Mutator transposable element (Mu) has been used as an effective tool to clone maize (Zea mays L.) genes. One opaque endosperm mutant (miol6) was identified in a pool of Mu inserted mutants. A modified method, te...Mutator transposable element (Mu) has been used as an effective tool to clone maize (Zea mays L.) genes. One opaque endosperm mutant (miol6) was identified in a pool of Mu inserted mutants. A modified method, termed the double selected amplification of insertion flanking fragments (DSAIFF), was employed to isolate the Mu flanking fragments (MFFs) of miol6. The target site duplications (TSDs) isolated from the Msp I and Mse I digested MFFs had a same 9-bp sequence and were confirmed to be the flanking sequence of one identically inserted gene. Co-segregation analysis suggested that the MFFs were associated with the mutant opaque endosperm, and miol6 was mapped in silico onto the physical position ranged from 229 965 021 to 229 965 409 bp of the maize chromosome 4.09 bin. The full-length cDNA of the wild-type gene was obtained by an RT-PCR primer-scanning technique, and Mio16 was found to putatively encode a homolog of the Arabidopsis MAP3K delta-1 protein kinase. RT-PCR result the mRNA expression of miol6 region anchored by primers Mu20 and af276 was not interrupted by Mu insertion. Further researches will be done to elucidate how the expression of miol6 is alternated by Mu insertion.展开更多
Transposable elements have been utilized as mutagens to create mutant libraries for functional genomics. Isolation of genomic segments flanking the insertion Mutator (Mu) is a key step in insertion mutagenesis studi...Transposable elements have been utilized as mutagens to create mutant libraries for functional genomics. Isolation of genomic segments flanking the insertion Mutator (Mu) is a key step in insertion mutagenesis studies. Herein, we adopted a modified AFLP method to identify and isolate Mu-flanking fragments from maize. The method consists of the following steps: 1) double-digestion of genomic DNA with Bgl II/Msp I and ligation of digested fragments to the Bgl II- and Msp I-adaptors; 2) enrichment of a subset of Bgl II/Msp I fragments followed by selective amplification of the Mu-flanking fragments; 3) simultaneous display of AFLP bands derived from the flanking regions for both insert and native Mu transposons; 4) identification and isolation of AFLP bands resulting from Mu insertions by comparing the banding profiles between Mu-induced mutants and their parental lines; and 5) confirmation of flanking fragments related to these Mu insertions. Using this approach, we have isolated flanking fragment(s) resulting from Mu insertion for every Mu-induced mutant, and one such fragment, M196-FF, is found to contain a partial sequence of the DNA topoisomerase I gene Topl. Moreover, the modified AFLP method including all restriction enzymes, adaptors and primers has been optimized in this study. The modified AFLP method has been proved to be simple and efficient in the isolation of Mu-flanking fragments and will find its usefulness in the functional genomics of maize.展开更多
Magnesium alloys have many advantages as lightweight materials for engineering applications,especially in the fields of automotive and aerospace.They undergo extensive cutting or machining while making products out of...Magnesium alloys have many advantages as lightweight materials for engineering applications,especially in the fields of automotive and aerospace.They undergo extensive cutting or machining while making products out of them.Dry cutting,a sustainable machining method,causes more friction and adhesion at the tool-chip interface.One of the promising solutions to this problem is cutting tool surface texturing,which can reduce tool wear and friction in dry cutting and improve machining performance.This paper aims to investigate the impact of dimple textures(made on the flank face of cutting inserts)on tool wear and chip morphology in the dry machining of AZ31B magnesium alloy.The results show that the cutting speed was the most significant factor affecting tool flank wear,followed by feed rate and cutting depth.The tool wear mechanism was examined using scanning electron microscope(SEM)images and energy dispersive X-ray spectroscopy(EDS)analysis reports,which showed that at low cutting speed,the main wear mechanism was abrasion,while at high speed,it was adhesion.The chips are discontinuous at low cutting speeds,while continuous at high cutting speeds.The dimple textured flank face cutting tools facilitate the dry machining of AZ31B magnesium alloy and contribute to ecological benefits.展开更多
The thirty-three 5′ flanking conserved sequences of the known low-molecular-weight subunit (LMW-GS) genes have been divided into eight clusters, which was in agreement with the classification based on the deduced N-t...The thirty-three 5′ flanking conserved sequences of the known low-molecular-weight subunit (LMW-GS) genes have been divided into eight clusters, which was in agreement with the classification based on the deduced N-terminal protein sequences. The DNA polymorphism between the eight clusters was obtained by sequence alignment, and a total of 34 polymorphic positions were observed in the approximately 200 bp regions, among which 18 polymorphic positions were candidate SNPs. Seven cluster-specific primer sets were designed for seven out of eight clusters containing cluster-specific bases, with which the genomic DNA of the ditelosomic lines of group 1 chromosomes of a wheat variety ‘Chinese Spring’ was employed to carry out chromosome assignment. The subse-quent cloning and DNA sequencing of PCR fragments validated the sequences specificity of the 5′ flanking conserved sequences between LMW-GS gene groups in different genomes. These results suggested that the coding and 5′ flanking regions of LMW-GS genes are likely to have evolved in a concerted fashion. The seven primer sets developed in this study could be used to isolate the com-plete ORFs of seven groups of LMW-GS genes, respectively, and therefore possess great value for further research in the contributions of a single LMW-GS gene to wheat quality in the complex genetic background and the efficient selections of quality-related components in breeding programs.展开更多
