Chitin is the most widespread amino polysaccharide in nature.Chitin synthase (CHS) plays an important role in chitin formation in the cuticle and the peritrophic membrane (PM) lining the midgut.Total RNA was isolated ...Chitin is the most widespread amino polysaccharide in nature.Chitin synthase (CHS) plays an important role in chitin formation in the cuticle and the peritrophic membrane (PM) lining the midgut.Total RNA was isolated from the cuticle of Mamestra brassicae (L.) fourth instar larva.cDNA sequence was cloned by RT-PCR and Rapid Amplifi cation of cDNA Ends (RACE).cDNA,5 220 bp in length,contained an open reading frame of 4 704 bp coding for a polypeptide of 1 567 amino acid residues with a predicted molecular weight of 178.3 ku and its pI was 6.42.The deduced amino acid sequence from M.brassicae (L.) shared the high level of identity with chitin synthase sequences from other insects,especially lepidopteran insects.cDNA sequence has been deposited with GenBank under accession No.GQ281761.展开更多
Degenerate primers were designed based on the conserved sequences of the Actin gene from other plants. Total RNA was extracted from the leaves of Iris lacteal var.chinensis Fisch.Koidz. Actin gene fragment was obtaine...Degenerate primers were designed based on the conserved sequences of the Actin gene from other plants. Total RNA was extracted from the leaves of Iris lacteal var.chinensis Fisch.Koidz. Actin gene fragment was obtained by reverse transcription polymerase chain reaction(RT-PCR) and cloned into pMD18-T vector. The positive clone identified by PCR was sequenced. The sequencing result showed that the Actin gene fragment from Iris lacteal var.chinensis Fisch.Koidz contained about 598 bp, encoding199 amino acids. Homology comparison with Actin gene sequences of other plants in the GenBank showed that it shared over 82%nucleotide sequence homology and 90% amino acid sequence homology. It indicated that this was the Actin gene. Because of the stability expression of Actin gene, it usually cited as the internal reference to study the expression and regulation of foundation in other genes of Iris lacteal var.chinensis Fisch.Koidz well.展开更多
[Objectives]The paper was to explore the genetic information and evolution of CymMV,and to provide an important scientific basis for monitoring and early warning of orchid virus disease and anti-virus genetic engineer...[Objectives]The paper was to explore the genetic information and evolution of CymMV,and to provide an important scientific basis for monitoring and early warning of orchid virus disease and anti-virus genetic engineering of orchid in Guangdong Province.[Methods]RT-PCR and DASELISA were used to detect and identify CymMV from leaves with suspected virus disease of Cymbidium sinense collected from Guangzhou area.The genome sequence assembly,annotation,phylogeny and selection pressure analysis of CymMV isolates were performed with related molecular biology software.[Results]Two CymMV isolates(GZV013 and ZC29)were found in Guangdong Province for the first time in this study.The genome of both GZV013 and ZC29 were 6227 nt in length,encoding 5 functional proteins.The similarity analysis of the full sequence showed that the nucleotide sequence identity of GZV013 and Taiwan isolate M2 was 97.03% and that of ZC29 and Nanjing isolate NJ-1 was 97.11%.The complete genome sequence identity among CymMV isolates ranged from 86.85% to 98.31%,and the differentiation of diverse populations was closely related to host species and geographical isolation.Each region of CymMV genome was affected by negative selection and conformed to the neutral evolution model.The genes encoding RdRp,TGB1 and TGB2 had the highest mutation rates in the genome.[Conclusions]GZV013 was most closely related to Taiwan isolate M2,and ZC29 was most closely related to Nanjing isolate NJ-1,belonging to the same branch of a family.展开更多
The method described in this work provides a sensitive and fast technique for investigating the primary structure of peptides with molecular weight up to 3340 amu. Usually, the metastable ion kinetic energy spectra (M...The method described in this work provides a sensitive and fast technique for investigating the primary structure of peptides with molecular weight up to 3340 amu. Usually, the metastable ion kinetic energy spectra (MIKES) and collisional activated decomposition (CAD) spectra provide complementary information for the FAB mass spectra, the MIKES and CAD spectra generally contain high-mass sequence ions.展开更多
Objective Tuberculosis remains one of the most serious infectious diseases in the world. In this study, a scheme of Mycobacterium tuberculosis (M. tuberculosis) multilocus sequence analysis (MLSA) was established for ...Objective Tuberculosis remains one of the most serious infectious diseases in the world. In this study, a scheme of Mycobacterium tuberculosis (M. tuberculosis) multilocus sequence analysis (MLSA) was established for the phylogenetic and epidemiology analysis. Methods To establish the scheme of M. tuberculosis MLSA, the genome of H37Rv, CCDC5079 and CCDC5180 were compared, and some variable genes were chosen to be the MLSA typing scheme. 44 M. tuberculosis clinical isolates were typed by MLSA, IS6110-RFLP, and soligotyping, to evaluate the MLSA methods. Results After comparison of the genome, seven high discrimination gene loci (recX, rpsL, rmlC, rpmG1, mprA, gcvH, ideR) were chosen to be the MLSA typing scheme finally. 11 variable SNP sites of those seven genes were found among the 44 M. tuberculosis isolate strains and 11 sequence types (STs) were identified. Based on the Hunter-Gaston Index (HGI), MLSA typing was not as good for discrimination at the strain level as IS6110-RFLP, but the HGI was much better than that of spoligotyping. In addition, the MEGA analysis result of MLSA data was similar to spoligotyping/PGG lineage, showing a strong phylogenetic signal in the modern strains of M. tuberculosis. The MLSA data analysis by eBURST revealed that 4 sequence types (ST) came into a main cluster, showing the major clonal complexes in those 44 strains. Conclusion MLSA genotyping not only can be used for molecular typing, but also is an ideal method for the phylogenetic analysis for M. tuberculosis.展开更多
