To investigate the existence of the major outer membrane protein (MOMP) gene LipL32 in 15 dominant Chinese strains of 15 serogroups of Leptospira interrogans and 2 international strains of 2 serogroups of Leptospira b...To investigate the existence of the major outer membrane protein (MOMP) gene LipL32 in 15 dominant Chinese strains of 15 serogroups of Leptospira interrogans and 2 international strains of 2 serogroups of Leptospira biflexa, and to clone and construct the expression system as well as to identify the recombinant proteins, genomic DNAs from strains of leptospira were prepared by routine phenol-chloroform method, and the fragments of the LipL32 gene with the whole length from the strains were amplified with high fidelity PCR. The target amplification products were sequenced after T-A cloning, and the expression system for the genes were thereby constructed. Expression of the recombinant proteins was identified by using SDS-PAGE after induction with IPTG at different dosages. Western blot assays with rabbit antiserum against the whole cell of TR/PatocⅠ of Leptospira and immunized serum with rMOMPs were used to determine the immunoreactivity and immunogenicity of the recombinant proteins. Microscopic agglutination test was used to determine the cross- agglutination titres in rabbit sera immunized with rMOMPs, and the cell adherence model of Leptospira was used to examine the blocking effects of rabbit antisera against these rMOMPs. It was found that the LipL32 gene could be found in all the 17 strains of Leptospira mentioned above with two different genotypes, i.e. LipL32/1 and LipL32/2. Amounts of expressions of rMOMP1 and rMOMP2 after IPTG accounted for 40% and 10% of the total bacterial proteins respectively. Both rMOMP1 and rMOMP2 could combine with the rabbit antiserum against leptospiral TR/PatocⅠ, and could induce the production of agglutination antibodies to these 17 strains of Leptospira with 1∶2 to 1∶64 MAT titres. The rabbit anti-rMOMP1 and anti-MOMP2 antibodies at 1∶2 to 1∶16 dilutions could efficiently block adherence of Leptospira. It concludes that all the Leptospira tested in the present study possess LipL32/1 or LipL32/2 genes, and the constructed expression system can express the rMOMP1 and rMOMP2. These recombinant proteins are showed to have good immunogenicity and satisfactory immunoreactivity.展开更多
Non-specific lipid transfer proteins(nsLTPs) are small, basic proteins that are characterized by an eight-cysteine motif. The biological functions of these proteins have been reported to involve plant reproduction and...Non-specific lipid transfer proteins(nsLTPs) are small, basic proteins that are characterized by an eight-cysteine motif. The biological functions of these proteins have been reported to involve plant reproduction and biotic or abiotic stress response. With the completion of the barley genome sequence, a genome-wide analysis of nsLTPs in barley(Hordeum vulgare L.)(HvLTPs) will be helpful for understanding the function of nsLTPs in plants. We performed a genome-wide analysis of the nsLTP gene family in barley and identified 70 nsLTP genes,which can be divided into five types(1, 2, C, D, and G). Each type of nsLTPs shares similar exon and intron gene structures. Expression analysis showed that barley nsLTPs have diverse expression patterns, revealing their various roles. Our results shed light on the phylogenetic relationships and potential functions of barley nsLTPs and will be useful for future studies of barley development and molecular breeding.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
[ Objective ] This study aimed to clone and analyze the gene sequence encoding carbonic anhydrase-related protein lO-like ( CARP X) from Apis mellif era. [Method] The cDNA sequence of CARPX gene was cloned through R...[ Objective ] This study aimed to clone and analyze the gene sequence encoding carbonic anhydrase-related protein lO-like ( CARP X) from Apis mellif era. [Method] The cDNA sequence of CARPX gene was cloned through RT-PCR, and then analyzed with bioinformatic method. [Result] The full-length cDNA sequence of CARPX was 972 bp long and encoded 324 amino acid residues, including a signal peptide and two transmembrane domains. The predicted molecular mass was 37.1 kDa and the predicted isoeleetric point was 7.458. The CARP X from A. mellifera shared close relationship with proteins from Apisflorae, Bombtas impatiens, Bombus terrestris, Nasonia vitripennis and Acyrthosiphon pisum. The insect CARP X family may include two subfamilies. [ Conclusion] The results pro- vide basis for studying CARPs family.展开更多
