base-pairs difference, which resulted in changes of two deduced amino acids between the cloned Sbel cDNA and the reported sbe1 (Genbank Accession No. D11082). The cloned Sbe1 and Sbe3 cDNAs make it possible to improve rice starch quality through genetic engineering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
Aim: To clone two splicing products of the mouse mLRG-cDNA and to express mLRG protein. Methods: The sequence obtained was compared human lrg to mouse genome with a comparative BLAST genome search and found completely...Aim: To clone two splicing products of the mouse mLRG-cDNA and to express mLRG protein. Methods: The sequence obtained was compared human lrg to mouse genome with a comparative BLAST genome search and found completely identical. We spliced some fragments to a whole mouse lrg-cDNA sequence and designed a pair of primers at completely homologous fragments in 5’-UTR and 3’-UTR, we amplified mouse lrg-cDNA by RT-PCR. Then the sequence encoding the mLRG protein was amplified by RT-PCR from the total RNA of NIH3T3 cell stimulated by lps (lipopolysaccharide), and we got two splicing products of mLRG (mLRGW, mLRGS) and two sequences encoding protein were cloned into the prokaryotic expression vector pTAT so as to construct the recombinant expression vector pTAT-MLRGW and pTAT-MLRGS. The proteins were expressed in E. coli BL21 (DE3). Results: We got a cDNA fragment with the length of 1905 bp. Its location is at chromosome X qF4 site and we amplified two encoding regions covered 1554 bp and 1404 bp respectively (mlrgW mlrgS). His-TAT-mLRGW and His-TAT-mLRGS fusion protein were expressed successfully. mlrgW is consist of 10 exons and 9 introns;mlrgS is consist of 11exons and 10 introns. Conclusion: Cloning of two splicing products of mouse novel gene MLRG and prokaryotic protein expressions are of help in the further study of this gene.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
[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
文摘Two starch-branching enzyme (SBE) in rice, is known to be a key enzyme in amylopectin biosynthesis. The cDNA of two SBE(starch-branching enzyme) genes Sbe1 and Sbc3 encoding SBE Ⅰ and SBE Ⅲ (two major isoforms in rice) were cloned by an improved RT-PCR technique, from a template cDNA library derived from the total mRNAs extracted from the immature seeds of a japonica rice Wuyunjing 7. DNA sequence analysis showed that the size of the cloned Sbe1 and Sbe3 cDNAs were 2490 and 2481 bp long, respectively, including their entire coding sequences. Comparison analysis indicated that the nucleotide sequence of Sbe3 was the same as that of sbc3 (Genbank Accession No. D16201) as reported previously. There were only four base-pairs difference, which resulted in changes of two deduced amino acids between the cloned Sbel cDNA and the reported sbe1 (Genbank Accession No. D11082). The cloned Sbe1 and Sbe3 cDNAs make it possible to improve rice starch quality through genetic engineering
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Aim: To clone two splicing products of the mouse mLRG-cDNA and to express mLRG protein. Methods: The sequence obtained was compared human lrg to mouse genome with a comparative BLAST genome search and found completely identical. We spliced some fragments to a whole mouse lrg-cDNA sequence and designed a pair of primers at completely homologous fragments in 5’-UTR and 3’-UTR, we amplified mouse lrg-cDNA by RT-PCR. Then the sequence encoding the mLRG protein was amplified by RT-PCR from the total RNA of NIH3T3 cell stimulated by lps (lipopolysaccharide), and we got two splicing products of mLRG (mLRGW, mLRGS) and two sequences encoding protein were cloned into the prokaryotic expression vector pTAT so as to construct the recombinant expression vector pTAT-MLRGW and pTAT-MLRGS. The proteins were expressed in E. coli BL21 (DE3). Results: We got a cDNA fragment with the length of 1905 bp. Its location is at chromosome X qF4 site and we amplified two encoding regions covered 1554 bp and 1404 bp respectively (mlrgW mlrgS). His-TAT-mLRGW and His-TAT-mLRGS fusion protein were expressed successfully. mlrgW is consist of 10 exons and 9 introns;mlrgS is consist of 11exons and 10 introns. Conclusion: Cloning of two splicing products of mouse novel gene MLRG and prokaryotic protein expressions are of help in the further study of this gene.
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。