Objective:To develop the current single primer PCR-based genome-walking method with Scenedesmus sp.Methods:The unknown 5’and/or 3’flanking regions for a specific conserved sequence were optimized and the current sin...Objective:To develop the current single primer PCR-based genome-walking method with Scenedesmus sp.Methods:The unknown 5’and/or 3’flanking regions for a specific conserved sequence were optimized and the current single primer PCR-based genome-walking method were developed.Alignment was between the related species of microalga and Scenedesmus sp.For 18S rDNA,we selected the species Scenedesmus sp.,Chlorella sp.,and Chlamydomonas sp.For the rbcL gene from the chloroplast genome,alignment was done between Scenedesmus sp.,and Chlamydomonas sp.Results:Obtaining a small conserved sequence for any gene family is something that can be achieved quite easily.However,identifying the whole gene is often difficult.After investigating and testing,some of the current protocols using to get the unknown 5’and/or 3’flanking regions for a specific conserved sequence,we developed the current single primer PCR-based genome-walking method.We performed two consecutive PCR reactions;band extraction and the PCR product were sequenced.We got our results by testing the method on three genes from the total DNA of Scenedesmus sp.;two genes had a fully known sequence in gene bank(18S rDNA and rbcL),but the third one has not yet been identified(rbcS).We designed our primers based on the alignment between the related species and to each other.We also tested two different DNA polymerases Ex Taq and TLA polymerase.Conclusions:Results from our study suggest that Ex Taq is the most suitable polymerase for the current protocol.展开更多
Flanking transmission has significant effects on the overall sound insulation of two adjacent rooms within a residence building. This study investigates the mechanism of the sound flanking transmission by dividing it ...Flanking transmission has significant effects on the overall sound insulation of two adjacent rooms within a residence building. This study investigates the mechanism of the sound flanking transmission by dividing it into several subsystems with the statistical energy analysis method. The sound energy equations of these subsystems are obtained first, and then,the sound transmissions on each flanking path are predicted and the dominant sound transmission path is determined by solving these equations and calculating the total loss factors of the subsystems and coupling loss factors between subsystems. With respect to a masonry building with heavy-weight homogeneous structure, the results show that:(1) the flanking transmission paths instead of the separating wall may become the dominant ones at low frequencies;(2) all sound transmissions on the flanking paths tend to be consistent at medium and high frequencies, so the sound insulation between two adjacent rooms depends on the direct path of the separating wall;(3) heavy-weight separating walls can be used to reduce the frequency range of the flanking transmission.展开更多
On June 282022 Sweden and Finland were invited to join NATO.During a symbolic ceremony at the NATO Madrid Summit,all the central actors gathered in front of the cameras,as if to replicate a“family photo”in mini form...On June 282022 Sweden and Finland were invited to join NATO.During a symbolic ceremony at the NATO Madrid Summit,all the central actors gathered in front of the cameras,as if to replicate a“family photo”in mini format:representatives from Sweden,Finland,and Turkey,as well as Secretary General Stoltenberg.The atmosphere was mixed:tense because of the dramatic backdrop of the Summit,relief because of the speedy process that had taken the process further.It was finally happening:Sweden and Finland would join the Atlantic Pact.But how did they get there?Over a few hectic months,two countries strongly associated with neutrality had moved their policies from emphasizing continuity to altering the very foundations of their security.Not only NATO critics were taken by surprise.Many NATO supporters were astonished by the rapid pace and the turnaround of the Swedish Social Democratic party.The relatively quick turnaround in Stockholm also meant that questions about the future and the consequences of NATO membership were postponed,not least because the Swedish election campaign put a lid on debate about strategic consequences:what journey awaits in the future and what strategic consequences does a future NATO membership have?This article discusses these questions in the belief that the temporal perspectives are connected:Sweden’s historical collaboration with NATO had significance for the country’s partner relationship,and in turn this influenced how Sweden became a member and what challenges the country faces together with its allies on a new northern flank in Europe.展开更多
[Objective] To explore the polymorphism of the 3′ flank region of equine IGF-Ⅰ gene. [Method] The 3′ flank region sequences of IGF-Ⅰ gene were amplified from genomic DNA of 270 horses, which included 4 types of Mo...[Objective] To explore the polymorphism of the 3′ flank region of equine IGF-Ⅰ gene. [Method] The 3′ flank region sequences of IGF-Ⅰ gene were amplified from genomic DNA of 270 horses, which included 4 types of Mongolian horse, Sanhe horse and Thoroughbred, and then analyzed by PCR-SSCP. [Result] Three genotypes (AA, BB and AB) were detected by PCR-SSCP and the distribution of genotypes of all research objects except Xinihe horse and Baerhu horse were in line with the "Hardy-Weinberg Law". [Conclusion] There was a polymorphic locus in the 3′ flank region of IGF-Ⅰ gene, which might affect the equine growth and development mechanism. The study is of important theoretical and practical significance to improve the performance and to develop equine industry.展开更多
Agrobactedum tumefac/ens-mediated transformation (ATMT) is an efficient tool for insertional mutagenesis and is used in a wide variety of plants. This paper reports a promoter trapping method to generate mutants in ...Agrobactedum tumefac/ens-mediated transformation (ATMT) is an efficient tool for insertional mutagenesis and is used in a wide variety of plants. This paper reports a promoter trapping method to generate mutants in the filamentous fungus, Colletotrichum gloeosporioides, by ATMT insertion of a trapping vector (pCAHPH) that carries a promoterless hygromycin phosphotransferase (hph) gone. Transformants were selected on the media containing 200 ~mL hy^omycin B, and screened for pathogenicity-related gene mdtants. Their pathogenicity-related mutants T-DNA flanking sequences were then cloned and analyzed. Hph genes were amplified from mutant genomic DNA but not from wild-type DNA, indicating that the phenotypic alternations of these mutants were the results of T-DNA inser- tion. T-DNA flanking sequences were obtained using modified themud asymmetric interlaced PCR. Two right-sided flanking sequences were highly homologous to proteins from other species.展开更多
