Two pairs of degenerate primers were designed based on nucleotide-binding site (NBS) and serine/threonine kinase domain. PCR was performed with the primers and cDNA from the Triticum aestivum-Haynaldia villosa translo...Two pairs of degenerate primers were designed based on nucleotide-binding site (NBS) and serine/threonine kinase domain. PCR was performed with the primers and cDNA from the Triticum aestivum-Haynaldia villosa translocation line 6VS/6AL. Amplified products were cloned and sequenced. Nine clones with NBS and one with serine/threonine kinase domain were obtained. The NBS clones were classified to six groups according to their nucleotide sequence identities (90% or higher). These resistance gene analogs (RGAs) all have open reading frames (ORF), and their amino acid sequences show high similarity to Yr10 in wheat, Mla1 and Mla6 in barley, RPS2 in Arabidopsis and other resistance (R) genes with conserved motifs. They were preliminarily mapped on the chromosomes of homoeologous groups 1, 2 and 5 of common wheat by nulli-tetrasomic analysis. The 5'-end sequence of an RGA N5 was obtained by 5'-RACE PCR. It encodes six leucine zipper (LZ) and has high sequence similarity to RPS2.展开更多
In the present study, microdissection of 6VS and the cloning of the resistance gene analogs(RGA)from them were reported. The 6VS were microdissected with needle and 10 types of resistance gene analogs were obtained by...In the present study, microdissection of 6VS and the cloning of the resistance gene analogs(RGA)from them were reported. The 6VS were microdissected with needle and 10 types of resistance gene analogs were obtained by PCR with degenerate oligonucleotide primer designed according to resistance genes. They were designated as Hvrgak1-Hvrgak10, GenBank accession numbers are AF387113-AF387121, AY040671- AY040672. Identity among RGAs was about 10-50%, and identity with cloned R gene from plants was 5-20%. Southern hybridization analysis results showed 3 RGAs, Hvrgak2, Hvrgak4, and Hvr-gak5 were linked with wheat powdery mildew resistance. These RGAs may be used as direct entrance or probes for cloning the disease resistance genes.展开更多
Diseases caused by fungal pathogens account for approximately 50% of all soybean disease losses around the world. Conflicting results of fungal disease resistance QTLs from different populations often occurred. The ob...Diseases caused by fungal pathogens account for approximately 50% of all soybean disease losses around the world. Conflicting results of fungal disease resistance QTLs from different populations often occurred. The objectives of this study were to: (i) evaluate evidence for reported fungal disease resistance QTLs associations in soybean and (ii) extract relatively reliable and useful information from the "real" QTLs and mine putative genes in soybean. An integrated map of fungal disease resistance QTLs in soybean was established with soymap 2 published in 2004 as a reference map. QTLs of fungal disease resistance developed from each of separate populations in recent 10 years were integrated into a combinative map for gene cloning and marker assisted selection in soybean. 107 QTLs from different maps were integrated and projected to the reference map with the software BioMercator 2.1. A method of meta-analysis was used to narrow down the confidence interval, and 23 "real" QTLs and their corresponding markers were obtained from 12 linkage groups (LG), respectively. Two published R genes were found in these "real" QTLs intervals. Sequences in the "real" QTLs intervals were predicted by GENSCAN, and these predicted genes were annotated in Goblet. 228 resistance gene analogs (RGAs) in 12 different terms were mined. The results will lay the foundation for a bioinformatics platform combining abundant QTLs, and offer the basis for marker assisted selection and gene cloning in soybean.展开更多
