Isolation of the Flanking Sequences Adjacent to Transgenic T-DNA in Brassica napus Genome by an Improved Inverse PCR Method

[Objective] The research aimed to isolate flanking sequences adjacent to the transgenic T-DNA in Brassica napus by an improved inverse PCR method.[Method] Using single clone of transgenic FS4 in Brassica napus as the research materials,total DNA was extracted from transgenic Brassica napus by using modified CTAB method.After enzyme digestion and purification,self-joining was made.Two circles of nested PCR and the sequence alignment were carried out.[Result] A fragement with the size of 4.0 kb was amplified ...

Isolating the Mutator Transposable Element Insertional Mutant Gene mio16 of Maize Using Double SelectedAmplification of Insertion Flanking Fragments (DSAIFF)

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.

Ac/Ds Transposition Activity in Transgenic Rice Population and DNA Flanking Sequence of Ds Insertion Sites

The Agrobacterium mediated transgenic rice ( Oryza saliva L.) population with inserts of maize transposon Activator/Dissociation (Ac/Ds) was investigated. DNA sequences flanking the T-DNA were analyzed with inverse PCR. Results showed that 65.4% of the T-DNA was integrated in different locations of rice genome, and some T-DNA flanking sequences were located on certain chromosomes. A number of T-DNA was found to have inserted into protein coding regions. In order to induce transposition of the inserted Ds elements, 354 crosses of Ac x Ds and Ds x Ac were constructed. The excision frequency of Ds element trans-activated by Ac transposase was 22.7% in the F-2 populations, and the transposition was confirmed with analyses of DNA sequences flanking the Ds elements. In addition to the transposition due to 'cut-paste' mechanism, Ds can replicate itself and integrate into a new locus, and inaccurate excisions were also found. A proportion of DNA segments flanking the Ds elements showed no homologies to sequences published in GenBank, of which two were registered under the accession numbers AF355153 and AF355770. The strategy of using transposon tagging for rice genomics study was discussed.

Analysis of the Flanking Sequence and EventSpecific Detection of Transgenic Line W-4 of Brassica napus

The genetically modified high-oleic rapeseed （Brassica napus L.） line W-4 was obtained by transforming a binary vector which harbored an inverted repeat expression cassette of fad2 gene into the rapeseed cultivar Westar.The transformation was mediated by Agrobacterium.The flanking sequences to both the left and right borders of T-DNA insertion site were amplified by thermal asymmetric interlaced PCR （TAIL-PCR） from the genomic DNA of the transgenic rapeseed line W-4.The flanking sequences to the right border was 290 bp in length and the nucleotide composition was 31.27％ for G＋C content while 68.73％ for A＋T content.The flanking sequence to the left border was 365 bp in length and the G＋C content was 32.6％ and the A＋T content was 67.4％,indicating that the T-DNA was integrated in the A/T-rich region.Further more,sequence alignment analysis showed a deletion of 62 bp including the right border of pCNFIRnos and the integration of the whole left border except a change of G to A.That was to say,the integration of the T-DNA in the transgenic line W-4 not involved in the vector sequences.Based on both flanking sequences as well as the left and right borders of the T-DNA sequences,two pairs of specific primers TLF/TLR and TRF/TRR were designed.Using the primers the event-specific PCR detection method for transgenic rapeseed line W-4 was established.By the PCR,two fragments of 485 and 405 bp were amplified from the W-4 genomic DNA as expected,while no products were amplified from the genomic DNA of other transgenic rapeseed lines and non-transgenic rapeseed line.And by the PCR it is possible to detect the W-4 genomic DNA from a mixed sample of genomic DNA.The limit of the detection for the qualitative PCR assay was 0.1％.The method developed in this work is highly specific,sensitive and suitable for event-specific detection of the transgenic rapeseed line W-4.

Identification and isolation of Mu-flanking fragments from maize

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.