Rice production and quality are seriously affected by the lepidopteran pest,striped stem borer(SSB),in Northeast China.In this study,a synthetic cry1 C gene encoding Bacillus thuringiensis(Bt)δ-endotoxin,which is tox...Rice production and quality are seriously affected by the lepidopteran pest,striped stem borer(SSB),in Northeast China.In this study,a synthetic cry1 C gene encoding Bacillus thuringiensis(Bt)δ-endotoxin,which is toxic to lepidopteran pest,was transformed into a japonica rice variety(Jigeng 88)in Northeast China by Agrobacterium-mediated transformation.Through molecular detection and the Basta resistance germination assay,a total of 16 single-copy homozygous transgenic lines were obtained from 126 independent transformants expressing cry1 C.Finally,four cry1 C-transgenic lines(JL16,JL23,JL41,and JL42)were selected by evaluation of the Cry1 C protein level,insect-resistance and agronomic traits.The cry1 C-transgenic lines had higher resistance to SSB and higher yield compared with non-transgenic(NT)control plants.T-DNA flanking sequence analysis of the transgenic line JL42 showed that the cry1 C gene was inserted into the intergenic region of chromosome 11,indicating that its insertion may not interfere with the genes near insertion site.In summary,this study developed four cry1 C-transgenic japonica rice lines with high insect resistance and high yield.They can be used as insect-resistant germplasm materials to overcome the problem of rice yield reduction caused by SSB and reduce the use of pesticides in Northeast China.展开更多
TaqMan quantitative PCR technique was used to detect the copies of exogenous CaMV35S flanks sequence in transgenic soybean. With soybean lectin as the endogenous reference gene, and gene complex DNA in non-GMO soybean...TaqMan quantitative PCR technique was used to detect the copies of exogenous CaMV35S flanks sequence in transgenic soybean. With soybean lectin as the endogenous reference gene, and gene complex DNA in non-GMO soybeans as the endogenous reference standard, the gradient dilution method was used to separately calculate Ct value of endogenous reference gene and plasmid DNA and correlation standard curve equation of logarithm of copies, and then to calculate the copies of samples through substituting thus-obtained Ct into the standard curve equation. The standard curve equation of endogenous reference gene was y =–3.422x+35.201, R2=0.998; the standard curve equation of exogenous gene was y =–3.495x+35.303, R2=0.999. The sample copies was got by putting Ct value into the standard curve equation, and it was the ratio of exogenous gene and reference gene. We found that CaMV35S gene in transgenic soy was single copy.展开更多
Even though the nitrate assimilation operon has been extensively studied in Phormidium laminosum, some aspects still remain unclear. The genetic manipulation of this cyanobacterium is problematic that hinders the eluc...Even though the nitrate assimilation operon has been extensively studied in Phormidium laminosum, some aspects still remain unclear. The genetic manipulation of this cyanobacterium is problematic that hinders the elucidation of further aspects of nitrogen metabolism. To circumvent this, Thermosynechococcus elongatus BP-1 was selected as a surrogate host and its nirA gene was substituted by the homologous gene of P. laminosum. This process, based on Long Flanking Homology Polymerase Chain Reaction and the natural competence of T. elongatus BP-1, required an intermediate T. elongatus BP-1 ΔnirA::kat mutant, which carries a gene encoding a thermostable kanamycin nucleotidyl transferase in place of nirA_Te. In the presence of nirA_Pl, nirA defective mutants of T. elongatus BP-1 recovered the ability to grow with nitrate as the sole nitrogen source, and showed a phenotype similar to that observed in wild-type cells. The procedure could be useful to substitute other genes from T. elongatus BP-1 with the homologues from P. laminosum in order to study this particular operon. Furthermore, it may be used as a general tool to explore phenotypic changes due to the exchange of a single gene between cyanobacteria.展开更多
Mitochondrial DNA sequences transferred to the nucleus give rise to the so-called nuclear mitochondrial DNA (numt). In the GenBank database, 244 numts have been found in six orders of birds (Anseriformes, Columbifo...Mitochondrial DNA sequences transferred to the nucleus give rise to the so-called nuclear mitochondrial DNA (numt). In the GenBank database, 244 numts have been found in six orders of birds (Anseriformes, Columbiformes, Falconiformes, Charadriiformes, Galliformes and Passeriformes). Sequences alignment (NCBI-BLASTN) was carded out with mitochondrial and corresponding nuclear genome sequences in nine vertebrate species. The sequences with high homology were considered as numts. The number of numts ranged from 15 in chicken to 159 in chimpanzee. The sequences of numts in macaque, chimpanzee, and human spanned 100% of the entire mammalian mitochondrial genome. The reconstructed frequency of the mitochondrial gene transferred to the nucleus demonstrated that the rRNA genes had high frequencies than other mitochondrial genes. Using the RepeatMasker program, the transposable elements were detected in the flanking regions of each numt. The results showed that less than 5% of the flanking sequences were made up of repetitive elements in chicken. The GC content of 5'- and 3'-flanking regions of numts in nine species was less than 44%. The analysis of the flanking sequences provided a valuable understanding for future study on mechanism of mitochondrial gene transfer to the nucleus and the site of numt integration.展开更多
