In order to isolate the total cDNA of rice (Oryza sativa L.) epsps gene, RT-PCR was carried out with template of rice first-strand cDNA and primers designed according to rice EPSP synthase genomic sequence obtained in...In order to isolate the total cDNA of rice (Oryza sativa L.) epsps gene, RT-PCR was carried out with template of rice first-strand cDNA and primers designed according to rice EPSP synthase genomic sequence obtained in previous study. A 1 585-bp cDNA fragment was amplified and cloned. The 1 585-bp cDNA contains an open reading frame (ORF) comprising of 1 533 nucleotides (nt) which encodes a 511 residue polypepetides, including 67 amino acids chloroplast transit peptide and 444 amino acids EPSP synthase mature peptide. A comparison between the EPSP synthase of different sources indicates that the mature peptide shows more than 51% identity except for the fungi EPSP synthase and the transit peptide shows considerably less sequence conservation. The copy number of rice epsps gene is estimated to be one copy per haploid rice genome using southern blot. RT-PCR indicated that rice epsps gene is expressed in rice leaves, endosperms and roots and has the highest expression level in leaves.展开更多
Two acetylcholinesterase (AChE) genes cDNA fragments,Ag.acel and Ag.ace2,have been cloned from cotton aphid,Aphis gossypii Glover using degenerate primers with RT-PCR technique.Ag.acel gene cDNA fragment is of 282?bp ...Two acetylcholinesterase (AChE) genes cDNA fragments,Ag.acel and Ag.ace2,have been cloned from cotton aphid,Aphis gossypii Glover using degenerate primers with RT-PCR technique.Ag.acel gene cDNA fragment is of 282?bp encoding 94 amino acids,and Ag.ace2 gene cDNA fragment is of 264?bp encoding 88 amino acids.Both two putative AChE genes cDNA fragments share numerous similarities with those cloned from other insects.This is the first report of two AChE cDNA fragment sequences in the insect species,which provided the direct evidence of multiple AChE existence in insects.展开更多
[ Objective] To clone and analyze the sequence of Adiponectin receptor 1 （ AdipoR1 ） and receptor 2 （AdipoR2） cDNA of Guangxi Bama mini-pig. [Method] The Adiponectin receptors cDNAs were amplified by RT-PCR using ...[ Objective] To clone and analyze the sequence of Adiponectin receptor 1 （ AdipoR1 ） and receptor 2 （AdipoR2） cDNA of Guangxi Bama mini-pig. [Method] The Adiponectin receptors cDNAs were amplified by RT-PCR using skeletal muscle total RNA as template and then ligated into pMD18-T vector after purification. The recombinant pMD18-T vector was transformed into the E. coil DH5α for identification and sequencing. And the results were compared with the cDNA sequence from other species. [Result] The fragments, 1 128 bp and 1 161 bp in size, were amplified by RT-PCR and respectively consistent with the coding sequence of AdipoR1 gene and AdipoR2 gene. The homology analysis showed that the sequences of AdipoR1 gene and AdipoR2 gene were respectively 99.8% and 99.7% homologous to the sequence of domestic pig reported in GenBank with one base and three base missense mutations correspondingly. [ Conclusion] The AdipoR1 gene and AdipoR2. gene were successfully amplified from Guangxi Bama mini-pig, laying the foundation for the further study of the biological function of AdipoR genes and the design of novel drugs with AdipoR as target.展开更多
Total mRNA was extracted from a venom gland of snake Daboia russellii siamensis following the manufacturer＇s protocol of the PolyATtract System 1000 kit purchased from Promega Biotech. cDNAs encoding C-type lectins w...Total mRNA was extracted from a venom gland of snake Daboia russellii siamensis following the manufacturer＇s protocol of the PolyATtract System 1000 kit purchased from Promega Biotech. cDNAs encoding C-type lectins were amplified by RT-PCR and subcloned into a pMD18-T vector. Fourteen positive clones were selected for nucleotide sequencing and seven cDNAs encoding various snake venom C-type lectin-like protein precursors, designated as DRS-L1, DRS-L2, DRS-L3, DRS-L4 DRS-L5, DRS-L6 and DRS-L7, were obtained. Amino acid sequences of these proteins were deduced and each contains a carbohydrate recognition domain. Of all the deduced protein sequences, only DRS-L1 seemed to represent a closer sequence similarity to α subunits of other known snake venom C-type lectin-like proteins using the BLAST program. Homology comparison combined with analysis of cysteine position indicate that DRS-L1 and DRS-L2 are probably the light chain LC2 and LC1 of factor X activator from Daboia russellii siamensis venom, respectively. DRS-L3 and DRS-L4 might be the β subunits of higher molecular weight C-type lectin-like proteins while DRS-L5 and DRS-L6 might be β subunits of lower molecular weight C-type lectin-like proteins. DRS-L7 might be the β subunit of a platelet membrane glycoprotein Ib-binding protein similar to echicetin.展开更多