Genomic structure analysis of SNC6, a progesterone-receptor associated protein gene, and cloning and characterization of its 5＇-flanking region

Objective: To analyze the genomic structure of SNC6, a progesterone\|receptor associated protein gene and its regulatory elements in its 5'\|flanking region. Methods: Genomic sequence from GenBank database (accession number: Z98048) covering the whole SNC6 gene was used to analyze the genomic structure of SNC6 and design primers for PCR amplification of its 5'\|flanking region. A 1894 bp fragment of the 5'\|flanking region \{(-1814\} to +75) was cloned by PCR using genomic DNA from a healthy donor peripheral blood lymphocyte as template. This fragment, as well as 3 shorter derivative fragments (1423 bp, 632 bp and 416 bp, which correspond to -1344 to +75, -552 to +75 and -337 to +75 respectively), were subcloned into pGL2 series luciferase reporter vectors. These constructs were introduced into colorectal cancer cell line SW620 for transient expression of reporter gene and luciferase activities were measured. Results: The genomic structure analysis showed there are 12 exons for SNC6 gene, which spans 32017 bp (nt71529 to nt39513 in Z98048 sequence). All transfected SW620 cells with the above 5\|flanking region\|containing constructs showed luciferase activities. The highest luciferase activities were measured in transfected cells with vectors containing 1894 bp fragments, and the lowest luciferase activities were measured in transfected cells with vectors containing 416 bp fragments. Luciferase activities were higher in transfected cells with vectors containing 632 bp fragments than that in transfected cells with vectors containing 1423 bp fragments. Conclusion: The basic transcription\|promoting element (promoter) for SNC6 expression resides between 0 to -337, and two transcription\|enhancing elements (enhancer) resides between -337 to -552 and -1344 to -1814, whereas one transcription\|inhibiting element (silencer) exists between -552 to -1344.

Isolation of T-DNA flanking plant DNA from T-DNAinsertional embryo-lethal mutants of Arabidopsis thaliana by plasmid rescue technique

Three T-DNA insertional embryonic lethal mutants from NASC (The Nottingham Arabidopsis Stock Center)were first checked with their segregation ratio of abortive and normal seeds and the copy number of T-DNA insertion. The N4081 mutant has a segregation ratio of 1:3.04in average and one T-DNA insertion site according to our assay It was therefore chosen for further analysis. To isolate the joint fragment of T-DNA and plan DNA, the plasmid rescue technique waJs used. pEL-7, one of plasmids from left border of T-DNA, which contained pBR322 was selected from ampicillin plate. The T-DNA fragment of pEL-7 was checked by restriction enzyme analysis and Southern Blot. Restriction analysis confirmed the presence of known sites of EcoRI, PstI and PvuII on it.For confirming the presence of flanking plant DNA in this plasmid, pEL-7 DNA was labeled and hybridized with wild type and mutant plant DNA. The Southern Blot indicated the hybridization band in both of them. Furthermore, the junction of T-DNA/plant DNA was subcloned into bluescript SK+ and sequenced by Applied Biosystem 373A Sequencer. The results showed the 822 bp fragment contained a 274 bp sequence, which is 99.6%homolog (273bp/274 bp) to Ti plasmid pTi 15955 DNA.Ten bp of left 25 bp border repeat were also found in the juction of T-DNA and Plant DNA.Taken together, pEL-7 should contain a joint fragment of T-DNA and flanking plant DNA. This plasmid DNA could be used for the isolation of plant gene, which will be helpful to elucidate the relationship between gene function and plant embryo development.