In this study, full-length CDS sequence of IGFBP-7 gene was cloned from Liangshan semi-fine wool sheep with RT-PCR method and analyzed with bioinformatics methods. The results showed that the full-length CDS sequence ...In this study, full-length CDS sequence of IGFBP-7 gene was cloned from Liangshan semi-fine wool sheep with RT-PCR method and analyzed with bioinformatics methods. The results showed that the full-length CDS sequence of IGFBP-7 gene in Liangshan semi-fine wool sheep was 846 bp in length, encoding 282 amino acids. The CDS sequence shared 99%, 95% and 90% homology with bovine, human and rat, respectively; the amino acid sequence shared 98%, 93% and 89%, respectively. The GenBank accession number was FJ589640.1. The amino acid molecular weight of IGFBP-7 was 29.0 kD, and the theoretical isoelec- tric point (pl) was 8.25. The result of phylogenetic analysis showed that IGFBP-7 gone in Liangshan semi-fine wool sheep exhibited close phylogenetic relationships with bovine, goat and other mammals, and distant phylogenetic relationships with Danio rerio and Haliotis diversicolor. IGFBP-7 gene had uniformly distributed hy- drophobic and hydrophilic regions, harboring one signal peptide, two transmembrane regions, 16 phosphorylation sites, four N-glycosylation sites and one O-glyco- sylation site. The result of secondary structure analysis showed that the random coil, or-helix and β-sheet regions accounted for 64.89%, 19.86% and 15.25%, respectively. The result of tertiary structure analysis showed that IGFBP-7 harbors an IGFBP_N domain and an Ig-like domain. This study provided scientific basis for further investigating the function of IGFBP-7 gene in sheep.展开更多
PCR amplification and sequencing of whole blood DNA from an individual with hereditary spastic paraplegia, as well as family members, revealed a fragment of proteolipid protein 1 (PLP1) gene exon 1, which excluded t...PCR amplification and sequencing of whole blood DNA from an individual with hereditary spastic paraplegia, as well as family members, revealed a fragment of proteolipid protein 1 (PLP1) gene exon 1, which excluded the possibility of isomer 1 expression for this family. The fragment sequence of exon 3 and exon 5 was consistent with the proteolipid protein 1 sequence at NCBI. In the proband samples, a PLP1 point mutation in exon 4 was detected at the basic group of position 844, T→C, phenylalanine→leucine. In proband samples from a male cousin, the basic group at position 844 was C, but gene sequencing signals revealed mixed signals of T and C, indicating possible mutation at this locus. Results demonstrated that changes in PLP1 exon 4 amino acids were associated with onset of hereditary spastic paraplegia.展开更多
An open reading frame (lcn61) of lymphocystis disease virus China (LCDV-cn), probably responsible for encoding putative zinc-finger proteins was amplified and inserted into pET24a (+) vector. Then it expressed in E. c...An open reading frame (lcn61) of lymphocystis disease virus China (LCDV-cn), probably responsible for encoding putative zinc-finger proteins was amplified and inserted into pET24a (+) vector. Then it expressed in E. coli BL21 (DE3), and His-tag fusion protein of high yield was obtained. It was found that the fusion protein existed in E. coli mainly as inclusion bodies. The bioinformatics analysis indicates that LCN61 is C2H2 type zinc-finger protein containing four C2H2 zinc-finger motifs. This work provides a theory for functional research of lcn61 gene.展开更多
The total RNA was isolated from pokeweed (Phytolacca americana) leaves using the method of guanidine isothiocyanite and used as a template to amplify the deleted mutant pokeweed antiviral protein (PAP) gene by RT-PCR ...The total RNA was isolated from pokeweed (Phytolacca americana) leaves using the method of guanidine isothiocyanite and used as a template to amplify the deleted mutant pokeweed antiviral protein (PAP) gene by RT-PCR and then the gene was cloned into the pGEMR-T vector. The sequencing results showed that the PAP gene consisted of 711nt, which was 99.6% identical to the PAP gene reported by Lin et al (1991). The IPTG-inducible expression vector containing the PAP gene was constructed and transferred into the E. coli strain BL21 (DE3)-plysS. A specific protein was produced after induction with 0.4m mol/L IPTG and its molecular weight was 26ku. The results of the double diffusion on the agar plate and the western blotting test showed that the protein produced in E. coli was highly identical with the PAP extracted by a Frenchman from French pokeweed leaves. These revealed that PAP gene was actually achieved and exactly expressed in E. coli.展开更多