The partial sequence of the rbcL from Bryopsis hypnoides, including the sequences of the upstream, extron and partial intron, was amplified by PCR and their sequences were determined. With Spinacia oleracea as the out...The partial sequence of the rbcL from Bryopsis hypnoides, including the sequences of the upstream, extron and partial intron, was amplified by PCR and their sequences were determined. With Spinacia oleracea as the outgroup, neighbor-joining method and maximum parsimony method were used respectively to build phylogenetic trees according to the rbcL exon sequence among 13 species that were the typical species of six phyla. Two kinds of trees showed clearly that there were two groups among those species, the green lineage and the non-green lineage. And the relationships of algae in the green lineage were similar in the two trees but those in the non-green lineage were not consistent. Analysis of codon preference indicated that the codon preference of the rbcL exon of Bryopsis hypnoides distinctly differed from that of the relevant sequence of photosynthetic bacteria.展开更多
In the subtribe Maydeae, Tripsacum and Zea are closely related genera. Tripsacum is a horticultural crop widely used as pasture forage. Previous studies suggested that Tripsacum might play an important role in maize o...In the subtribe Maydeae, Tripsacum and Zea are closely related genera. Tripsacum is a horticultural crop widely used as pasture forage. Previous studies suggested that Tripsacum might play an important role in maize origin and evolution. However, our understanding of the genomics and the evolution of Tripsacum remains limited. In this study, two diploids,T. dactyloides var. meridionale(2n = 36, MR) and T. dactyloides(2n = 36, DD), and one tetraploid,T. dactyloides(2n = 72, DL) were sequenced by low-coverage genome sequencing followed by graph-based cluster analysis. The results showed that 63.23%, 59.20%, and 61.57% of the respective genome of MR, DD, and DL were repetitive DNA sequence. The proportions of different repetitive sequences varied greatly among the three species. Fluorescence in situ hybridization(FISH) analysis of mitotic metaphase chromosomes with satellite repeats as the probes showed that the FISH signal patterns of DL were more similar to that of DD than to that of MR. Comparative analysis of the repeats also showed that DL shared more common repeat families with DD than with MR. Phylogenetic analysis of internal transcribed spacer region sequences further supported the evolutionary relationship among the three species. Repetitive sequences comparison showed that Tripsacum shared more repeat families with Zea than with Coix and Sorghum. Our study sheds new light on the genomics of Tripsacum and differential speciation in the Poaceae family.展开更多
Using PCR degenerate primers, designed with reference to the sequences of the conserved amino acids of known expansins, to amplify cDNA fragments in litchi fruit by RT-PCR, two different cDNA fragments, named as Lc-Ex...Using PCR degenerate primers, designed with reference to the sequences of the conserved amino acids of known expansins, to amplify cDNA fragments in litchi fruit by RT-PCR, two different cDNA fragments, named as Lc-Expl and Lc-Exp2, were cloned. Lc-Expl and Lc-Exp2 was respectively composed of 531 bp encoding 177 amino acids and 537 bp encoding 179 amino acids. Eight cysteine residues and three tryptopban residues, which is supposed to be the characteristics of expansins, are conserved in both Lc-Expl and e-Exp2. In addition, the homology between the two expansins is 71.6% at nucleotide acid sequences and 76.3% at amino acid sequences. The homology of Lc-Expl with Fa-Exp2 or Pp-Expl was 92.7% or 92.1%, but that of Lc-Exp2 with Fa-Exp2 or Pp-Expl was only 77.4% or 76.3% at amino acid sequences.展开更多
The full length cDNA sequence of CBF3(CRT/DRE-binding factor) was cloned from Vitis amurensis by reverse transcription polymerase chain reaction(RT-PCR) using the primers designed based on CBF genes available in GenBa...The full length cDNA sequence of CBF3(CRT/DRE-binding factor) was cloned from Vitis amurensis by reverse transcription polymerase chain reaction(RT-PCR) using the primers designed based on CBF genes available in GenBank.Sequence analysis showed that the gene had 854 bp long and its coding sequence contained 720 bp,encoding a protein with 239 amino acids and an AP2 structural domain.The molecular mass of CBF3 was predicted to be 25.9 kDa and its theoretical isoelectric point was 7.02 and aliphatic index was 59.29.The average hydropathicity of the protein was -0.551.The tertiary structures of CBF3 were also analyzed.The prokaryotic expression vector pGEX-4T-CBF3 containing CBF3 gene was constructed and CBF3 fusion protein(52 kDa) was produced in Escherichia coli after induction with 1 mmol L-1 IPTG.Further studies are needed to evaluate its potential application for improving plant resistance to cold and other stress condition such as drought and salinity.展开更多
A novel HMW glutenin subunit gene 1Dy10.1 was isolated and characterized from Xinjiang wheat (Triticum petropavlovskyi. Udacz. et Migusch) accession Daomai 2. The complete open reading frame (ORF) of 1Dy10.1 was 1 965...A novel HMW glutenin subunit gene 1Dy10.1 was isolated and characterized from Xinjiang wheat (Triticum petropavlovskyi. Udacz. et Migusch) accession Daomai 2. The complete open reading frame (ORF) of 1Dy10.1 was 1 965 bp, encoding 655 amino acids. The numbers and distribution of cysteines in 1Dy10.1 were similar to those of 1Dy10 and other y-type subunits. In the N-terminal of 1Dy10.1, an amino acid was changed from L (leucine) to P (proline) at position 55. The repetitive domain of 1Dy10.1 differed from those of known HMW subunits by substitutions, insertions or/and deletions involving single or more amino acid residues. In the repetitive domain of subunit 1Dy10.1, the deletion of tripeptide GQQ in the consensus unit PGQGQQ resulted in the appearance of the motif PGQ that have not been observed in other known y-type HMW subunits. In comparison with the subunit 1Dy12, a deletion of dipeptide GQ, which occurred in subunit 1Dy10, was also observed in subunit 1Dy10.1. The cloned 1Dy10.1 gene had been successfully expressed in Escherichia coli, and the expressed protein had the identical mobility with the endogenous subunit 1Dy10.1 from seed.展开更多
