Cinnamoyl-CoA reductase (CCR) is responsible for the first committed reaction in monolignol biosynthesis, which diverts phenylpropanoid-derived metabolites into the biosynthesis of lignin. To gain a better understandi...Cinnamoyl-CoA reductase (CCR) is responsible for the first committed reaction in monolignol biosynthesis, which diverts phenylpropanoid-derived metabolites into the biosynthesis of lignin. To gain a better understanding of the lion biosynthesis in wheat development, two cDNAs encoding CCR were identified from wheat (Triticum aestivum L. cv. H4564). DNA sequence analyses indicated that the two cDNAs represent two classes of CCR. RT-PCR and Northern blot hybridization demonstrated that one of them, W-cr6, was expressed actively in stem and leaf tissue, the other one, W-cr19, was expressed in root and stem tissue. The results suggested that there are at least two genes encoded for CCR existing in wheat genome.展开更多
The nucleotide sequences of the open reading frames of cDNAs for selenoprotein W from skeletal muscle of rat, mouse, sheep, rhesus monkey and human are reported. Theoretical translation of the coding sequences indicat...The nucleotide sequences of the open reading frames of cDNAs for selenoprotein W from skeletal muscle of rat, mouse, sheep, rhesus monkey and human are reported. Theoretical translation of the coding sequences indicated highly similar proteins of 88 (mouse and rat) or 87 (human, monkey and sheep) amino acids. In 73 of 88 positions the specified amino acids are identical for all five proteins. TGA encoding selenocysteine is the 13th codon of all the cDNAs. The rnouse, rat and sheep open reading frames terminate with TGA but the human and rhesus monkey coding regions terminate with TAA. The encoded amino acid sequences are identical for the rat and mouse proteins, and for the human and monkey proteins. The similarity of the cDNAs continues in the 3' noncoding regions through the putative selenocysteine insertion sequence (SEClS) elements which are required for correct interpretation of the selenocysteine codon. The region between the SECIS elements and the polyadenylation signals showed much lower similarity. The cloned rat gene for selenoprotein W is 5000 bases long,with the 663 bases of the cDNA in six exons. The transcription start site was identified by nuclease protection assay to be 16 bases upstream of the longest cDNA clone. A canonical TATA box occurs 150 bases upstream, but the assay did not indicate the presence of longer mRNAs展开更多
Heavy metal contamination of soil has become a major environmental concern worldwide over the past several decades,the exploitation and application of heavy metal hyperaccumulating plants will defend the safety of foo...Heavy metal contamination of soil has become a major environmental concern worldwide over the past several decades,the exploitation and application of heavy metal hyperaccumulating plants will defend the safety of food and environment on which human life relied,whereas using natural hyperaccumulators is insufficient,due展开更多
<abstract>Aim: Identification of the rodent counterparts of human and canine epididymal cDNAs HE3, HE4 and Ce8/Ly6G5C by sequence homology and analysis of their expression patterns and regulation level in the ra...<abstract>Aim: Identification of the rodent counterparts of human and canine epididymal cDNAs HE3, HE4 and Ce8/Ly6G5C by sequence homology and analysis of their expression patterns and regulation level in the rat. Methods: 'Electronic screening' of Expressed Sequence Tag (EST) and genomic databases, followed by RT-PCR and Northern blot analysis. Results: Rodent ESTs and genomic sequences homologous to HE3, HE4 and Ce8/Ly6G5C were identified in the public databases and the 'full-length' rat cDNAs cloned. To emphasise their homology to the human and canine genes, they were named Me3/Re3, Me4/Re4 and Re8 for mouse and rat counterparts, respectively. mRNA expression patterns were analysed in rats, including rat HEl and HE5/CD52 counterparts as controls. Re3 and Re8 mRNAs were only found in the rat epididymis, while Re4 showed a broader tissue distribution. Within the epididymis, Re3 and Re4 mRNAs were detected in all regions; Re8, on the other hand, was restricted to the caput. During postnatal development, Re3 and control mRNAs were found from the earliest stages investigated, while Re8 mRNA was observed only from day 24 postnatum, corresponding to the onset of spermatogenesis in the prepubertal testis. Castration and testosterone supplementation of adult male rats suggested that none of the cloned mRNAs was directly androgen-regulated. Efferent duct ligation, however, showed that Re8 mRNA levels depended on testicular factors other than androgens. Conclusion: The novel rodent cDNAs can now be used to monitor epididymal gene expression more closely and to set up various regulatory and functional studies.展开更多