Objective: To explore a common B-and T-cell epitope-based vaccine that can elicit an immune response against encephalitis causing genus Henipaviruses, Hendra virus(He V) and Nipah virus(Ni V). Methods: Membrane protei...Objective: To explore a common B-and T-cell epitope-based vaccine that can elicit an immune response against encephalitis causing genus Henipaviruses, Hendra virus(He V) and Nipah virus(Ni V). Methods: Membrane proteins F, G and M of He V and Ni V were retrieved from the protein database and subjected to different bioinformatics tools to predict antigenic B-cell epitopes. Best B-cell epitopes were then analyzed to predict their T-cell antigenic potentiality. Antigenic B-and T-cell epitopes that shared maximum identity with He V and Ni V were selected. Stability of the selected epitopes was predicted. Finally, the selected epitopes were subjected to molecular docking simulation with HLA-DR to confirm their antigenic potentiality in silico. Results: One epitope from G proteins, one from M proteins and none from F proteins were selected based on their antigenic potentiality. The epitope from the G proteins was stable whereas that from M was unstable. The M-epitope was made stable by adding flanking dipeptides. The 15-mer G-epitope(VDPLRVQWRNNSVIS) showed at least 66% identity with all Ni V and He V G protein sequences, while the 15-mer M-epitope(GKLEFRRNNAIAFKG) with the dipeptide flanking residues showed 73% identity with all Ni V and He V M protein sequences available in the database. Molecular docking simulation with most frequent MHC class-II(MHC II) and class-I(MHC I) molecules showed that these epitopes could bind within HLA binding grooves to elicit an immune response. Conclusions: Data in our present study revealed the notion that the epitopes from G and M proteins might be the target for peptide-based subunit vaccine design against He V and Ni V. However, the biochemical analysis is necessary to experimentally validate the interaction of epitopes individually with the MHC molecules through elucidation of immunity induction.展开更多
Growth hormone gene (GH) ofRhinopithecus roxellanae was amplified by PCR based on the sequences of the reported mammalian growth hormone gene for the first time. The amplified fragment was about 1.8 kb. It was cloned ...Growth hormone gene (GH) ofRhinopithecus roxellanae was amplified by PCR based on the sequences of the reported mammalian growth hormone gene for the first time. The amplified fragment was about 1.8 kb. It was cloned and its upper stream was sequenced. This sequencing region consists of a 5′flanking regulatory region, exon I and part of exon II, intron I of growth hormone gene. Comparing the corresponding sequences of growth hormone gene betweenRhinopithecus roxellanae and the porcine, we concluded that the homology reached 81% in the region, and there was high conservation in the 5′flanking sequence. The kinds of amino acids of exon I and exon II for about 90% were the same to those in pig. Many mutations occurred in the degenerate site of the triplet code. In the nucleotides of intron I, there were only 72% homologies with those in pig. It means that introns and 3′flanking sequence maybe play an important part in growth hormone gene regulation of the different animals.展开更多
The scope of this study is to investigate flanking noise transmission through joints between prefabricated concrete elements in Hellenic school buildings;such joints apparently are non-existent in the wholesome struct...The scope of this study is to investigate flanking noise transmission through joints between prefabricated concrete elements in Hellenic school buildings;such joints apparently are non-existent in the wholesome structure of ordinary concrete buildings.Sound insulation measurements were carried out in two prefabricated concrete buildings of the cell type;the latter involves demountable/reusable concrete elements(the cells);in this case,apparently,flanking noise transmission at joints,may be even more pronounced.A sample of seventeen fa?ade and interior partitions of classrooms was tested.Sound insulation was also predicted based on classical theory.Analysis of the measured data confirms,by and large,the satisfactory sound insulation performance of the test partitions,with the exception of composite partitions which involve door and window openings.The latter were identified to be a major source of sound insulation deterioration.The importance of meticulous sealing of joints is demonstrated.展开更多
In this study, the event-specific real-time fluorescence quantitative PCR method established by Siehuan Academy of Agricultural Sciences was employed to detect the content of flanking fragment (specific fragment) in...In this study, the event-specific real-time fluorescence quantitative PCR method established by Siehuan Academy of Agricultural Sciences was employed to detect the content of flanking fragment (specific fragment) in samples containing 2% genetically modified maize event NK603. The uncertainty of detection results was evaluated based on various uncertainty sources, such as PCR amplification system, data analysis and micropipette. The results showed that A-type uncertainty ( uA ), B-type uncertainty ( uB ), combined standard uncertainty ( uC ) and expanded uncertainty ( U95 ) were 0. 000 8,0.1301,0. 001 and 0. 002, respectively; the final detection result was 1.9% ±0.002. Thus, the main uncertainty in detecting flanking fragment of genetically modified maize event NK603 with realtime fluorescence quantitative PCR method was derived from the random effect in the experimental process.展开更多
[ Objective ] The aim of this study was to develop a quantitative PCR detection method for genetically modified maize event NK603, so as to provide ba- sis for quantitative analysis of event NK603. [ Methods ] A quant...[ Objective ] The aim of this study was to develop a quantitative PCR detection method for genetically modified maize event NK603, so as to provide ba- sis for quantitative analysis of event NK603. [ Methods ] A quantitative PCR detection method for genetically modified maize event NK603 was developed using primers and Taqman probe designed according to the flanking sequence of event NK603, which was then adopted to detect the samples containing 2% NK603 stand- ard (with uncertain quantity of 10% ). [ Results ] The slope of standard curve ranged between -3.6 and -3.1, and the correlation coefficient was higher than 0. 99. The amplification efficiency of this method reached 100.2%, fallen between 90% and 110%. The detected quantity of the experimental sample was 1.9%, closer to the true quantity (2%). [ Conclusion] This quantitative PCR detection method for genetically modified maize event NK603 is very precise and can be a- dopted in routine testing analysis.展开更多
Short tool life and rapid tool wear in micromachining of hard-to-machine materials remain a barrier to the process being economically viable. In this study, standard procedures and conditions set by the ISO for tool l...Short tool life and rapid tool wear in micromachining of hard-to-machine materials remain a barrier to the process being economically viable. In this study, standard procedures and conditions set by the ISO for tool life testing in milling were used to analyze the wear of tungsten carbide micro-end-milling tools through slot milling conducted on titanium alloy Ti-6 Al-4 V. Tool wear was characterized by flank wear rate,cutting-edge radius change, and tool volumetric change. The effect of machining parameters, such as cutting speed and feedrate, on tool wear was investigated with reference to surface roughness and geometric accuracy of the finished workpiece. Experimental data indicate different modes of tool wear throughout machining, where nonuniform flank wear and abrasive wear are the dominant wear modes. High cutting speed and low feedrate can reduce the tool wear rate and improve the tool life during micromachining.However, the low feedrate enhances the plowing effect on the cutting zone, resulting in reduced surface quality and leading to burr formation and premature tool failure. This study concludes with a proposal of tool rejection criteria for micro-milling of Ti-6 Al-4 V.展开更多