The diseased samples collected from Guangdong Province were inoculated to Marc-145 cells,PRRSV-specific cytopathogenic effects( CPE) such as cell aggregation,cell shrinkage and cell exfoliation were observed and a PRR...The diseased samples collected from Guangdong Province were inoculated to Marc-145 cells,PRRSV-specific cytopathogenic effects( CPE) such as cell aggregation,cell shrinkage and cell exfoliation were observed and a PRRSV strain XH-GD was obtained. By using primers designed in accordance with the published gene sequences of PRRSV in GenBank,NSP2,ORF3 and ORF5 fragments were amplified by reverse transcription polymerase chain reaction and analyzed by sequence alignment. The results demonstrated that PRRSV strain XH-GD belonged to the North American genotype and the NSP2 gene contained discontinuous deletions of 30 amino acids. NSP2,ORF3 and ORF5 genes of XH-GD shared 83. 3%- 98. 9%,88. 6%- 99. 2% and 88. 1%- 99. 2% nucleotide homology,and76. 8%- 98. 3%,83. 7%- 98. 8% and 88. 1%- 99. 2% amino acid homology with HUB1,NX06,BJsy06,VR-2332,HB-1( sh) 2002,HB-2( sh) 2002,CH-la and RespPRRS MLV,respectively; however,NSP2,ORF3 and ORF5 genes of XH-GD shared only 52. 9%,65. 0% and 64. 3% nucleotide homology,and31. 3%,57. 5% and 58. 9% amino acid homology with LV,respectively. Phylogenetic analysis revealed that XH-GD had a closer relationship with HUB1,NX06,BJsy06,VR-2332,HB-1( sh) 2002,HB-2( sh) 2002,CH-la and RespPRRS MLV strains compared with LV strain.展开更多
A combination method of the usual-PCR and reverse-PCR for the cloning of a novel lipase gene directly from the total genomic DNA of strain lip35 (Pseudomonas sp.) is described, whereby a lipase gene (lip) was cloned d...A combination method of the usual-PCR and reverse-PCR for the cloning of a novel lipase gene directly from the total genomic DNA of strain lip35 (Pseudomonas sp.) is described, whereby a lipase gene (lip) was cloned directly from genomic DNA. The sequence data have been deposited in the GenBank and EMBL data bank with the accession number EU414288. The nucleotide sequence showed a major open reading frame encoding a 59-kDa protein of 566 amino acid residues, which contained a lipase consensus sequence GXSXG. The lipase lip had 74 and 70% homologies with the lipases of an uncultured bacterium and P. fluorescens PfO-1, respectively, but it did not show any overall homology with lipases from other origins. The functional lipase was obtained when the lip gene was expressed in Pichia pastoris GS115.展开更多
The sequences of the ITS(internal transcribed spacer) and 5.8S rDNA of three cultivated strains of Porphyra haitanensis thalli(NB,PT and ST) were amplified,sequenced and analyzed.In addition,the phylogenic relationshi...The sequences of the ITS(internal transcribed spacer) and 5.8S rDNA of three cultivated strains of Porphyra haitanensis thalli(NB,PT and ST) were amplified,sequenced and analyzed.In addition,the phylogenic relationships of the sequences identified in this study with those of other Porphyra retrieved from GenBank were evaluated.The results are as follows:the sequences of the ITS and 5.8S rDNA were essentially identical among the three strains.The sequences of ITS1 were 331 bp to 334 bp,while those of the 5.8S rDNA were 158 bp and the sequences of ITS2 ranged from 673 bp to 681 bp.The sequences of the ITS had a high level of homology(up to 99.5%) with that of P.haitanensis(DQ662228) retrieved from GenBank,but were only approximately 50% homologous with those of other species of Porphyra.The results obtained when a phylogenetic tree was constructed coincided with the results of the homology analysis.These results suggest that the three cultivated strains of P.haitanensis evolved conservatively and that the ITS showed evolutionary consistency.However,the sequences of the ITS and 5.8S rDNA of different Porphyra species showed great variations.Therefore,the relationship of Porphyra interspecies phyletic evolution could be judged,which provides the proof for Porphyra identification study.However,proper classifications of the subspecies and the populations of Porphyra should be determined through the use of other molecular techniques to determine the genetic variability and rational phylogenetic relationships.展开更多
The aim of this study was to isolate the full-length cDNA sequences of an unusually highly conserved olfactory receptor(orthologue to the Drosophila melanogaster DOR83b) from the antennae of Spodoptera exigua and S.li...The aim of this study was to isolate the full-length cDNA sequences of an unusually highly conserved olfactory receptor(orthologue to the Drosophila melanogaster DOR83b) from the antennae of Spodoptera exigua and S.litura by using reverse transcription-polymerase chain reaction(RT-PCR) and rapid amplification of cDNA ends(RACE) methods.Bioinformatics methods were used to further analyze the cDNA sequences and putative amino acid sequences.The two full-length cDNA sequences from olfactory receptor(OR) of male S.exigua and S.litura were named as SexiOR2 and SlitOR2,respectively.SexiOR2 and SlitOR2 consisted of nucleotide sequence of 1 906 and 2 483 bp,respectively,and both with deduced amino acid sequences of 473 residues.The sequence analysis indicated that the deduced amino acid sequences of the cDNA shared the high homologies with OR83b orthologue chemoreceptor sequences from previously reported moths,implying that the cDNA sequences were of OR83b orthologue chemoreceptor genes.展开更多