AIM: To develop and optimize cDNA representationaldifference analysis (cDNA RDA) method and to identify andclone garlic up-regulated genes in human gastric cancer(HGC) cells.METHODS: We performed cDNA RDA method by us...AIM: To develop and optimize cDNA representationaldifference analysis (cDNA RDA) method and to identify andclone garlic up-regulated genes in human gastric cancer(HGC) cells.METHODS: We performed cDNA RDA method by usingabundant double-stranded cDNA messages provided by twoself-constructed cDNA libraries (Allitridi-trested and paternalHGC cell line BGC823 cells cDNA libraries respectively).BamH Ⅰ and Xho I restriction sites harbored in the libraryvector were used to select representations. Northern andSlot blots analyses were employed to identify the obtaineddifference products.RESJLTS: Fragments released from the cDNA library vectorafter restriction endonuclease digestion acted as goodmarker indicating the appropriate digestion degree for libraryDNA. Two novel expressed sequence tags (ESTs) and arecombinant gene were obtained. Slot blots result showed a8-fold increase of gila-derived nexin/protease nexin 1 (GDN/PN1 ) gene expression level and 4-fold increase of hepatitis Bvirus x-interacting protein (XIP) mRNA level in BGC823 cellsafter Allitridi treatment for 72 h.CONCLUSION: Elevated levels of GDN/PN1 and XIP mRNAsinduced by Allitridi provide valuable molecular evidence forelucidating the garlic' s efficacies against neurodegenerativeand inflammatory diseases. Isolation of a recombinant geneand two novel ESTs further show cDNA RDA based on cDNAlibraries to be a powerful method with high specificity andreproducibility in cloning differentially expressed genes.展开更多
Salinity causes a detrimental impact on plant growth,particularly when the stress occurs during germination and early development stages.Barley is one of the most salt-tolerant crops;previously we mapped two quantitat...Salinity causes a detrimental impact on plant growth,particularly when the stress occurs during germination and early development stages.Barley is one of the most salt-tolerant crops;previously we mapped two quantitative trait loci(QTL)for salinity tolerance during germination on the short arm of chromosome 2 H using a CM72/Gairdner doubled haploid(DH)population.Here,we narrowed down the major QTL to a region of 0.341 or 0.439 Mb containing 9 or 24 candidate genes belonging to 6 or 20 functional gene families according to barley reference genomes v1 and v3 respectively,using two DH populations of CM72/Gairdner and Skiff/CM72,F_(2)and F;generations of CM72/Gairdner/;Spartacus CL,Two Receptorlike kinase 4(RLPK4)v1 or Receptor-like kinase(RLK)v3 could be the candidates for enhanced germination under salinity stress because of their upregulated expression in salt-tolerant variety CM72.Besides,several insertion/deletion polymorphisms were identified within the 3 rd exon of the genes between CM72 and Gairdner.The sequence variations resulted in shifted functional protein domains,which may be associated with differences in salinity tolerance.Two molecular markers were designed for selecting the locus with receptor-like protein kinase 4,and one was inside HORVU2 Hr1 G111760.1 or HORVU.MOREX.r3.2 HG0202810.1.The diagnostic markers will allow for pyramiding of 2 H locus in barley varieties and facilitate genetic improvement for saline soils.Further,validation of the genes to elucidate the mechanisms involved in enhancing salinity tolerance at germination and designing RLPK4 specific markers is proposed.For this publication,all the analysis was based on barley reference genome of2017(v1),and it was used throughout for consistence.However,the positions of the markers and genes identified were updated according to new genome(v3)for reference.展开更多