[Objective] cDNA of growth hormone releasing hormone （GHRH） receptor gene from Wuzhishan miniature pig was cloned and its sequence was also analyzed. [Method] Using genomic DNA extracted from porcine ear tissues of ...[Objective] cDNA of growth hormone releasing hormone （GHRH） receptor gene from Wuzhishan miniature pig was cloned and its sequence was also analyzed. [Method] Using genomic DNA extracted from porcine ear tissues of Wuzhishan miniature pig as the template, three pairs of primers were designed by the reported cDNA sequence of porcine GHRH, and cDNA was also amplified by RT-PCR. After being recovered and purified, PCR products were ligated to pMD18-T and then transformed into Escherichia coli DH5a. The transformation products were analyzed by PCR and double enzyme digestion to screen positive clones, and the positive clones were sequenced after identification in LB liquid medium. [ Result] cDNA of Wuzhishan miniature pig GHRH receptor gene was obtained successfully, and its length was 1 577 bp coding 423 amino acids. BLAST analysis showed that there were only 23 nuoleotides in difference between this fragment and pomine GHRH receptor gene, and its homology was 98%. However, both GHRH receptor genes were constituted by 423 amino acids with the sequence homology of 96%. [ Conclusion] This study provides theoretical basis for further studies on the dwarf mechanism of Wuzhishan miniature pig.展开更多
The effect of cold stress on the gene expression of cold-inducible RNA-binding protein （CIRP） in the intravital animals has not been reported till now. Compared with their organism cells, there were much more compli...The effect of cold stress on the gene expression of cold-inducible RNA-binding protein （CIRP） in the intravital animals has not been reported till now. Compared with their organism cells, there were much more complicated regulatory mechanisms for cold stress response in the organisms. The BALB/C mice with cold treatment were used as experimental animals for this study. The cDNA of CIRP was firstly cloned from the testis tissues of the BALB/C mice treated by cold stress The results indicated that CIRP in the organisms could be induced at low temperature and may protect the organisms from the cold damage. The amino acid sequence deduced via cDNA clone was 100%, 99.4%, 95.5%, 67.4%, 76.9%, 79.1% and 58.4% identical with that of CIRP in mice, rats, human, bullfrog, Xenopus and axolotl cells, respectively. These results showed that the CIRP was highly conserved in the evolution process and may be involved in various physiological functions. Therefore, this study will establish a systematic model for experiments and provide a new foundation for exploring the molecular mechanisms of human and other animals under cold stress.展开更多
A cDNA library containing 2×10￣6 recombinant plasmids was constructed using one week old young sorghum leaves as starting material and λgt10 as vector. In in situ hybridization, 2 positive clones were selected ...A cDNA library containing 2×10￣6 recombinant plasmids was constructed using one week old young sorghum leaves as starting material and λgt10 as vector. In in situ hybridization, 2 positive clones were selected using as probe RAcl cDNA from rice . One of them was subjected to Southern blot analysis and the recombinant containing the cDNA of SoAcl thus found was subcloned into pBluescript SK for sequencing. As shown by sequencing,SoAcl is composed of 1445 bps and 377 amino acids are encoded by the 1131 bp encoding region. It is the first report on it and has been included in the EMBO BANK (regist. no. X79378). High homology is found between it and other plant actin genes so far reported. The homology of amino acids sequency with RAcl is as high as 97.5%. Besides, highly homologous structural domains are found when it is compared with other plant and animal actins. These structural domains, thought to he sites for interaction among actins and/or between actins and their binding proteins, may prove to be important in the functioning of actin.(1 PhD candidate in Beijing University 2 Visiting Research Fellow of College of Oral Medicine of Beijing Medical University 3 Research Fellow of Institute of Developmental Biology, to whom correspondence is addressed 3 Reseach Fellow of Institute of Developmental Biology,to whom correspondence is addressed)展开更多