One resistance gene analog fragment named RGA-CIN14 was isolated from TcLr19 wheat,which contains kinase-2,kinase-3a,and the GLPL motif of the NBS-spanning region,using degenerated primers according to the nucleotide ...One resistance gene analog fragment named RGA-CIN14 was isolated from TcLr19 wheat,which contains kinase-2,kinase-3a,and the GLPL motif of the NBS-spanning region,using degenerated primers according to the nucleotide binding site (NBS) conserved domain.Based on the RGA-CIN14,a full-length cDNA,CIN14,which was 2 987 bp encoding 880 amino acids,was obtained by using the method of the rapid amplification cDNA ends (RACE).Bioinformatics analysis showed that the deduced amino acids of CIN14 protein consisted of a NB-ARC conserved domain and many leucine-rich repeats (LRR) domains.The phylogenetic tree analysis indicated a considerable identity of the protein encoded by CIN14 with that of wheat leaf rust resistance gene Lr1,but a lower similarity with Lr21.The expression profile of the CIN14 gene detected by semi-quantitative RT-PCR showed that the CIN14 gene was not induced by Puccinia triticina and it was a constitutive gene with low abundance in the wheat leaf tissue.The resistance homology sequence was successfully obtained,which provides the shortcut for cloning of the resistance gene in TcLr19 wheat.展开更多
A menaquinone-7(MK-7) high-producing strain needs to be isolated to increase MK-7 production, in order to meet a requirement of MK-7 given the low MK-7 content in food products. This article focuses on developing MK...A menaquinone-7(MK-7) high-producing strain needs to be isolated to increase MK-7 production, in order to meet a requirement of MK-7 given the low MK-7 content in food products. This article focuses on developing MK-7 high-producing strains via screening and mutagenesis by an atmospheric and room temperature plasma(ARTP) mutation breeding system. We isolated an MK-7-producing strain Y-2 and identified it as Bacillus amyloliquefaciens, which produced(7.1±0.5) mg/L of MK-7 with maize meal hydrolysate as carbon source. Then, an MK-7 highproducing strain B. amyloliquefaciens H.β.D.R.-5 with resistance to 1-hydroxy-2-naphthoic acid, β-2-thienylalanine, and diphenylamine was obtained from the mutation of the strain Y-2 using an ARTP mutation breeding system. Using strain H.β.D.R.-5, efficient production of MK-7 was achieved((30.2±2.7) mg/L). In addition, the effects of nitrogen sources, prenyl alcohols, and MgSO_4 on MK-7 production were investigated, suggesting that soymeal extract combined with yeast extract, isopentenol, and MgSO4 was beneficial. Under the optimized condition, the MK-7 production and biomass-specific yield reached(61.3±5.2) mg/L and 2.59 mg/L per OD600 unit respectively in a 7-L fermenter. These results demonstrated that strain H.β.D.R.-5 has the capacity to produce MK-7 from maize meal hydrolysate, which could reduce the substrate cost.展开更多
AIM To report naturally occurring mutations in the reverse transcriptase region(RT) of hepatitis B virus(HBV) polymerase from treatment na?ve Korean chronic patients infected with genotype C2.METHODS Here, full-length...AIM To report naturally occurring mutations in the reverse transcriptase region(RT) of hepatitis B virus(HBV) polymerase from treatment na?ve Korean chronic patients infected with genotype C2.METHODS Here, full-length HBV reverse transcriptase RT sequences were amplified and sequenced from 131 treatment na?ve Korean patients chronically infected with hepatitis B genotype C2. The patients had two distinct clinical statuses: 59 patients with chronic hepatitis(CH) and 72 patients with hepatocellular carcinoma(HCC). The deduced amino acids(AAs) at42 previously reported potential nucleos(t)ide analog resistance(NAr) mutation positions in the RT region were analyzed. RESULTS Potential NAr mutations involving 24 positions were found in 79 of the 131 patients(60.3%). Notably, AA substitutions at 2 positions(rt184 and rt204) involved in primary drug resistance and at 2 positions(rt80 and rt180) that functioned as secondary/compensatory mutations were detected in 10 patients(1 CH patient and 9 HCC patients) and 7 patients(1 CH and 6 HCC patients), respectively. The overall mutation frequencies in the HCC patients(3.17%, 96/3024 mutations) were significantly higher than the frequencies in the CH patients(2.09%, 52/2478 mutations)(P = 0.003). In addition, a total of 3 NAr positions, rt80, rt139 and rt204 were found to be significantly related to HCC from treatment na?ve Korean patients. CONCLUSION Our data showed that naturally occurring NAr mutations in South Korea might contribute to liver disease progression(particularly HCC generation) in chronic patients with genotype C2 infections.展开更多