MCM10 protein is an essential replication factor involved in the initiation of DNA replication. A mcm10 mutan (mcm10-1) of budding yeast shows a growth arrest at 37 C. In the present work, we have isolated a mcm10-1 s...MCM10 protein is an essential replication factor involved in the initiation of DNA replication. A mcm10 mutan (mcm10-1) of budding yeast shows a growth arrest at 37 C. In the present work, we have isolated a mcm10-1 suppressor strain, which grows at 37 C. Interestingly, this mcm10-1 suppressor undergoes cell Cycle arrest at 14 C. A novel gene, YLR003c, is identified by high-copy complementation of this suppressor. We called it as Cms1 (Complementation of Mcm 10 Suppressor). Furthermore, the experiments of trans- formation show that cells of mcm10-1 suppressor with high-copy plasmid but not low-copy plasmid grow at 14 C, indicating that overexpression of Cms1 can rescue the growth arrest of this mcm10 suppressor at non-permissive temperature. These results suggest that CMS1 protein may functionally interact with MCM10 protein and play a role in the regulation of DNA replication and cell cycle control.展开更多
To find Schistosoma japonicum(S.j)new antigen gene thus provide more useful vaccine candidates, the cDNA library of S.j adult worm was screened with sera of rabbits immunized with the membrane antigens of Schistosoma ...To find Schistosoma japonicum(S.j)new antigen gene thus provide more useful vaccine candidates, the cDNA library of S.j adult worm was screened with sera of rabbits immunized with the membrane antigens of Schistosoma japonicum hepato-portal schistosomula (SjHmAg). The positive clones were amplified by PCR and sequenced, then the sequences of clones were compared with all sequences in GenBank database using Blast process. The new clones were submitted to GenBank for accession numbers. Fifteen positive clones were obtained after three rounds of immunoscreening. The size of S.j cDNA fragments in positive clones ranged from 0.7?kb-3.0?kb after automatically excised with the helper phage. Sequence analysis revealed that partial sequence of clone M5 had significant homology with S.j mitochondria mRNA, the other positive clones were new S.j genes. M2 clone sequence (GenBank accession number AF502579) was 730?bp long it had a 117?bp open reading frame (ORF). The sequence of M15 (GenBank accession number AF502582) has no transmembrane region and encodes 92 amino acids, and its protein contains a ferredoxins iron-sulfur binding region signature and two VWFC signal regions. The size of M1、M8、M9、M12(GenBank accession numbers: AF502578, AF502580, AF500622, AF502581) ranges from 402?bp to 766?bp. It concluded that the sera from rabbit immunized with SjHmAg could recognize S.j specific antigens molecules, and these antigens may induce the protective immunity against S.j infection.展开更多
文摘To investigate the existence of the major outer membrane protein (MOMP) gene LipL32 in 15 dominant Chinese strains of 15 serogroups of Leptospira interrogans and 2 international strains of 2 serogroups of Leptospira biflexa, and to clone and construct the expression system as well as to identify the recombinant proteins, genomic DNAs from strains of leptospira were prepared by routine phenol-chloroform method, and the fragments of the LipL32 gene with the whole length from the strains were amplified with high fidelity PCR. The target amplification products were sequenced after T-A cloning, and the expression system for the genes were thereby constructed. Expression of the recombinant proteins was identified by using SDS-PAGE after induction with IPTG at different dosages. Western blot assays with rabbit antiserum against the whole cell of TR/PatocⅠ of Leptospira and immunized serum with rMOMPs were used to determine the immunoreactivity and immunogenicity of the recombinant proteins. Microscopic agglutination test was used to determine the cross- agglutination titres in rabbit sera immunized with rMOMPs, and the cell adherence model of Leptospira was used to examine the blocking effects of rabbit antisera against these rMOMPs. It was found that the LipL32 gene could be found in all the 17 strains of Leptospira mentioned above with two different genotypes, i.e. LipL32/1 and LipL32/2. Amounts of expressions of rMOMP1 and rMOMP2 after IPTG accounted for 40% and 10% of the total bacterial proteins respectively. Both rMOMP1 and rMOMP2 could combine with the rabbit antiserum against leptospiral TR/PatocⅠ, and could induce the production of agglutination antibodies to these 17 strains of Leptospira with 1∶2 to 1∶64 MAT titres. The rabbit anti-rMOMP1 and anti-MOMP2 antibodies at 1∶2 to 1∶16 dilutions could efficiently block adherence of Leptospira. It concludes that all the Leptospira tested in the present study possess LipL32/1 or LipL32/2 genes, and the constructed expression system can express the rMOMP1 and rMOMP2. These recombinant proteins are showed to have good immunogenicity and satisfactory immunoreactivity.