文摘In order to isolate the total cDNA of rice (Oryza sativa L.) epsps gene, RT-PCR was carried out with template of rice first-strand cDNA and primers designed according to rice EPSP synthase genomic sequence obtained in previous study. A 1 585-bp cDNA fragment was amplified and cloned. The 1 585-bp cDNA contains an open reading frame (ORF) comprising of 1 533 nucleotides (nt) which encodes a 511 residue polypepetides, including 67 amino acids chloroplast transit peptide and 444 amino acids EPSP synthase mature peptide. A comparison between the EPSP synthase of different sources indicates that the mature peptide shows more than 51% identity except for the fungi EPSP synthase and the transit peptide shows considerably less sequence conservation. The copy number of rice epsps gene is estimated to be one copy per haploid rice genome using southern blot. RT-PCR indicated that rice epsps gene is expressed in rice leaves, endosperms and roots and has the highest expression level in leaves.
文摘Two acetylcholinesterase (AChE) genes cDNA fragments,Ag.acel and Ag.ace2,have been cloned from cotton aphid,Aphis gossypii Glover using degenerate primers with RT-PCR technique.Ag.acel gene cDNA fragment is of 282?bp encoding 94 amino acids,and Ag.ace2 gene cDNA fragment is of 264?bp encoding 88 amino acids.Both two putative AChE genes cDNA fragments share numerous similarities with those cloned from other insects.This is the first report of two AChE cDNA fragment sequences in the insect species,which provided the direct evidence of multiple AChE existence in insects.
基金Supported by Guangxi Science Foundation (0542025)~~
文摘[ Objective] To clone and analyze the sequence of Adiponectin receptor 1 （ AdipoR1 ） and receptor 2 （AdipoR2） cDNA of Guangxi Bama mini-pig. [Method] The Adiponectin receptors cDNAs were amplified by RT-PCR using skeletal muscle total RNA as template and then ligated into pMD18-T vector after purification. The recombinant pMD18-T vector was transformed into the E. coil DH5α for identification and sequencing. And the results were compared with the cDNA sequence from other species. [Result] The fragments, 1 128 bp and 1 161 bp in size, were amplified by RT-PCR and respectively consistent with the coding sequence of AdipoR1 gene and AdipoR2 gene. The homology analysis showed that the sequences of AdipoR1 gene and AdipoR2 gene were respectively 99.8% and 99.7% homologous to the sequence of domestic pig reported in GenBank with one base and three base missense mutations correspondingly. [ Conclusion] The AdipoR1 gene and AdipoR2. gene were successfully amplified from Guangxi Bama mini-pig, laying the foundation for the further study of the biological function of AdipoR genes and the design of novel drugs with AdipoR as target.
文摘Total mRNA was extracted from a venom gland of snake Daboia russellii siamensis following the manufacturer＇s protocol of the PolyATtract System 1000 kit purchased from Promega Biotech. cDNAs encoding C-type lectins were amplified by RT-PCR and subcloned into a pMD18-T vector. Fourteen positive clones were selected for nucleotide sequencing and seven cDNAs encoding various snake venom C-type lectin-like protein precursors, designated as DRS-L1, DRS-L2, DRS-L3, DRS-L4 DRS-L5, DRS-L6 and DRS-L7, were obtained. Amino acid sequences of these proteins were deduced and each contains a carbohydrate recognition domain. Of all the deduced protein sequences, only DRS-L1 seemed to represent a closer sequence similarity to α subunits of other known snake venom C-type lectin-like proteins using the BLAST program. Homology comparison combined with analysis of cysteine position indicate that DRS-L1 and DRS-L2 are probably the light chain LC2 and LC1 of factor X activator from Daboia russellii siamensis venom, respectively. DRS-L3 and DRS-L4 might be the β subunits of higher molecular weight C-type lectin-like proteins while DRS-L5 and DRS-L6 might be β subunits of lower molecular weight C-type lectin-like proteins. DRS-L7 might be the β subunit of a platelet membrane glycoprotein Ib-binding protein similar to echicetin.