A sulfate reducing bacteria was isolated from mining sewage of Daqing Oilfield by Hungate anaerobic technology. Physiological-biochemical analysis showed that the strain could utilize polyacrylamide as sole carbon and...A sulfate reducing bacteria was isolated from mining sewage of Daqing Oilfield by Hungate anaerobic technology. Physiological-biochemical analysis showed that the strain could utilize polyacrylamide as sole carbon and nitrogen source. The sequence analysis of 16S rDNA illustrated that the similarity of F8 and Desulfovibrio desulfuricans (AF192153) was 99%, and the similarity sequence of dissimilatory sulfite reductase gene (DSR) cloned from the strain and Desulfovibrio desulfuricans (AF273034) was 98%. Their phylogenitic analysis was basically anastomosed, and thus temporarily named as Desulfovibrio desulfuricans F8. The DSR cloned from F8 strain was 2740 bp in length consisting of three ORF, DSRA, DSRB and DSRD as a single operon (DSRABD) regulated by the same operator. DSRA contained typical conservative box of sulfate—sulfite reducing enzyme (SiteⅠand SiteⅡ), which could bind siroheme and [Fe4S4]. DSRB retained a [Fe4S4] binding site, with an uncomplimentary structure for siroheme binding. There was no conservative box in DSRD. Sequence analysis of DSR will provide a theoretical basis for quantitative detection, metabolic pathway modification through gene engineering, and sulfate reducing bacteria (SRB) suppression.展开更多
A technique of direct sequence analysis of β-globin gene with the products of amplifi-cation by polymerase chain reaction (PCR) was reported and a case of β-thalassemia with therare mutation in Chinese,‘codon 14/15...A technique of direct sequence analysis of β-globin gene with the products of amplifi-cation by polymerase chain reaction (PCR) was reported and a case of β-thalassemia with therare mutation in Chinese,‘codon 14/15 (+G)’ was detected by this method.After the se-quence of the mutation site was determined,an analysis of the restriction map of the gene anddot blot hybridization with radioactive allele specific oligonucleotide probe was designed to con-firm the result of DNA sequencing.展开更多
Aequorea taiwanensis,a new hydrozoan species from the Taiwan Strait was described using morphological and molecular characteristics.Both morphological and mitochondrial cytochrome oxidase subunit I(mtCOI) data support...Aequorea taiwanensis,a new hydrozoan species from the Taiwan Strait was described using morphological and molecular characteristics.Both morphological and mitochondrial cytochrome oxidase subunit I(mtCOI) data supported A.taiwanensis n.sp.as a valid species.Sequence divergence and genetic distance of A.taiwanensis n.sp.,A.papillata and A.conica were analysed based on the mtCOI gene sequences.The mtCOI sequences from these three species of the genus Aequorea showed high variation frequency,with sequence divergences ranging from 9.10% to 11.9%,and pairwise genetic distances ranging from 0.097 to 0.130.MtCOI sequence analysis provided diagnostic molecular systematic characteristics for accurate identification and discrimination of the species of Aequorea or their populations,and will be used to resolve evolutionary relationships among them.It was suggested that 10%-20% pairwise mtCOI sequence differences indicated the species-level divergence among congeneric species in the Hydromedusae.展开更多
Telomeric repeat amplification protocol (TRAP) is now a conventional assay for detecting telomerase activity. However, this method presents problems owing to tedious quantitation, radioisotopic handling. In order to a...Telomeric repeat amplification protocol (TRAP) is now a conventional assay for detecting telomerase activity. However, this method presents problems owing to tedious quantitation, radioisotopic handling. In order to alleviate these inconveniences, a novel telomerase DNA sequencing assay together with TRAP to detect human telomerase activity was developed. It was used to detect telomerase activity in Hela, HLF, MCF, K562, SMMC 7721 cells, Leukocytes and RNase pretreated or heat treated cells as control. Telomerase activity assayed by this method was positive when the number of K562 cells examined was 102,103, and 104. The telomerase activity depended on the number of K562 cells used in the assay. Telomerase activity of Rnase pretreated cells or heat treated cells, and human normal peripheral blood leukocyte(Leu) were negative. The result of this method was available within a few hours and was handled without radioisotope. Further studies should be taken to detect telomerase activity in quantitation.展开更多
Based on conserved regions among genomic RNA of tobamoviruses, a pair of primers spanning the sequence encoding the movement protein were synthesized. A cDNA fragment of 1700bp was thus amplified by RT-PCR(reverse tra...Based on conserved regions among genomic RNA of tobamoviruses, a pair of primers spanning the sequence encoding the movement protein were synthesized. A cDNA fragment of 1700bp was thus amplified by RT-PCR(reverse transcription-polymerase chain reaction). The fragment was cloned into pGEM-T easy vector and sequenced. DNA sequence analysis showed that the fragment contained a region of 768 nucleotides encoding protein of 256 amino acid of frangipani mosaic virus (FMV) and also partial sequence corresponding to 180ku and 17. 5ku protein.展开更多
基金Supported by Natural Science Foundation of Heilongjiang Province (C2007-7)Scientific and Technical Innovation Fund of Harbin (RC2006QN002027)Northeast Agricultural University Research Fund (2005)
文摘Chitin is the most widespread amino polysaccharide in nature.Chitin synthase (CHS) plays an important role in chitin formation in the cuticle and the peritrophic membrane (PM) lining the midgut.Total RNA was isolated from the cuticle of Mamestra brassicae (L.) fourth instar larva.cDNA sequence was cloned by RT-PCR and Rapid Amplifi cation of cDNA Ends (RACE).cDNA,5 220 bp in length,contained an open reading frame of 4 704 bp coding for a polypeptide of 1 567 amino acid residues with a predicted molecular weight of 178.3 ku and its pI was 6.42.The deduced amino acid sequence from M.brassicae (L.) shared the high level of identity with chitin synthase sequences from other insects,especially lepidopteran insects.cDNA sequence has been deposited with GenBank under accession No.GQ281761.