基金supported by funds from the National Key Research and Development Program of China (2016YFD0100903)
文摘Non-specific lipid transfer proteins(nsLTPs) are small, basic proteins that are characterized by an eight-cysteine motif. The biological functions of these proteins have been reported to involve plant reproduction and biotic or abiotic stress response. With the completion of the barley genome sequence, a genome-wide analysis of nsLTPs in barley(Hordeum vulgare L.)(HvLTPs) will be helpful for understanding the function of nsLTPs in plants. We performed a genome-wide analysis of the nsLTP gene family in barley and identified 70 nsLTP genes,which can be divided into five types(1, 2, C, D, and G). Each type of nsLTPs shares similar exon and intron gene structures. Expression analysis showed that barley nsLTPs have diverse expression patterns, revealing their various roles. Our results shed light on the phylogenetic relationships and potential functions of barley nsLTPs and will be useful for future studies of barley development and molecular breeding.
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.
基金Supported by the Science and Technology Project of Education Commission of Shaanxi Province (11JK0618)
文摘[ Objective ] This study aimed to clone and analyze the gene sequence encoding carbonic anhydrase-related protein lO-like ( CARP X) from Apis mellif era. [Method] The cDNA sequence of CARPX gene was cloned through RT-PCR, and then analyzed with bioinformatic method. [Result] The full-length cDNA sequence of CARPX was 972 bp long and encoded 324 amino acid residues, including a signal peptide and two transmembrane domains. The predicted molecular mass was 37.1 kDa and the predicted isoeleetric point was 7.458. The CARP X from A. mellifera shared close relationship with proteins from Apisflorae, Bombtas impatiens, Bombus terrestris, Nasonia vitripennis and Acyrthosiphon pisum. The insect CARP X family may include two subfamilies. [ Conclusion] The results pro- vide basis for studying CARPs family.
基金Supported by Special Fund for Agro-scientific Research in the Public Interest from the Ministry of Agriculture(201003061)Key Project of Livestock and Poultry Breeding of Sichuan Province(01NG029-18)
文摘In this study, full-length CDS sequence of IGFBP-7 gene was cloned from Liangshan semi-fine wool sheep with RT-PCR method and analyzed with bioinformatics methods. The results showed that the full-length CDS sequence of IGFBP-7 gene in Liangshan semi-fine wool sheep was 846 bp in length, encoding 282 amino acids. The CDS sequence shared 99%, 95% and 90% homology with bovine, human and rat, respectively; the amino acid sequence shared 98%, 93% and 89%, respectively. The GenBank accession number was FJ589640.1. The amino acid molecular weight of IGFBP-7 was 29.0 kD, and the theoretical isoelec- tric point (pl) was 8.25. The result of phylogenetic analysis showed that IGFBP-7 gone in Liangshan semi-fine wool sheep exhibited close phylogenetic relationships with bovine, goat and other mammals, and distant phylogenetic relationships with Danio rerio and Haliotis diversicolor. IGFBP-7 gene had uniformly distributed hy- drophobic and hydrophilic regions, harboring one signal peptide, two transmembrane regions, 16 phosphorylation sites, four N-glycosylation sites and one O-glyco- sylation site. The result of secondary structure analysis showed that the random coil, or-helix and β-sheet regions accounted for 64.89%, 19.86% and 15.25%, respectively. The result of tertiary structure analysis showed that IGFBP-7 harbors an IGFBP_N domain and an Ig-like domain. This study provided scientific basis for further investigating the function of IGFBP-7 gene in sheep.