In an attempt to isolate and identify the target genes relevant to salt tolerance in a mangrove plant （Sesuvium portulacastrum L.）, a subtracted cDNA library was constructed via suppressive subtractive hybridization...In an attempt to isolate and identify the target genes relevant to salt tolerance in a mangrove plant （Sesuvium portulacastrum L.）, a subtracted cDNA library was constructed via suppressive subtractive hybridization （SSH）, in which the poly（A）＋RNA isolated from salt-tolerant S. portulacastrum leaves was used as a tester, whereas the driver was poly（A）＋RNA, derived from salt-sensitive S. portulacastrum leaves. Screening of this subtracted cDNA library revealed five clones, of which the expression levels in the salt-tolerant plant were markedly higher than those observed in the salt-sensitive plant, indicating that these candidate clones may be involved in salt-tolerance pathways. Among the clones isolated, P66, P175, and P233 are novel because no significant similarity was obtained upon alignment with the GenBank database. Clone P89 demonstrated high homology with NADPH of Arabidopsis thaliana, whereas clone P152 was highly homologous with the gene encoding late embryogenesis abundant （LEA） protein of A. thaliana. The full-length gene of clone P152, with a predicated 344 amino acid residues, was shown to bear LEA-2 domains, a signature motif for proteins that have been enriched under salty and drought conditions. It is thus implied that clone P152 would be a salt-tolerance gene of S. portulacastrum. In addition, we have also developed a strategy for the extraction of total RNA from mangrove plants.展开更多
The meiotic stage of pollen mother cell is a very important stage in controlling the development and formation of pollen. In order to clone the rice cDNA(s) of this stage, a normal rice, Annong N and its thermosensiti...The meiotic stage of pollen mother cell is a very important stage in controlling the development and formation of pollen. In order to clone the rice cDNA(s) of this stage, a normal rice, Annong N and its thermosensitive mutant, Annong S-1 were used as the plant material. The mRNA has been extracted from the young panicle at the meiotic stage. By using the cDNA subtraction hybridization technique, three cDNA fragments, RP-1, RP-2 and RP-3 have展开更多
Diatraea saccharalis is a major corn borer pest. Midgut serine proteinases are essential for insect growth and development. Alteration of midgut proteinases is re- sponsible for Bt resistance development in some speci...Diatraea saccharalis is a major corn borer pest. Midgut serine proteinases are essential for insect growth and development. Alteration of midgut proteinases is re- sponsible for Bt resistance development in some species. To clone midgut trypsin and chymotrypsin cDNAs and to test if the CrylAb resistance in D. saccharalis is associ- ated with changes in midgut proteinases, total midgut tryptic and chymotryptic activities, cDNA sequences, and gene expressions of three trypsin and three chymotrypsin genes were comparatively examined between Cry 1 Ab-susceptible （Cry 1 Ab- S S） and Cry 1 Ab-resistant （Cry 1 Ab-RR） strains. Full-length cDNAs encoding three trypsin- and three chymotrypsin- like proteinases were sequenced from CrylAb-SS and CrylAb-RR larvae. These cDNAs code for active forms of midgut serine proteinases with all fimctional motifs, includ- ing signal peptide, conserved His-Asp-Ser for the catalytic triad, three pairs of cysteines for disulfide bridge configurations, and conserved substrate specificity determination residues. In general, cDNA and putative protein sequences are highly similar between CrylAb-SS and CrylAb-RR strains, except for a few nucleotide and predicted amino acid substitutions, whose function need to be further clarified. Total trypsin and chymotrypsin activities were also similar between CrylAb-SS and CrylAb-RR strains. Transcriptional levels of the trypsin and chymotrypsin genes had numerical difference between Cry 1 Ab-SS and CrylAb-RR strains, but the difference was not statistically significant. Data suggest that the development of CrylAb resistance in D. saccharalis was not significantly as- sociated with these trypsins and chymotrypsins. Results clarified the role of six midgut proteinases and provided a foundation for continuing examination of potential involvement of other midgut proteinases in Bt resistance development and other important biochemical processes.展开更多