基金Supported by the Postdoctoral Scientific Research Foundation of Heilongjiang Province(LBH-Q10144)the Natural Science Foundation of Heilongjiang Province(C201112)Northeast Agricultural University Doctoral Research Fund(200830)
文摘Degenerate primers were designed based on the conserved sequences of the Actin gene from other plants. Total RNA was extracted from the leaves of Iris lacteal var.chinensis Fisch.Koidz. Actin gene fragment was obtained by reverse transcription polymerase chain reaction(RT-PCR) and cloned into pMD18-T vector. The positive clone identified by PCR was sequenced. The sequencing result showed that the Actin gene fragment from Iris lacteal var.chinensis Fisch.Koidz contained about 598 bp, encoding199 amino acids. Homology comparison with Actin gene sequences of other plants in the GenBank showed that it shared over 82%nucleotide sequence homology and 90% amino acid sequence homology. It indicated that this was the Actin gene. Because of the stability expression of Actin gene, it usually cited as the internal reference to study the expression and regulation of foundation in other genes of Iris lacteal var.chinensis Fisch.Koidz well.
基金Supported by Science and Technology Project of Guangdong Province(2019B030316033,2021KJ121,C2024900075,C2024900210)Science and Technology Project of Guangzhou City(202102020809)。
文摘[Objectives]The paper was to explore the genetic information and evolution of CymMV,and to provide an important scientific basis for monitoring and early warning of orchid virus disease and anti-virus genetic engineering of orchid in Guangdong Province.[Methods]RT-PCR and DASELISA were used to detect and identify CymMV from leaves with suspected virus disease of Cymbidium sinense collected from Guangzhou area.The genome sequence assembly,annotation,phylogeny and selection pressure analysis of CymMV isolates were performed with related molecular biology software.[Results]Two CymMV isolates(GZV013 and ZC29)were found in Guangdong Province for the first time in this study.The genome of both GZV013 and ZC29 were 6227 nt in length,encoding 5 functional proteins.The similarity analysis of the full sequence showed that the nucleotide sequence identity of GZV013 and Taiwan isolate M2 was 97.03% and that of ZC29 and Nanjing isolate NJ-1 was 97.11%.The complete genome sequence identity among CymMV isolates ranged from 86.85% to 98.31%,and the differentiation of diverse populations was closely related to host species and geographical isolation.Each region of CymMV genome was affected by negative selection and conformed to the neutral evolution model.The genes encoding RdRp,TGB1 and TGB2 had the highest mutation rates in the genome.[Conclusions]GZV013 was most closely related to Taiwan isolate M2,and ZC29 was most closely related to Nanjing isolate NJ-1,belonging to the same branch of a family.
文摘The method described in this work provides a sensitive and fast technique for investigating the primary structure of peptides with molecular weight up to 3340 amu. Usually, the metastable ion kinetic energy spectra (MIKES) and collisional activated decomposition (CAD) spectra provide complementary information for the FAB mass spectra, the MIKES and CAD spectra generally contain high-mass sequence ions.