基金supported by the High-Tech R&D Program of China(2006AA10A106)the open funds of the National Key Laboratory of Crop Genetic Improvement and China National Fundamental Fund of Personnel Training (J0730649)
文摘Mutator transposable element (Mu) has been used as an effective tool to clone maize (Zea mays L.) genes. One opaque endosperm mutant (miol6) was identified in a pool of Mu inserted mutants. A modified method, termed the double selected amplification of insertion flanking fragments (DSAIFF), was employed to isolate the Mu flanking fragments (MFFs) of miol6. The target site duplications (TSDs) isolated from the Msp I and Mse I digested MFFs had a same 9-bp sequence and were confirmed to be the flanking sequence of one identically inserted gene. Co-segregation analysis suggested that the MFFs were associated with the mutant opaque endosperm, and miol6 was mapped in silico onto the physical position ranged from 229 965 021 to 229 965 409 bp of the maize chromosome 4.09 bin. The full-length cDNA of the wild-type gene was obtained by an RT-PCR primer-scanning technique, and Mio16 was found to putatively encode a homolog of the Arabidopsis MAP3K delta-1 protein kinase. RT-PCR result the mRNA expression of miol6 region anchored by primers Mu20 and af276 was not interrupted by Mu insertion. Further researches will be done to elucidate how the expression of miol6 is alternated by Mu insertion.
文摘Transposable elements have been utilized as mutagens to create mutant libraries for functional genomics. Isolation of genomic segments flanking the insertion Mutator (Mu) is a key step in insertion mutagenesis studies. Herein, we adopted a modified AFLP method to identify and isolate Mu-flanking fragments from maize. The method consists of the following steps: 1) double-digestion of genomic DNA with Bgl II/Msp I and ligation of digested fragments to the Bgl II- and Msp I-adaptors; 2) enrichment of a subset of Bgl II/Msp I fragments followed by selective amplification of the Mu-flanking fragments; 3) simultaneous display of AFLP bands derived from the flanking regions for both insert and native Mu transposons; 4) identification and isolation of AFLP bands resulting from Mu insertions by comparing the banding profiles between Mu-induced mutants and their parental lines; and 5) confirmation of flanking fragments related to these Mu insertions. Using this approach, we have isolated flanking fragment(s) resulting from Mu insertion for every Mu-induced mutant, and one such fragment, M196-FF, is found to contain a partial sequence of the DNA topoisomerase I gene Topl. Moreover, the modified AFLP method including all restriction enzymes, adaptors and primers has been optimized in this study. The modified AFLP method has been proved to be simple and efficient in the isolation of Mu-flanking fragments and will find its usefulness in the functional genomics of maize.
文摘Magnesium alloys have many advantages as lightweight materials for engineering applications,especially in the fields of automotive and aerospace.They undergo extensive cutting or machining while making products out of them.Dry cutting,a sustainable machining method,causes more friction and adhesion at the tool-chip interface.One of the promising solutions to this problem is cutting tool surface texturing,which can reduce tool wear and friction in dry cutting and improve machining performance.This paper aims to investigate the impact of dimple textures(made on the flank face of cutting inserts)on tool wear and chip morphology in the dry machining of AZ31B magnesium alloy.The results show that the cutting speed was the most significant factor affecting tool flank wear,followed by feed rate and cutting depth.The tool wear mechanism was examined using scanning electron microscope(SEM)images and energy dispersive X-ray spectroscopy(EDS)analysis reports,which showed that at low cutting speed,the main wear mechanism was abrasion,while at high speed,it was adhesion.The chips are discontinuous at low cutting speeds,while continuous at high cutting speeds.The dimple textured flank face cutting tools facilitate the dry machining of AZ31B magnesium alloy and contribute to ecological benefits.
基金supported by the National High Technology Research and Development Program of China(863 Program)(Grant No.2003AA207100)National Natural Science Foundation of China(Grant Nos.30300219&30571163)+1 种基金the Foundation for the Author of National Excellent Doctoral Dissertation of China from Ministry of Education,China(Grant Nos.200357&200458)supported by the Program for Changjiang Scholars and Innovative Research Teams in University of China(Grant No.IRT0453)
文摘The thirty-three 5′ flanking conserved sequences of the known low-molecular-weight subunit (LMW-GS) genes have been divided into eight clusters, which was in agreement with the classification based on the deduced N-terminal protein sequences. The DNA polymorphism between the eight clusters was obtained by sequence alignment, and a total of 34 polymorphic positions were observed in the approximately 200 bp regions, among which 18 polymorphic positions were candidate SNPs. Seven cluster-specific primer sets were designed for seven out of eight clusters containing cluster-specific bases, with which the genomic DNA of the ditelosomic lines of group 1 chromosomes of a wheat variety ‘Chinese Spring’ was employed to carry out chromosome assignment. The subse-quent cloning and DNA sequencing of PCR fragments validated the sequences specificity of the 5′ flanking conserved sequences between LMW-GS gene groups in different genomes. These results suggested that the coding and 5′ flanking regions of LMW-GS genes are likely to have evolved in a concerted fashion. The seven primer sets developed in this study could be used to isolate the com-plete ORFs of seven groups of LMW-GS genes, respectively, and therefore possess great value for further research in the contributions of a single LMW-GS gene to wheat quality in the complex genetic background and the efficient selections of quality-related components in breeding programs.