Salicylic acid (SA) is very important in systemic acquired resistance and hypersensitive response in plant defense, and yet its role is not fully understood. This study seeks to clarify the mechanism of SA induced res...Salicylic acid (SA) is very important in systemic acquired resistance and hypersensitive response in plant defense, and yet its role is not fully understood. This study seeks to clarify the mechanism of SA induced resistance in cotton. Total RNA was extracted from low gossypol cultivated cotton seedlings treated with exogenous SA and subjected to fluorescent differential display PCR (FDD PCR). Seven cDNA fragments were selected from the total ten differential bands. Comparison with Genbank database shows that all seven cDNA sequences are newly discovered in cotton. However, they share high amino acid identity to some registered cDNAs. Among them, three of the cDNAs could be predicted to encode basic chitinase, penicillin binding 6 b precursor and ATP dependent DNA helicase RecG, while the functions of the other four cDNAs are undetermined. Dot blot analysis demonstrates that the expression of five cDNAs in cotton seedlings is induced by SA, while SA induction has a negative effect on the transcript accumulation of the other two cDNAs (E13 and E14). Since SA was previously shown to enhance the resistance to cotton wilt disease, the finding of a basic chitinase gene in cotton expressed by SA induction will provide a new insight into induced disease resistance in cotton.展开更多
Partial cDNA sequences coding for antifreeze proteins in Tenebrio molitor were obtained by RT-PCR.Sequence analysis revealed nine putative cDNAs with a high degree of homology to Tenebrio molitor antifreeze protein ge...Partial cDNA sequences coding for antifreeze proteins in Tenebrio molitor were obtained by RT-PCR.Sequence analysis revealed nine putative cDNAs with a high degree of homology to Tenebrio molitor antifreeze protein genes published in GenBank.The recombinant pGEX-4T-1-tmafp-XJ430 was introduced into E.coli BL21 to induce a GST fusion protein by IPTG.SDS-PAGE analysis for the fusion protein shows a band of 38 kDa.pCDNA3-tmafp-XJ430 was injected into mice to generate antiserum which was later detected by indirect ELISA.The titer of the antibody was 1:2000.Western blotting analysis shows that the antiserum was specifically against the antifreeze protein.Our results laid the foundation for further studies on the properties and functions of insect antifreeze proteins.展开更多
We have developed a comprehensive software suite for bioinformatics research of cDNAs; it is aimed at rapid characterization of the features of genes and the proteins they code. Methods implemented include the detecti...We have developed a comprehensive software suite for bioinformatics research of cDNAs; it is aimed at rapid characterization of the features of genes and the proteins they code. Methods implemented include the detection of translation initia- tion and termination signals, statistical analysis of codon usage, comparative study of amino acid composition, comparative modeling of the structures of product proteins, prediction of alternative splice forms, and metabolic pathway reconstruction. The software package is freely available under the GNU General Public License at http： / /www.g-language.org/ data/cdna/.展开更多
文摘Cinnamoyl-CoA reductase (CCR) is responsible for the first committed reaction in monolignol biosynthesis, which diverts phenylpropanoid-derived metabolites into the biosynthesis of lignin. To gain a better understanding of the lion biosynthesis in wheat development, two cDNAs encoding CCR were identified from wheat (Triticum aestivum L. cv. H4564). DNA sequence analyses indicated that the two cDNAs represent two classes of CCR. RT-PCR and Northern blot hybridization demonstrated that one of them, W-cr6, was expressed actively in stem and leaf tissue, the other one, W-cr19, was expressed in root and stem tissue. The results suggested that there are at least two genes encoded for CCR existing in wheat genome.