基金supported by the fund of State Key Laboratory for Infectious Diseases Prevention and Control (2011SKLID208)the project "Transmission Mode of Tuberculosis"of National Key Program of Mega Infectious Diseases (2008ZX100/03-010)
文摘Objective Tuberculosis remains one of the most serious infectious diseases in the world. In this study, a scheme of Mycobacterium tuberculosis (M. tuberculosis) multilocus sequence analysis (MLSA) was established for the phylogenetic and epidemiology analysis. Methods To establish the scheme of M. tuberculosis MLSA, the genome of H37Rv, CCDC5079 and CCDC5180 were compared, and some variable genes were chosen to be the MLSA typing scheme. 44 M. tuberculosis clinical isolates were typed by MLSA, IS6110-RFLP, and soligotyping, to evaluate the MLSA methods. Results After comparison of the genome, seven high discrimination gene loci (recX, rpsL, rmlC, rpmG1, mprA, gcvH, ideR) were chosen to be the MLSA typing scheme finally. 11 variable SNP sites of those seven genes were found among the 44 M. tuberculosis isolate strains and 11 sequence types (STs) were identified. Based on the Hunter-Gaston Index (HGI), MLSA typing was not as good for discrimination at the strain level as IS6110-RFLP, but the HGI was much better than that of spoligotyping. In addition, the MEGA analysis result of MLSA data was similar to spoligotyping/PGG lineage, showing a strong phylogenetic signal in the modern strains of M. tuberculosis. The MLSA data analysis by eBURST revealed that 4 sequence types (ST) came into a main cluster, showing the major clonal complexes in those 44 strains. Conclusion MLSA genotyping not only can be used for molecular typing, but also is an ideal method for the phylogenetic analysis for M. tuberculosis.
基金This study was supported by the National Natural Science Foundation of China under contract Nos 39890390,30170499,40476059 and 30250003the Project of Scientific Innovation of Chinese Academy of Sciences under contract No.KZCX2-211+1 种基金the Project of Scientific Innovation of Institute of Oceanology,Chinese Academy of Sciences under contract No.2002-2005the“863”Project of China under contract No.2004AA603220.
文摘The partial sequence of the rbcL from Bryopsis hypnoides, including the sequences of the upstream, extron and partial intron, was amplified by PCR and their sequences were determined. With Spinacia oleracea as the outgroup, neighbor-joining method and maximum parsimony method were used respectively to build phylogenetic trees according to the rbcL exon sequence among 13 species that were the typical species of six phyla. Two kinds of trees showed clearly that there were two groups among those species, the green lineage and the non-green lineage. And the relationships of algae in the green lineage were similar in the two trees but those in the non-green lineage were not consistent. Analysis of codon preference indicated that the codon preference of the rbcL exon of Bryopsis hypnoides distinctly differed from that of the relevant sequence of photosynthetic bacteria.
基金supported by the National Natural Science Foundation of China (Nos. 31471499, 91535206)
文摘In the subtribe Maydeae, Tripsacum and Zea are closely related genera. Tripsacum is a horticultural crop widely used as pasture forage. Previous studies suggested that Tripsacum might play an important role in maize origin and evolution. However, our understanding of the genomics and the evolution of Tripsacum remains limited. In this study, two diploids,T. dactyloides var. meridionale(2n = 36, MR) and T. dactyloides(2n = 36, DD), and one tetraploid,T. dactyloides(2n = 72, DL) were sequenced by low-coverage genome sequencing followed by graph-based cluster analysis. The results showed that 63.23%, 59.20%, and 61.57% of the respective genome of MR, DD, and DL were repetitive DNA sequence. The proportions of different repetitive sequences varied greatly among the three species. Fluorescence in situ hybridization(FISH) analysis of mitotic metaphase chromosomes with satellite repeats as the probes showed that the FISH signal patterns of DL were more similar to that of DD than to that of MR. Comparative analysis of the repeats also showed that DL shared more common repeat families with DD than with MR. Phylogenetic analysis of internal transcribed spacer region sequences further supported the evolutionary relationship among the three species. Repetitive sequences comparison showed that Tripsacum shared more repeat families with Zea than with Coix and Sorghum. Our study sheds new light on the genomics of Tripsacum and differential speciation in the Poaceae family.
文摘Using PCR degenerate primers, designed with reference to the sequences of the conserved amino acids of known expansins, to amplify cDNA fragments in litchi fruit by RT-PCR, two different cDNA fragments, named as Lc-Expl and Lc-Exp2, were cloned. Lc-Expl and Lc-Exp2 was respectively composed of 531 bp encoding 177 amino acids and 537 bp encoding 179 amino acids. Eight cysteine residues and three tryptopban residues, which is supposed to be the characteristics of expansins, are conserved in both Lc-Expl and e-Exp2. In addition, the homology between the two expansins is 71.6% at nucleotide acid sequences and 76.3% at amino acid sequences. The homology of Lc-Expl with Fa-Exp2 or Pp-Expl was 92.7% or 92.1%, but that of Lc-Exp2 with Fa-Exp2 or Pp-Expl was only 77.4% or 76.3% at amino acid sequences.
基金supported by the Fundamental Research Funds for the Central Universities,China(DL09EAQ02)the Natural Science Foundation of Heilongjiang Province and Harbin City,China(C200606nd and 2006RFQN005)
文摘The full length cDNA sequence of CBF3(CRT/DRE-binding factor) was cloned from Vitis amurensis by reverse transcription polymerase chain reaction(RT-PCR) using the primers designed based on CBF genes available in GenBank.Sequence analysis showed that the gene had 854 bp long and its coding sequence contained 720 bp,encoding a protein with 239 amino acids and an AP2 structural domain.The molecular mass of CBF3 was predicted to be 25.9 kDa and its theoretical isoelectric point was 7.02 and aliphatic index was 59.29.The average hydropathicity of the protein was -0.551.The tertiary structures of CBF3 were also analyzed.The prokaryotic expression vector pGEX-4T-CBF3 containing CBF3 gene was constructed and CBF3 fusion protein(52 kDa) was produced in Escherichia coli after induction with 1 mmol L-1 IPTG.Further studies are needed to evaluate its potential application for improving plant resistance to cold and other stress condition such as drought and salinity.