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(2010-0013600).
文摘Objective:To develop the current single primer PCR-based genome-walking method with Scenedesmus sp.Methods:The unknown 5’and/or 3’flanking regions for a specific conserved sequence were optimized and the current single primer PCR-based genome-walking method were developed.Alignment was between the related species of microalga and Scenedesmus sp.For 18S rDNA,we selected the species Scenedesmus sp.,Chlorella sp.,and Chlamydomonas sp.For the rbcL gene from the chloroplast genome,alignment was done between Scenedesmus sp.,and Chlamydomonas sp.Results:Obtaining a small conserved sequence for any gene family is something that can be achieved quite easily.However,identifying the whole gene is often difficult.After investigating and testing,some of the current protocols using to get the unknown 5’and/or 3’flanking regions for a specific conserved sequence,we developed the current single primer PCR-based genome-walking method.We performed two consecutive PCR reactions;band extraction and the PCR product were sequenced.We got our results by testing the method on three genes from the total DNA of Scenedesmus sp.;two genes had a fully known sequence in gene bank(18S rDNA and rbcL),but the third one has not yet been identified(rbcS).We designed our primers based on the alignment between the related species and to each other.We also tested two different DNA polymerases Ex Taq and TLA polymerase.Conclusions:Results from our study suggest that Ex Taq is the most suitable polymerase for the current protocol.
基金supported by the National Natural Science Foundation of China(51568003)the Natural Science Foundation of Guangxi Province,China(2015GXNSFAA139254,2014GXNSFAA118017)
文摘Flanking transmission has significant effects on the overall sound insulation of two adjacent rooms within a residence building. This study investigates the mechanism of the sound flanking transmission by dividing it into several subsystems with the statistical energy analysis method. The sound energy equations of these subsystems are obtained first, and then,the sound transmissions on each flanking path are predicted and the dominant sound transmission path is determined by solving these equations and calculating the total loss factors of the subsystems and coupling loss factors between subsystems. With respect to a masonry building with heavy-weight homogeneous structure, the results show that:(1) the flanking transmission paths instead of the separating wall may become the dominant ones at low frequencies;(2) all sound transmissions on the flanking paths tend to be consistent at medium and high frequencies, so the sound insulation between two adjacent rooms depends on the direct path of the separating wall;(3) heavy-weight separating walls can be used to reduce the frequency range of the flanking transmission.
文摘On June 282022 Sweden and Finland were invited to join NATO.During a symbolic ceremony at the NATO Madrid Summit,all the central actors gathered in front of the cameras,as if to replicate a“family photo”in mini format:representatives from Sweden,Finland,and Turkey,as well as Secretary General Stoltenberg.The atmosphere was mixed:tense because of the dramatic backdrop of the Summit,relief because of the speedy process that had taken the process further.It was finally happening:Sweden and Finland would join the Atlantic Pact.But how did they get there?Over a few hectic months,two countries strongly associated with neutrality had moved their policies from emphasizing continuity to altering the very foundations of their security.Not only NATO critics were taken by surprise.Many NATO supporters were astonished by the rapid pace and the turnaround of the Swedish Social Democratic party.The relatively quick turnaround in Stockholm also meant that questions about the future and the consequences of NATO membership were postponed,not least because the Swedish election campaign put a lid on debate about strategic consequences:what journey awaits in the future and what strategic consequences does a future NATO membership have?This article discusses these questions in the belief that the temporal perspectives are connected:Sweden’s historical collaboration with NATO had significance for the country’s partner relationship,and in turn this influenced how Sweden became a member and what challenges the country faces together with its allies on a new northern flank in Europe.
文摘[Objective] To explore the polymorphism of the 3′ flank region of equine IGF-Ⅰ gene. [Method] The 3′ flank region sequences of IGF-Ⅰ gene were amplified from genomic DNA of 270 horses, which included 4 types of Mongolian horse, Sanhe horse and Thoroughbred, and then analyzed by PCR-SSCP. [Result] Three genotypes (AA, BB and AB) were detected by PCR-SSCP and the distribution of genotypes of all research objects except Xinihe horse and Baerhu horse were in line with the "Hardy-Weinberg Law". [Conclusion] There was a polymorphic locus in the 3′ flank region of IGF-Ⅰ gene, which might affect the equine growth and development mechanism. The study is of important theoretical and practical significance to improve the performance and to develop equine industry.
基金Supported by the Key Projects in Hainan Province(090141)National Natural Science Fund(31101408)
文摘Agrobactedum tumefac/ens-mediated transformation (ATMT) is an efficient tool for insertional mutagenesis and is used in a wide variety of plants. This paper reports a promoter trapping method to generate mutants in the filamentous fungus, Colletotrichum gloeosporioides, by ATMT insertion of a trapping vector (pCAHPH) that carries a promoterless hygromycin phosphotransferase (hph) gone. Transformants were selected on the media containing 200 ~mL hy^omycin B, and screened for pathogenicity-related gene mdtants. Their pathogenicity-related mutants T-DNA flanking sequences were then cloned and analyzed. Hph genes were amplified from mutant genomic DNA but not from wild-type DNA, indicating that the phenotypic alternations of these mutants were the results of T-DNA inser- tion. T-DNA flanking sequences were obtained using modified themud asymmetric interlaced PCR. Two right-sided flanking sequences were highly homologous to proteins from other species.