Low genetic diversity makes peanut (Arachis hypogaea L.) very vulnerable to plant pathogens, causing severe yield loss and reduced seed quality. Several hundred partial genomic DNA sequences as nucleotide-binding-si...Low genetic diversity makes peanut (Arachis hypogaea L.) very vulnerable to plant pathogens, causing severe yield loss and reduced seed quality. Several hundred partial genomic DNA sequences as nucleotide-binding-site leucine-rich repeat (NBS-LRR) resistance genes (R) have been identified, but a small portion with expressed transcripts has been found. We aimed to identify resistance gene analogs (RGAs) from peanut expressed sequence tags (ESTs) and to develop polymorphic markers. The protein sequences of 54 known R genes were used to identify homologs from peanut ESTs from public databases. A total of 1,053 ESTs corresponding to six different classes of known R genes were recovered, and assembled 156 contigs and 229 singletons as peanut-expressed RGAs. There were 69 that encoded for NBS-LRR proteins, 191 that encoded for protein kinases, 82 that encoded for LRR-PK/transmembrane proteins, 28 that encoded for Toxin reductases, 11 that encoded for LRR-domain containing proteins and four that encoded for TM-domain containing proteins. Twenty-eight simple sequence repeats (SSRs) were identified from 25 peanut expressed RGAs. One SSR polymorphic marker (RGA121) was identified. Two polymerase chain reaction-based markers (Ahsw-1 and Ahsw-2) developed from RGA013 were homologous to the Tomato Spotted Wilt Virus (TSWV) resistance gene. All three markers were mapped on the same linkage group AhIV. These expressed RGAs are the source for RGA-tagged marker development and identification of peanut resistance genes.展开更多
文摘Two pairs of degenerate primers were designed based on nucleotide-binding site (NBS) and serine/threonine kinase domain. PCR was performed with the primers and cDNA from the Triticum aestivum-Haynaldia villosa translocation line 6VS/6AL. Amplified products were cloned and sequenced. Nine clones with NBS and one with serine/threonine kinase domain were obtained. The NBS clones were classified to six groups according to their nucleotide sequence identities (90% or higher). These resistance gene analogs (RGAs) all have open reading frames (ORF), and their amino acid sequences show high similarity to Yr10 in wheat, Mla1 and Mla6 in barley, RPS2 in Arabidopsis and other resistance (R) genes with conserved motifs. They were preliminarily mapped on the chromosomes of homoeologous groups 1, 2 and 5 of common wheat by nulli-tetrasomic analysis. The 5'-end sequence of an RGA N5 was obtained by 5'-RACE PCR. It encodes six leucine zipper (LZ) and has high sequence similarity to RPS2.
文摘In the present study, microdissection of 6VS and the cloning of the resistance gene analogs(RGA)from them were reported. The 6VS were microdissected with needle and 10 types of resistance gene analogs were obtained by PCR with degenerate oligonucleotide primer designed according to resistance genes. They were designated as Hvrgak1-Hvrgak10, GenBank accession numbers are AF387113-AF387121, AY040671- AY040672. Identity among RGAs was about 10-50%, and identity with cloned R gene from plants was 5-20%. Southern hybridization analysis results showed 3 RGAs, Hvrgak2, Hvrgak4, and Hvr-gak5 were linked with wheat powdery mildew resistance. These RGAs may be used as direct entrance or probes for cloning the disease resistance genes.