基金This work was supported by the National High Technology Research and Development Program of China （2003AA207100）the grants from the National Natural Science Foundation of China （30300219, 30370882, and 30571163） the Foundation for the Author of National Excellent Doctoral Dissertation of China （200357 and 200458）.
文摘A novel HMW glutenin subunit gene 1Dy10.1 was isolated and characterized from Xinjiang wheat (Triticum petropavlovskyi. Udacz. et Migusch) accession Daomai 2. The complete open reading frame (ORF) of 1Dy10.1 was 1 965 bp, encoding 655 amino acids. The numbers and distribution of cysteines in 1Dy10.1 were similar to those of 1Dy10 and other y-type subunits. In the N-terminal of 1Dy10.1, an amino acid was changed from L (leucine) to P (proline) at position 55. The repetitive domain of 1Dy10.1 differed from those of known HMW subunits by substitutions, insertions or/and deletions involving single or more amino acid residues. In the repetitive domain of subunit 1Dy10.1, the deletion of tripeptide GQQ in the consensus unit PGQGQQ resulted in the appearance of the motif PGQ that have not been observed in other known y-type HMW subunits. In comparison with the subunit 1Dy12, a deletion of dipeptide GQ, which occurred in subunit 1Dy10, was also observed in subunit 1Dy10.1. The cloned 1Dy10.1 gene had been successfully expressed in Escherichia coli, and the expressed protein had the identical mobility with the endogenous subunit 1Dy10.1 from seed.
基金Supported by National Natural Science Foundation of China(31272564)Joint Fund of NSFC-Guangdong of China(U0931003)+1 种基金China Agriculture Research System(CARS-36)Hainan Provincial Research Foundation for public institutions(Qiong Cai Yu[2013]No.131)
文摘The diseased samples collected from Guangdong Province were inoculated to Marc-145 cells,PRRSV-specific cytopathogenic effects( CPE) such as cell aggregation,cell shrinkage and cell exfoliation were observed and a PRRSV strain XH-GD was obtained. By using primers designed in accordance with the published gene sequences of PRRSV in GenBank,NSP2,ORF3 and ORF5 fragments were amplified by reverse transcription polymerase chain reaction and analyzed by sequence alignment. The results demonstrated that PRRSV strain XH-GD belonged to the North American genotype and the NSP2 gene contained discontinuous deletions of 30 amino acids. NSP2,ORF3 and ORF5 genes of XH-GD shared 83. 3%- 98. 9%,88. 6%- 99. 2% and 88. 1%- 99. 2% nucleotide homology,and76. 8%- 98. 3%,83. 7%- 98. 8% and 88. 1%- 99. 2% amino acid homology with HUB1,NX06,BJsy06,VR-2332,HB-1( sh) 2002,HB-2( sh) 2002,CH-la and RespPRRS MLV,respectively; however,NSP2,ORF3 and ORF5 genes of XH-GD shared only 52. 9%,65. 0% and 64. 3% nucleotide homology,and31. 3%,57. 5% and 58. 9% amino acid homology with LV,respectively. Phylogenetic analysis revealed that XH-GD had a closer relationship with HUB1,NX06,BJsy06,VR-2332,HB-1( sh) 2002,HB-2( sh) 2002,CH-la and RespPRRS MLV strains compared with LV strain.
基金The article was a part of the research program financed by the Science and Technology Bureau of Hebei Province, China (06220106D)
文摘A combination method of the usual-PCR and reverse-PCR for the cloning of a novel lipase gene directly from the total genomic DNA of strain lip35 (Pseudomonas sp.) is described, whereby a lipase gene (lip) was cloned directly from genomic DNA. The sequence data have been deposited in the GenBank and EMBL data bank with the accession number EU414288. The nucleotide sequence showed a major open reading frame encoding a 59-kDa protein of 566 amino acid residues, which contained a lipase consensus sequence GXSXG. The lipase lip had 74 and 70% homologies with the lipases of an uncultured bacterium and P. fluorescens PfO-1, respectively, but it did not show any overall homology with lipases from other origins. The functional lipase was obtained when the lip gene was expressed in Pichia pastoris GS115.
基金Supported by the National Natural Science Foundation of China (No 40576074)the Key Laboratory of Experimental Marine Biology,Institute of Oceanology,Chinese Academy of Sciences (No KFN92007NO1)
文摘The sequences of the ITS(internal transcribed spacer) and 5.8S rDNA of three cultivated strains of Porphyra haitanensis thalli(NB,PT and ST) were amplified,sequenced and analyzed.In addition,the phylogenic relationships of the sequences identified in this study with those of other Porphyra retrieved from GenBank were evaluated.The results are as follows:the sequences of the ITS and 5.8S rDNA were essentially identical among the three strains.The sequences of ITS1 were 331 bp to 334 bp,while those of the 5.8S rDNA were 158 bp and the sequences of ITS2 ranged from 673 bp to 681 bp.The sequences of the ITS had a high level of homology(up to 99.5%) with that of P.haitanensis(DQ662228) retrieved from GenBank,but were only approximately 50% homologous with those of other species of Porphyra.The results obtained when a phylogenetic tree was constructed coincided with the results of the homology analysis.These results suggest that the three cultivated strains of P.haitanensis evolved conservatively and that the ITS showed evolutionary consistency.However,the sequences of the ITS and 5.8S rDNA of different Porphyra species showed great variations.Therefore,the relationship of Porphyra interspecies phyletic evolution could be judged,which provides the proof for Porphyra identification study.However,proper classifications of the subspecies and the populations of Porphyra should be determined through the use of other molecular techniques to determine the genetic variability and rational phylogenetic relationships.