基金supported by grants from the Jilin Provincial Agricultural Science and Technology Innovation Project in China(CXGC2021TD014)the National Major Project of Breeding for Genetically Modified Organisms in China(2016ZX08001001-001-007)。
文摘Rice production and quality are seriously affected by the lepidopteran pest,striped stem borer(SSB),in Northeast China.In this study,a synthetic cry1 C gene encoding Bacillus thuringiensis(Bt)δ-endotoxin,which is toxic to lepidopteran pest,was transformed into a japonica rice variety(Jigeng 88)in Northeast China by Agrobacterium-mediated transformation.Through molecular detection and the Basta resistance germination assay,a total of 16 single-copy homozygous transgenic lines were obtained from 126 independent transformants expressing cry1 C.Finally,four cry1 C-transgenic lines(JL16,JL23,JL41,and JL42)were selected by evaluation of the Cry1 C protein level,insect-resistance and agronomic traits.The cry1 C-transgenic lines had higher resistance to SSB and higher yield compared with non-transgenic(NT)control plants.T-DNA flanking sequence analysis of the transgenic line JL42 showed that the cry1 C gene was inserted into the intergenic region of chromosome 11,indicating that its insertion may not interfere with the genes near insertion site.In summary,this study developed four cry1 C-transgenic japonica rice lines with high insect resistance and high yield.They can be used as insect-resistant germplasm materials to overcome the problem of rice yield reduction caused by SSB and reduce the use of pesticides in Northeast China.
基金Supported by the Program of Technology Bureau of Harbin (2010RFQXN101)the Subproject of Transgenic Significant Specific Project (20112X08004-002-002-004)
文摘TaqMan quantitative PCR technique was used to detect the copies of exogenous CaMV35S flanks sequence in transgenic soybean. With soybean lectin as the endogenous reference gene, and gene complex DNA in non-GMO soybeans as the endogenous reference standard, the gradient dilution method was used to separately calculate Ct value of endogenous reference gene and plasmid DNA and correlation standard curve equation of logarithm of copies, and then to calculate the copies of samples through substituting thus-obtained Ct into the standard curve equation. The standard curve equation of endogenous reference gene was y =–3.422x+35.201, R2=0.998; the standard curve equation of exogenous gene was y =–3.495x+35.303, R2=0.999. The sample copies was got by putting Ct value into the standard curve equation, and it was the ratio of exogenous gene and reference gene. We found that CaMV35S gene in transgenic soy was single copy.
文摘Even though the nitrate assimilation operon has been extensively studied in Phormidium laminosum, some aspects still remain unclear. The genetic manipulation of this cyanobacterium is problematic that hinders the elucidation of further aspects of nitrogen metabolism. To circumvent this, Thermosynechococcus elongatus BP-1 was selected as a surrogate host and its nirA gene was substituted by the homologous gene of P. laminosum. This process, based on Long Flanking Homology Polymerase Chain Reaction and the natural competence of T. elongatus BP-1, required an intermediate T. elongatus BP-1 ΔnirA::kat mutant, which carries a gene encoding a thermostable kanamycin nucleotidyl transferase in place of nirA_Te. In the presence of nirA_Pl, nirA defective mutants of T. elongatus BP-1 recovered the ability to grow with nitrate as the sole nitrogen source, and showed a phenotype similar to that observed in wild-type cells. The procedure could be useful to substitute other genes from T. elongatus BP-1 with the homologues from P. laminosum in order to study this particular operon. Furthermore, it may be used as a general tool to explore phenotypic changes due to the exchange of a single gene between cyanobacteria.
基金the National Natural Science Foundation of China (No. 30470936)
文摘Mitochondrial DNA sequences transferred to the nucleus give rise to the so-called nuclear mitochondrial DNA (numt). In the GenBank database, 244 numts have been found in six orders of birds (Anseriformes, Columbiformes, Falconiformes, Charadriiformes, Galliformes and Passeriformes). Sequences alignment (NCBI-BLASTN) was carded out with mitochondrial and corresponding nuclear genome sequences in nine vertebrate species. The sequences with high homology were considered as numts. The number of numts ranged from 15 in chicken to 159 in chimpanzee. The sequences of numts in macaque, chimpanzee, and human spanned 100% of the entire mammalian mitochondrial genome. The reconstructed frequency of the mitochondrial gene transferred to the nucleus demonstrated that the rRNA genes had high frequencies than other mitochondrial genes. Using the RepeatMasker program, the transposable elements were detected in the flanking regions of each numt. The results showed that less than 5% of the flanking sequences were made up of repetitive elements in chicken. The GC content of 5'- and 3'-flanking regions of numts in nine species was less than 44%. The analysis of the flanking sequences provided a valuable understanding for future study on mechanism of mitochondrial gene transfer to the nucleus and the site of numt integration.