基金supported by the funding from the National Natural Science Foundation of China(30971809)the National 973 Program of China(2004CB 117203-5)+2 种基金the National 948 Project of China[(2006-G1(A)]the National High-Tech R&D Program of China(863 Program, 2006AA100104-3)the Heilongjiang Foundation for University Key Teachers,China(1152G007)
文摘Diseases caused by fungal pathogens account for approximately 50% of all soybean disease losses around the world. Conflicting results of fungal disease resistance QTLs from different populations often occurred. The objectives of this study were to: (i) evaluate evidence for reported fungal disease resistance QTLs associations in soybean and (ii) extract relatively reliable and useful information from the "real" QTLs and mine putative genes in soybean. An integrated map of fungal disease resistance QTLs in soybean was established with soymap 2 published in 2004 as a reference map. QTLs of fungal disease resistance developed from each of separate populations in recent 10 years were integrated into a combinative map for gene cloning and marker assisted selection in soybean. 107 QTLs from different maps were integrated and projected to the reference map with the software BioMercator 2.1. A method of meta-analysis was used to narrow down the confidence interval, and 23 "real" QTLs and their corresponding markers were obtained from 12 linkage groups (LG), respectively. Two published R genes were found in these "real" QTLs intervals. Sequences in the "real" QTLs intervals were predicted by GENSCAN, and these predicted genes were annotated in Goblet. 228 resistance gene analogs (RGAs) in 12 different terms were mined. The results will lay the foundation for a bioinformatics platform combining abundant QTLs, and offer the basis for marker assisted selection and gene cloning in soybean.
基金funded by the National Natural Science Foundation of China (30771391,30700505)the Natural Science Foundation of Hebei Province,China (C2008000281)
文摘One resistance gene analog fragment named RGA-CIN14 was isolated from TcLr19 wheat,which contains kinase-2,kinase-3a,and the GLPL motif of the NBS-spanning region,using degenerated primers according to the nucleotide binding site (NBS) conserved domain.Based on the RGA-CIN14,a full-length cDNA,CIN14,which was 2 987 bp encoding 880 amino acids,was obtained by using the method of the rapid amplification cDNA ends (RACE).Bioinformatics analysis showed that the deduced amino acids of CIN14 protein consisted of a NB-ARC conserved domain and many leucine-rich repeats (LRR) domains.The phylogenetic tree analysis indicated a considerable identity of the protein encoded by CIN14 with that of wheat leaf rust resistance gene Lr1,but a lower similarity with Lr21.The expression profile of the CIN14 gene detected by semi-quantitative RT-PCR showed that the CIN14 gene was not induced by Puccinia triticina and it was a constitutive gene with low abundance in the wheat leaf tissue.The resistance homology sequence was successfully obtained,which provides the shortcut for cloning of the resistance gene in TcLr19 wheat.
基金supported by the Natural Science Foundation of Jiangsu Province(No.BK20150149),China
文摘A menaquinone-7(MK-7) high-producing strain needs to be isolated to increase MK-7 production, in order to meet a requirement of MK-7 given the low MK-7 content in food products. This article focuses on developing MK-7 high-producing strains via screening and mutagenesis by an atmospheric and room temperature plasma(ARTP) mutation breeding system. We isolated an MK-7-producing strain Y-2 and identified it as Bacillus amyloliquefaciens, which produced(7.1±0.5) mg/L of MK-7 with maize meal hydrolysate as carbon source. Then, an MK-7 highproducing strain B. amyloliquefaciens H.β.D.R.-5 with resistance to 1-hydroxy-2-naphthoic acid, β-2-thienylalanine, and diphenylamine was obtained from the mutation of the strain Y-2 using an ARTP mutation breeding system. Using strain H.β.D.R.-5, efficient production of MK-7 was achieved((30.2±2.7) mg/L). In addition, the effects of nitrogen sources, prenyl alcohols, and MgSO_4 on MK-7 production were investigated, suggesting that soymeal extract combined with yeast extract, isopentenol, and MgSO4 was beneficial. Under the optimized condition, the MK-7 production and biomass-specific yield reached(61.3±5.2) mg/L and 2.59 mg/L per OD600 unit respectively in a 7-L fermenter. These results demonstrated that strain H.β.D.R.-5 has the capacity to produce MK-7 from maize meal hydrolysate, which could reduce the substrate cost.