基金Supported by Natural Science Foundation of Hainan Province(30515)~~
文摘[Objective] cDNA of growth hormone releasing hormone （GHRH） receptor gene from Wuzhishan miniature pig was cloned and its sequence was also analyzed. [Method] Using genomic DNA extracted from porcine ear tissues of Wuzhishan miniature pig as the template, three pairs of primers were designed by the reported cDNA sequence of porcine GHRH, and cDNA was also amplified by RT-PCR. After being recovered and purified, PCR products were ligated to pMD18-T and then transformed into Escherichia coli DH5a. The transformation products were analyzed by PCR and double enzyme digestion to screen positive clones, and the positive clones were sequenced after identification in LB liquid medium. [ Result] cDNA of Wuzhishan miniature pig GHRH receptor gene was obtained successfully, and its length was 1 577 bp coding 423 amino acids. BLAST analysis showed that there were only 23 nuoleotides in difference between this fragment and pomine GHRH receptor gene, and its homology was 98%. However, both GHRH receptor genes were constituted by 423 amino acids with the sequence homology of 96%. [ Conclusion] This study provides theoretical basis for further studies on the dwarf mechanism of Wuzhishan miniature pig.
基金This research was supported by National Postdoctoral Science Foundation of China （No. 20060390241）.
文摘The effect of cold stress on the gene expression of cold-inducible RNA-binding protein （CIRP） in the intravital animals has not been reported till now. Compared with their organism cells, there were much more complicated regulatory mechanisms for cold stress response in the organisms. The BALB/C mice with cold treatment were used as experimental animals for this study. The cDNA of CIRP was firstly cloned from the testis tissues of the BALB/C mice treated by cold stress The results indicated that CIRP in the organisms could be induced at low temperature and may protect the organisms from the cold damage. The amino acid sequence deduced via cDNA clone was 100%, 99.4%, 95.5%, 67.4%, 76.9%, 79.1% and 58.4% identical with that of CIRP in mice, rats, human, bullfrog, Xenopus and axolotl cells, respectively. These results showed that the CIRP was highly conserved in the evolution process and may be involved in various physiological functions. Therefore, this study will establish a systematic model for experiments and provide a new foundation for exploring the molecular mechanisms of human and other animals under cold stress.
文摘A cDNA library containing 2×10￣6 recombinant plasmids was constructed using one week old young sorghum leaves as starting material and λgt10 as vector. In in situ hybridization, 2 positive clones were selected using as probe RAcl cDNA from rice . One of them was subjected to Southern blot analysis and the recombinant containing the cDNA of SoAcl thus found was subcloned into pBluescript SK for sequencing. As shown by sequencing,SoAcl is composed of 1445 bps and 377 amino acids are encoded by the 1131 bp encoding region. It is the first report on it and has been included in the EMBO BANK (regist. no. X79378). High homology is found between it and other plant actin genes so far reported. The homology of amino acids sequency with RAcl is as high as 97.5%. Besides, highly homologous structural domains are found when it is compared with other plant and animal actins. These structural domains, thought to he sites for interaction among actins and/or between actins and their binding proteins, may prove to be important in the functioning of actin.(1 PhD candidate in Beijing University 2 Visiting Research Fellow of College of Oral Medicine of Beijing Medical University 3 Research Fellow of Institute of Developmental Biology, to whom correspondence is addressed 3 Reseach Fellow of Institute of Developmental Biology,to whom correspondence is addressed)