文摘PCR amplification and sequencing of whole blood DNA from an individual with hereditary spastic paraplegia, as well as family members, revealed a fragment of proteolipid protein 1 (PLP1) gene exon 1, which excluded the possibility of isomer 1 expression for this family. The fragment sequence of exon 3 and exon 5 was consistent with the proteolipid protein 1 sequence at NCBI. In the proband samples, a PLP1 point mutation in exon 4 was detected at the basic group of position 844, T→C, phenylalanine→leucine. In proband samples from a male cousin, the basic group at position 844 was C, but gene sequencing signals revealed mixed signals of T and C, indicating possible mutation at this locus. Results demonstrated that changes in PLP1 exon 4 amino acids were associated with onset of hereditary spastic paraplegia.
基金Supported by High Technology Research and Development Program of China (863 Program, No. 2006AA100309)
文摘An open reading frame (lcn61) of lymphocystis disease virus China (LCDV-cn), probably responsible for encoding putative zinc-finger proteins was amplified and inserted into pET24a (+) vector. Then it expressed in E. coli BL21 (DE3), and His-tag fusion protein of high yield was obtained. It was found that the fusion protein existed in E. coli mainly as inclusion bodies. The bioinformatics analysis indicates that LCN61 is C2H2 type zinc-finger protein containing four C2H2 zinc-finger motifs. This work provides a theory for functional research of lcn61 gene.
基金supported by the“948”Project of the Ministry of Agriculture(991003).
文摘The total RNA was isolated from pokeweed (Phytolacca americana) leaves using the method of guanidine isothiocyanite and used as a template to amplify the deleted mutant pokeweed antiviral protein (PAP) gene by RT-PCR and then the gene was cloned into the pGEMR-T vector. The sequencing results showed that the PAP gene consisted of 711nt, which was 99.6% identical to the PAP gene reported by Lin et al (1991). The IPTG-inducible expression vector containing the PAP gene was constructed and transferred into the E. coli strain BL21 (DE3)-plysS. A specific protein was produced after induction with 0.4m mol/L IPTG and its molecular weight was 26ku. The results of the double diffusion on the agar plate and the western blotting test showed that the protein produced in E. coli was highly identical with the PAP extracted by a Frenchman from French pokeweed leaves. These revealed that PAP gene was actually achieved and exactly expressed in E. coli.
基金the Natural Scientific Foundation of China (NSFC3962526)National High-Technology Project-863 (102-10-01-04)
文摘AIM: To develop and optimize cDNA representationaldifference analysis (cDNA RDA) method and to identify andclone garlic up-regulated genes in human gastric cancer(HGC) cells.METHODS: We performed cDNA RDA method by usingabundant double-stranded cDNA messages provided by twoself-constructed cDNA libraries (Allitridi-trested and paternalHGC cell line BGC823 cells cDNA libraries respectively).BamH Ⅰ and Xho I restriction sites harbored in the libraryvector were used to select representations. Northern andSlot blots analyses were employed to identify the obtaineddifference products.RESJLTS: Fragments released from the cDNA library vectorafter restriction endonuclease digestion acted as goodmarker indicating the appropriate digestion degree for libraryDNA. Two novel expressed sequence tags (ESTs) and arecombinant gene were obtained. Slot blots result showed a8-fold increase of gila-derived nexin/protease nexin 1 (GDN/PN1 ) gene expression level and 4-fold increase of hepatitis Bvirus x-interacting protein (XIP) mRNA level in BGC823 cellsafter Allitridi treatment for 72 h.CONCLUSION: Elevated levels of GDN/PN1 and XIP mRNAsinduced by Allitridi provide valuable molecular evidence forelucidating the garlic' s efficacies against neurodegenerativeand inflammatory diseases. Isolation of a recombinant geneand two novel ESTs further show cDNA RDA based on cDNAlibraries to be a powerful method with high specificity andreproducibility in cloning differentially expressed genes.