文摘Objective: To explore a common B-and T-cell epitope-based vaccine that can elicit an immune response against encephalitis causing genus Henipaviruses, Hendra virus(He V) and Nipah virus(Ni V). Methods: Membrane proteins F, G and M of He V and Ni V were retrieved from the protein database and subjected to different bioinformatics tools to predict antigenic B-cell epitopes. Best B-cell epitopes were then analyzed to predict their T-cell antigenic potentiality. Antigenic B-and T-cell epitopes that shared maximum identity with He V and Ni V were selected. Stability of the selected epitopes was predicted. Finally, the selected epitopes were subjected to molecular docking simulation with HLA-DR to confirm their antigenic potentiality in silico. Results: One epitope from G proteins, one from M proteins and none from F proteins were selected based on their antigenic potentiality. The epitope from the G proteins was stable whereas that from M was unstable. The M-epitope was made stable by adding flanking dipeptides. The 15-mer G-epitope(VDPLRVQWRNNSVIS) showed at least 66% identity with all Ni V and He V G protein sequences, while the 15-mer M-epitope(GKLEFRRNNAIAFKG) with the dipeptide flanking residues showed 73% identity with all Ni V and He V M protein sequences available in the database. Molecular docking simulation with most frequent MHC class-II(MHC II) and class-I(MHC I) molecules showed that these epitopes could bind within HLA binding grooves to elicit an immune response. Conclusions: Data in our present study revealed the notion that the epitopes from G and M proteins might be the target for peptide-based subunit vaccine design against He V and Ni V. However, the biochemical analysis is necessary to experimentally validate the interaction of epitopes individually with the MHC molecules through elucidation of immunity induction.
文摘Growth hormone gene (GH) ofRhinopithecus roxellanae was amplified by PCR based on the sequences of the reported mammalian growth hormone gene for the first time. The amplified fragment was about 1.8 kb. It was cloned and its upper stream was sequenced. This sequencing region consists of a 5′flanking regulatory region, exon I and part of exon II, intron I of growth hormone gene. Comparing the corresponding sequences of growth hormone gene betweenRhinopithecus roxellanae and the porcine, we concluded that the homology reached 81% in the region, and there was high conservation in the 5′flanking sequence. The kinds of amino acids of exon I and exon II for about 90% were the same to those in pig. Many mutations occurred in the degenerate site of the triplet code. In the nucleotides of intron I, there were only 72% homologies with those in pig. It means that introns and 3′flanking sequence maybe play an important part in growth hormone gene regulation of the different animals.
文摘The scope of this study is to investigate flanking noise transmission through joints between prefabricated concrete elements in Hellenic school buildings;such joints apparently are non-existent in the wholesome structure of ordinary concrete buildings.Sound insulation measurements were carried out in two prefabricated concrete buildings of the cell type;the latter involves demountable/reusable concrete elements(the cells);in this case,apparently,flanking noise transmission at joints,may be even more pronounced.A sample of seventeen fa?ade and interior partitions of classrooms was tested.Sound insulation was also predicted based on classical theory.Analysis of the measured data confirms,by and large,the satisfactory sound insulation performance of the test partitions,with the exception of composite partitions which involve door and window openings.The latter were identified to be a major source of sound insulation deterioration.The importance of meticulous sealing of joints is demonstrated.
基金Supported by Youth Fund of Sichuan Academy of Agricultural Sciences(2009QNJJ-037)
文摘In this study, the event-specific real-time fluorescence quantitative PCR method established by Siehuan Academy of Agricultural Sciences was employed to detect the content of flanking fragment (specific fragment) in samples containing 2% genetically modified maize event NK603. The uncertainty of detection results was evaluated based on various uncertainty sources, such as PCR amplification system, data analysis and micropipette. The results showed that A-type uncertainty ( uA ), B-type uncertainty ( uB ), combined standard uncertainty ( uC ) and expanded uncertainty ( U95 ) were 0. 000 8,0.1301,0. 001 and 0. 002, respectively; the final detection result was 1.9% ±0.002. Thus, the main uncertainty in detecting flanking fragment of genetically modified maize event NK603 with realtime fluorescence quantitative PCR method was derived from the random effect in the experimental process.
基金Supported by Youth Science and Technology Program of Sichuan Academy of Agricultural Science(2009QNJJ-037)Program for Monitoring Invasive Species of Ministry of Agriculture
文摘[ Objective ] The aim of this study was to develop a quantitative PCR detection method for genetically modified maize event NK603, so as to provide ba- sis for quantitative analysis of event NK603. [ Methods ] A quantitative PCR detection method for genetically modified maize event NK603 was developed using primers and Taqman probe designed according to the flanking sequence of event NK603, which was then adopted to detect the samples containing 2% NK603 stand- ard (with uncertain quantity of 10% ). [ Results ] The slope of standard curve ranged between -3.6 and -3.1, and the correlation coefficient was higher than 0. 99. The amplification efficiency of this method reached 100.2%, fallen between 90% and 110%. The detected quantity of the experimental sample was 1.9%, closer to the true quantity (2%). [ Conclusion] This quantitative PCR detection method for genetically modified maize event NK603 is very precise and can be a- dopted in routine testing analysis.
基金the Engineering and Physical Sciences Research Council (EP/M020657/1) for the support for this work
文摘Short tool life and rapid tool wear in micromachining of hard-to-machine materials remain a barrier to the process being economically viable. In this study, standard procedures and conditions set by the ISO for tool life testing in milling were used to analyze the wear of tungsten carbide micro-end-milling tools through slot milling conducted on titanium alloy Ti-6 Al-4 V. Tool wear was characterized by flank wear rate,cutting-edge radius change, and tool volumetric change. The effect of machining parameters, such as cutting speed and feedrate, on tool wear was investigated with reference to surface roughness and geometric accuracy of the finished workpiece. Experimental data indicate different modes of tool wear throughout machining, where nonuniform flank wear and abrasive wear are the dominant wear modes. High cutting speed and low feedrate can reduce the tool wear rate and improve the tool life during micromachining.However, the low feedrate enhances the plowing effect on the cutting zone, resulting in reduced surface quality and leading to burr formation and premature tool failure. This study concludes with a proposal of tool rejection criteria for micro-milling of Ti-6 Al-4 V.