文摘The nucleotide sequences of the open reading frames of cDNAs for selenoprotein W from skeletal muscle of rat, mouse, sheep, rhesus monkey and human are reported. Theoretical translation of the coding sequences indicated highly similar proteins of 88 (mouse and rat) or 87 (human, monkey and sheep) amino acids. In 73 of 88 positions the specified amino acids are identical for all five proteins. TGA encoding selenocysteine is the 13th codon of all the cDNAs. The rnouse, rat and sheep open reading frames terminate with TGA but the human and rhesus monkey coding regions terminate with TAA. The encoded amino acid sequences are identical for the rat and mouse proteins, and for the human and monkey proteins. The similarity of the cDNAs continues in the 3' noncoding regions through the putative selenocysteine insertion sequence (SEClS) elements which are required for correct interpretation of the selenocysteine codon. The region between the SECIS elements and the polyadenylation signals showed much lower similarity. The cloned rat gene for selenoprotein W is 5000 bases long,with the 663 bases of the cDNA in six exons. The transcription start site was identified by nuclease protection assay to be 16 bases upstream of the longest cDNA clone. A canonical TATA box occurs 150 bases upstream, but the assay did not indicate the presence of longer mRNAs
文摘Heavy metal contamination of soil has become a major environmental concern worldwide over the past several decades,the exploitation and application of heavy metal hyperaccumulating plants will defend the safety of food and environment on which human life relied,whereas using natural hyperaccumulators is insufficient,due
文摘<abstract>Aim: Identification of the rodent counterparts of human and canine epididymal cDNAs HE3, HE4 and Ce8/Ly6G5C by sequence homology and analysis of their expression patterns and regulation level in the rat. Methods: 'Electronic screening' of Expressed Sequence Tag (EST) and genomic databases, followed by RT-PCR and Northern blot analysis. Results: Rodent ESTs and genomic sequences homologous to HE3, HE4 and Ce8/Ly6G5C were identified in the public databases and the 'full-length' rat cDNAs cloned. To emphasise their homology to the human and canine genes, they were named Me3/Re3, Me4/Re4 and Re8 for mouse and rat counterparts, respectively. mRNA expression patterns were analysed in rats, including rat HEl and HE5/CD52 counterparts as controls. Re3 and Re8 mRNAs were only found in the rat epididymis, while Re4 showed a broader tissue distribution. Within the epididymis, Re3 and Re4 mRNAs were detected in all regions; Re8, on the other hand, was restricted to the caput. During postnatal development, Re3 and control mRNAs were found from the earliest stages investigated, while Re8 mRNA was observed only from day 24 postnatum, corresponding to the onset of spermatogenesis in the prepubertal testis. Castration and testosterone supplementation of adult male rats suggested that none of the cloned mRNAs was directly androgen-regulated. Efferent duct ligation, however, showed that Re8 mRNA levels depended on testicular factors other than androgens. Conclusion: The novel rodent cDNAs can now be used to monitor epididymal gene expression more closely and to set up various regulatory and functional studies.
文摘In an attempt to isolate and identify the target genes relevant to salt tolerance in a mangrove plant （Sesuvium portulacastrum L.）, a subtracted cDNA library was constructed via suppressive subtractive hybridization （SSH）, in which the poly（A）＋RNA isolated from salt-tolerant S. portulacastrum leaves was used as a tester, whereas the driver was poly（A）＋RNA, derived from salt-sensitive S. portulacastrum leaves. Screening of this subtracted cDNA library revealed five clones, of which the expression levels in the salt-tolerant plant were markedly higher than those observed in the salt-sensitive plant, indicating that these candidate clones may be involved in salt-tolerance pathways. Among the clones isolated, P66, P175, and P233 are novel because no significant similarity was obtained upon alignment with the GenBank database. Clone P89 demonstrated high homology with NADPH of Arabidopsis thaliana, whereas clone P152 was highly homologous with the gene encoding late embryogenesis abundant （LEA） protein of A. thaliana. The full-length gene of clone P152, with a predicated 344 amino acid residues, was shown to bear LEA-2 domains, a signature motif for proteins that have been enriched under salty and drought conditions. It is thus implied that clone P152 would be a salt-tolerance gene of S. portulacastrum. In addition, we have also developed a strategy for the extraction of total RNA from mangrove plants.