基金Australian Grains Research and Development Corporation(GRDC)grant IDUmu00046Graduate Research Funds from Murdoch University。
文摘Salinity causes a detrimental impact on plant growth,particularly when the stress occurs during germination and early development stages.Barley is one of the most salt-tolerant crops;previously we mapped two quantitative trait loci(QTL)for salinity tolerance during germination on the short arm of chromosome 2 H using a CM72/Gairdner doubled haploid(DH)population.Here,we narrowed down the major QTL to a region of 0.341 or 0.439 Mb containing 9 or 24 candidate genes belonging to 6 or 20 functional gene families according to barley reference genomes v1 and v3 respectively,using two DH populations of CM72/Gairdner and Skiff/CM72,F_(2)and F;generations of CM72/Gairdner/;Spartacus CL,Two Receptorlike kinase 4(RLPK4)v1 or Receptor-like kinase(RLK)v3 could be the candidates for enhanced germination under salinity stress because of their upregulated expression in salt-tolerant variety CM72.Besides,several insertion/deletion polymorphisms were identified within the 3 rd exon of the genes between CM72 and Gairdner.The sequence variations resulted in shifted functional protein domains,which may be associated with differences in salinity tolerance.Two molecular markers were designed for selecting the locus with receptor-like protein kinase 4,and one was inside HORVU2 Hr1 G111760.1 or HORVU.MOREX.r3.2 HG0202810.1.The diagnostic markers will allow for pyramiding of 2 H locus in barley varieties and facilitate genetic improvement for saline soils.Further,validation of the genes to elucidate the mechanisms involved in enhancing salinity tolerance at germination and designing RLPK4 specific markers is proposed.For this publication,all the analysis was based on barley reference genome of2017(v1),and it was used throughout for consistence.However,the positions of the markers and genes identified were updated according to new genome(v3)for reference.
文摘MCM10 protein is an essential replication factor involved in the initiation of DNA replication. A mcm10 mutan (mcm10-1) of budding yeast shows a growth arrest at 37 C. In the present work, we have isolated a mcm10-1 suppressor strain, which grows at 37 C. Interestingly, this mcm10-1 suppressor undergoes cell Cycle arrest at 14 C. A novel gene, YLR003c, is identified by high-copy complementation of this suppressor. We called it as Cms1 (Complementation of Mcm 10 Suppressor). Furthermore, the experiments of trans- formation show that cells of mcm10-1 suppressor with high-copy plasmid but not low-copy plasmid grow at 14 C, indicating that overexpression of Cms1 can rescue the growth arrest of this mcm10 suppressor at non-permissive temperature. These results suggest that CMS1 protein may functionally interact with MCM10 protein and play a role in the regulation of DNA replication and cell cycle control.
文摘To find Schistosoma japonicum(S.j)new antigen gene thus provide more useful vaccine candidates, the cDNA library of S.j adult worm was screened with sera of rabbits immunized with the membrane antigens of Schistosoma japonicum hepato-portal schistosomula (SjHmAg). The positive clones were amplified by PCR and sequenced, then the sequences of clones were compared with all sequences in GenBank database using Blast process. The new clones were submitted to GenBank for accession numbers. Fifteen positive clones were obtained after three rounds of immunoscreening. The size of S.j cDNA fragments in positive clones ranged from 0.7?kb-3.0?kb after automatically excised with the helper phage. Sequence analysis revealed that partial sequence of clone M5 had significant homology with S.j mitochondria mRNA, the other positive clones were new S.j genes. M2 clone sequence (GenBank accession number AF502579) was 730?bp long it had a 117?bp open reading frame (ORF). The sequence of M15 (GenBank accession number AF502582) has no transmembrane region and encodes 92 amino acids, and its protein contains a ferredoxins iron-sulfur binding region signature and two VWFC signal regions. The size of M1、M8、M9、M12(GenBank accession numbers: AF502578, AF502580, AF500622, AF502581) ranges from 402?bp to 766?bp. It concluded that the sera from rabbit immunized with SjHmAg could recognize S.j specific antigens molecules, and these antigens may induce the protective immunity against S.j infection.