基金supported by funds from the National Natural Science Foundation of China(30800725 and30770278)the Central Public Research Institutes Basic Funds for Research and Development(Agro-Environmental Protection Institute,Ministry of Agriculture,China)
文摘The aim of this study was to isolate the full-length cDNA sequences of an unusually highly conserved olfactory receptor(orthologue to the Drosophila melanogaster DOR83b) from the antennae of Spodoptera exigua and S.litura by using reverse transcription-polymerase chain reaction(RT-PCR) and rapid amplification of cDNA ends(RACE) methods.Bioinformatics methods were used to further analyze the cDNA sequences and putative amino acid sequences.The two full-length cDNA sequences from olfactory receptor(OR) of male S.exigua and S.litura were named as SexiOR2 and SlitOR2,respectively.SexiOR2 and SlitOR2 consisted of nucleotide sequence of 1 906 and 2 483 bp,respectively,and both with deduced amino acid sequences of 473 residues.The sequence analysis indicated that the deduced amino acid sequences of the cDNA shared the high homologies with OR83b orthologue chemoreceptor sequences from previously reported moths,implying that the cDNA sequences were of OR83b orthologue chemoreceptor genes.
基金Sponsored by the National Basic Research and Development (973) Program of China(Grant No.2004CB418505)
文摘A sulfate reducing bacteria was isolated from mining sewage of Daqing Oilfield by Hungate anaerobic technology. Physiological-biochemical analysis showed that the strain could utilize polyacrylamide as sole carbon and nitrogen source. The sequence analysis of 16S rDNA illustrated that the similarity of F8 and Desulfovibrio desulfuricans (AF192153) was 99%, and the similarity sequence of dissimilatory sulfite reductase gene (DSR) cloned from the strain and Desulfovibrio desulfuricans (AF273034) was 98%. Their phylogenitic analysis was basically anastomosed, and thus temporarily named as Desulfovibrio desulfuricans F8. The DSR cloned from F8 strain was 2740 bp in length consisting of three ORF, DSRA, DSRB and DSRD as a single operon (DSRABD) regulated by the same operator. DSRA contained typical conservative box of sulfate—sulfite reducing enzyme (SiteⅠand SiteⅡ), which could bind siroheme and [Fe4S4]. DSRB retained a [Fe4S4] binding site, with an uncomplimentary structure for siroheme binding. There was no conservative box in DSRD. Sequence analysis of DSR will provide a theoretical basis for quantitative detection, metabolic pathway modification through gene engineering, and sulfate reducing bacteria (SRB) suppression.
文摘A technique of direct sequence analysis of β-globin gene with the products of amplifi-cation by polymerase chain reaction (PCR) was reported and a case of β-thalassemia with therare mutation in Chinese,‘codon 14/15 (+G)’ was detected by this method.After the se-quence of the mutation site was determined,an analysis of the restriction map of the gene anddot blot hybridization with radioactive allele specific oligonucleotide probe was designed to con-firm the result of DNA sequencing.
文摘Aequorea taiwanensis,a new hydrozoan species from the Taiwan Strait was described using morphological and molecular characteristics.Both morphological and mitochondrial cytochrome oxidase subunit I(mtCOI) data supported A.taiwanensis n.sp.as a valid species.Sequence divergence and genetic distance of A.taiwanensis n.sp.,A.papillata and A.conica were analysed based on the mtCOI gene sequences.The mtCOI sequences from these three species of the genus Aequorea showed high variation frequency,with sequence divergences ranging from 9.10% to 11.9%,and pairwise genetic distances ranging from 0.097 to 0.130.MtCOI sequence analysis provided diagnostic molecular systematic characteristics for accurate identification and discrimination of the species of Aequorea or their populations,and will be used to resolve evolutionary relationships among them.It was suggested that 10%-20% pairwise mtCOI sequence differences indicated the species-level divergence among congeneric species in the Hydromedusae.
基金LiaoningScience&TechnologyPlanFoundation No :993 0 5 0 0 1
文摘Telomeric repeat amplification protocol (TRAP) is now a conventional assay for detecting telomerase activity. However, this method presents problems owing to tedious quantitation, radioisotopic handling. In order to alleviate these inconveniences, a novel telomerase DNA sequencing assay together with TRAP to detect human telomerase activity was developed. It was used to detect telomerase activity in Hela, HLF, MCF, K562, SMMC 7721 cells, Leukocytes and RNase pretreated or heat treated cells as control. Telomerase activity assayed by this method was positive when the number of K562 cells examined was 102,103, and 104. The telomerase activity depended on the number of K562 cells used in the assay. Telomerase activity of Rnase pretreated cells or heat treated cells, and human normal peripheral blood leukocyte(Leu) were negative. The result of this method was available within a few hours and was handled without radioisotope. Further studies should be taken to detect telomerase activity in quantitation.
文摘Based on conserved regions among genomic RNA of tobamoviruses, a pair of primers spanning the sequence encoding the movement protein were synthesized. A cDNA fragment of 1700bp was thus amplified by RT-PCR(reverse transcription-polymerase chain reaction). The fragment was cloned into pGEM-T easy vector and sequenced. DNA sequence analysis showed that the fragment contained a region of 768 nucleotides encoding protein of 256 amino acid of frangipani mosaic virus (FMV) and also partial sequence corresponding to 180ku and 17. 5ku protein.