文摘The meiotic stage of pollen mother cell is a very important stage in controlling the development and formation of pollen. In order to clone the rice cDNA(s) of this stage, a normal rice, Annong N and its thermosensitive mutant, Annong S-1 were used as the plant material. The mRNA has been extracted from the young panicle at the meiotic stage. By using the cDNA subtraction hybridization technique, three cDNA fragments, RP-1, RP-2 and RP-3 have
文摘Diatraea saccharalis is a major corn borer pest. Midgut serine proteinases are essential for insect growth and development. Alteration of midgut proteinases is re- sponsible for Bt resistance development in some species. To clone midgut trypsin and chymotrypsin cDNAs and to test if the CrylAb resistance in D. saccharalis is associ- ated with changes in midgut proteinases, total midgut tryptic and chymotryptic activities, cDNA sequences, and gene expressions of three trypsin and three chymotrypsin genes were comparatively examined between Cry 1 Ab-susceptible （Cry 1 Ab- S S） and Cry 1 Ab-resistant （Cry 1 Ab-RR） strains. Full-length cDNAs encoding three trypsin- and three chymotrypsin- like proteinases were sequenced from CrylAb-SS and CrylAb-RR larvae. These cDNAs code for active forms of midgut serine proteinases with all fimctional motifs, includ- ing signal peptide, conserved His-Asp-Ser for the catalytic triad, three pairs of cysteines for disulfide bridge configurations, and conserved substrate specificity determination residues. In general, cDNA and putative protein sequences are highly similar between CrylAb-SS and CrylAb-RR strains, except for a few nucleotide and predicted amino acid substitutions, whose function need to be further clarified. Total trypsin and chymotrypsin activities were also similar between CrylAb-SS and CrylAb-RR strains. Transcriptional levels of the trypsin and chymotrypsin genes had numerical difference between Cry 1 Ab-SS and CrylAb-RR strains, but the difference was not statistically significant. Data suggest that the development of CrylAb resistance in D. saccharalis was not significantly as- sociated with these trypsins and chymotrypsins. Results clarified the role of six midgut proteinases and provided a foundation for continuing examination of potential involvement of other midgut proteinases in Bt resistance development and other important biochemical processes.
基金Supported by the National Natural Science Foundationof China (No. 39780 0 30 ) the High- TechnologyDevelopm ent Program of China(No.2 0 0 1AA2 2 2 0 5 3)and the National Plant Transgenic Research andIndustrialization Special Foundation of Chin
文摘Salicylic acid (SA) is very important in systemic acquired resistance and hypersensitive response in plant defense, and yet its role is not fully understood. This study seeks to clarify the mechanism of SA induced resistance in cotton. Total RNA was extracted from low gossypol cultivated cotton seedlings treated with exogenous SA and subjected to fluorescent differential display PCR (FDD PCR). Seven cDNA fragments were selected from the total ten differential bands. Comparison with Genbank database shows that all seven cDNA sequences are newly discovered in cotton. However, they share high amino acid identity to some registered cDNAs. Among them, three of the cDNAs could be predicted to encode basic chitinase, penicillin binding 6 b precursor and ATP dependent DNA helicase RecG, while the functions of the other four cDNAs are undetermined. Dot blot analysis demonstrates that the expression of five cDNAs in cotton seedlings is induced by SA, while SA induction has a negative effect on the transcript accumulation of the other two cDNAs (E13 and E14). Since SA was previously shown to enhance the resistance to cotton wilt disease, the finding of a basic chitinase gene in cotton expressed by SA induction will provide a new insight into induced disease resistance in cotton.
文摘Partial cDNA sequences coding for antifreeze proteins in Tenebrio molitor were obtained by RT-PCR.Sequence analysis revealed nine putative cDNAs with a high degree of homology to Tenebrio molitor antifreeze protein genes published in GenBank.The recombinant pGEX-4T-1-tmafp-XJ430 was introduced into E.coli BL21 to induce a GST fusion protein by IPTG.SDS-PAGE analysis for the fusion protein shows a band of 38 kDa.pCDNA3-tmafp-XJ430 was injected into mice to generate antiserum which was later detected by indirect ELISA.The titer of the antibody was 1:2000.Western blotting analysis shows that the antiserum was specifically against the antifreeze protein.Our results laid the foundation for further studies on the properties and functions of insect antifreeze proteins.
基金This research was supported by the Japan Society for the Promotion of Science(JSPS)a grant from the Ministry of Education,Culture,Sports,Science and Technology of Japan(The 21st Century COE Program).
文摘We have developed a comprehensive software suite for bioinformatics research of cDNAs; it is aimed at rapid characterization of the features of genes and the proteins they code. Methods implemented include the detection of translation initia- tion and termination signals, statistical analysis of codon usage, comparative study of amino acid composition, comparative modeling of the structures of product proteins, prediction of alternative splice forms, and metabolic pathway reconstruction. The software package is freely available under the GNU General Public License at http： / /www.g-language.org/ data/cdna/.