基金Supported by Basic Science Research Program through the National Research Foundation of Korea(NRF)funded by the Ministry of Education,Science and Technology No.NRF-2015R1C1A1A02037267Korea Health Technology R&D Project through the Korea Health Industry Development Institute,funded by the Ministry of Health and Welfare,South Korea,No.HI14C0955
文摘AIM To report naturally occurring mutations in the reverse transcriptase region(RT) of hepatitis B virus(HBV) polymerase from treatment na?ve Korean chronic patients infected with genotype C2.METHODS Here, full-length HBV reverse transcriptase RT sequences were amplified and sequenced from 131 treatment na?ve Korean patients chronically infected with hepatitis B genotype C2. The patients had two distinct clinical statuses: 59 patients with chronic hepatitis(CH) and 72 patients with hepatocellular carcinoma(HCC). The deduced amino acids(AAs) at42 previously reported potential nucleos(t)ide analog resistance(NAr) mutation positions in the RT region were analyzed. RESULTS Potential NAr mutations involving 24 positions were found in 79 of the 131 patients(60.3%). Notably, AA substitutions at 2 positions(rt184 and rt204) involved in primary drug resistance and at 2 positions(rt80 and rt180) that functioned as secondary/compensatory mutations were detected in 10 patients(1 CH patient and 9 HCC patients) and 7 patients(1 CH and 6 HCC patients), respectively. The overall mutation frequencies in the HCC patients(3.17%, 96/3024 mutations) were significantly higher than the frequencies in the CH patients(2.09%, 52/2478 mutations)(P = 0.003). In addition, a total of 3 NAr positions, rt80, rt139 and rt204 were found to be significantly related to HCC from treatment na?ve Korean patients. CONCLUSION Our data showed that naturally occurring NAr mutations in South Korea might contribute to liver disease progression(particularly HCC generation) in chronic patients with genotype C2 infections.
基金supported by funds provided by the Georgia Agricultural Commodity Commission for Peanuts,the National Peanut Board and the Peanut Foundation
文摘Low genetic diversity makes peanut (Arachis hypogaea L.) very vulnerable to plant pathogens, causing severe yield loss and reduced seed quality. Several hundred partial genomic DNA sequences as nucleotide-binding-site leucine-rich repeat (NBS-LRR) resistance genes (R) have been identified, but a small portion with expressed transcripts has been found. We aimed to identify resistance gene analogs (RGAs) from peanut expressed sequence tags (ESTs) and to develop polymorphic markers. The protein sequences of 54 known R genes were used to identify homologs from peanut ESTs from public databases. A total of 1,053 ESTs corresponding to six different classes of known R genes were recovered, and assembled 156 contigs and 229 singletons as peanut-expressed RGAs. There were 69 that encoded for NBS-LRR proteins, 191 that encoded for protein kinases, 82 that encoded for LRR-PK/transmembrane proteins, 28 that encoded for Toxin reductases, 11 that encoded for LRR-domain containing proteins and four that encoded for TM-domain containing proteins. Twenty-eight simple sequence repeats (SSRs) were identified from 25 peanut expressed RGAs. One SSR polymorphic marker (RGA121) was identified. Two polymerase chain reaction-based markers (Ahsw-1 and Ahsw-2) developed from RGA013 were homologous to the Tomato Spotted Wilt Virus (TSWV) resistance gene. All three markers were mapped on the same linkage group AhIV. These expressed RGAs are the source for RGA-tagged marker development and identification of peanut resistance genes.