Conceptual design and aerodynamic evaluation of hypersonic airplane with double flanking air inlets

To aim at design requirements of high lift-to-drag ratio as well as high volumetric efficiency of next generation hypersonic airplanes,a body-wing-blending configuration with double flanking air inlets layout is presented.Moreover,a novel forebody design methodology which by rotating and assembling two waverider-based surfaces is firstly introduced in this paper.Some typical configurations are designed and their aerodynamic performances are evaluated by computational fluid dynamics.The results for forebodies analysis show that large volumetric efficiency,high lift-to-drag ratio,and uniformly distributed flowfield at the inlet cross section can be assured simultaneously.Furthermore,results of numerical simulation of four integrated configurations with various leading edge shapes,including three power-law curves and a cosine curve clearly show the advantage of high lift-to-drag ratio.Besides,the high pressure generated by the side wall of the airframe can be partly captured by the reasonably designed wings in the condition of small flight attack angle.Then the order of lift-to-drag ratio of four configurations at 0 degree flight attack angle is completely different from the condition of 4-degree flight attack angle.This result demonstrates that the curve shape of the leading edge is very important for the lift-to-drag ratio of the aircraft,and it should be further optimized under the cruising attack angle in future work.

Analysis and validation of genome-specific DNA variations in 5′flanking conserved sequences of wheat low-molecular-weight glutenin subunit genes

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,; 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 subsequent cloning; 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; 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 complete ORFs of seven groups of LMW-GS genes, respectively,; therefore possess great value for further research in the contributions of a single LMW-GS gene to wheat quality in the complex genetic background; the efficient selections of quality-related components in breeding programs.

Roles of flanking sequences in the binding between unimolecular parallel-stranded G-quadruplexes and ligands

G-quadruplexes attract more and more attention in recent years.Numerous small molecules which can induce or stabilize the formation of G-quadruplexes have been investigated on the purpose of anticancer drug development.As a motif existed in physiological condition,flanking sequences are an important part of G-quadruplexes but the study on the impact of flanking sequences on (G-quadruplex)-ligand binding is rarely reported.In this paper,the effects of flanking sequences on binding affinity between a series of unimolecular parallel-stranded G-quadruplex sequences derived from c-myc oncogene promoter (termed as c-myc G-quadruplexes) and their ligands are discussed in detail.The results showed that the flanking sequences on c-myc G-quadruplexes play key roles in (G-quadruplex)-ligand interaction.When a c-myc G-quadruplex is bound to its ligands,the flanking sequences might form a binding cavity above the terminal G-quartet,which could provide a suitable site for ligands to dock in.Moreover,the bases on flanking sequences could interact with ligand through π-π stacking,and finally form a sandwich-stacking mode (terminal G-quartet,ligand and bases on the flanking sequence).This mode could stabilize the (G-quadruplex)-ligand complex effectively and enhance the binding affinity dramatically.However,flanking sequences are also found to exhibit steric hindrance effect which could impede the (G-quadruplex)-ligand binding.

An easy PCR-based genome-walking method for getting the unknown 5’flanking region of a Scenedesmus sp.

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.

Prediction of sound flanking transmission through two adjacent rooms within a residence building

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.

On dry machining of AZ31B magnesium alloy using textured cutting tool inserts

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.

Cutting Path Planning for Ruled Surface Impellers

At present, most commercial computer-aided manufacturing （CAM） systems are deficient in efficiency and performances on generating tool path during machining impellers. To solve the problem, this article develops a special software to plan cutting path for ruled surface impellers. An approximation algorithm to generate cutting path for machining integral ruled surface impellers is proposed. By fitting sampling data points of an impeller blade into a curve, a model of ruled surface blade of an impeller is built up. Furthermore, by calculating the points where the cutter axis vector intersects the free-form hub surface of an impeller, problems about, for instance, the ambiguity in calculation and machining the wide blade surface with a short flute cutter are solved. Finally, an integral impeller cutting path is planned by way of an integrated cutter location control algorithm. Simulation and machining tests with an impeller are performed on a 5-axis computer numerically controlled （CNC） mill machine, which shows the feasibility of the proposed algorithm.

Polymorphism Analysis of the 3′ Flank Region of Equine IGF-Ⅰ Gene by PCR-SSCP

[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.

Role of gastrin-peptides in Barrett's and colorectal carcinogenesis

Gastrin is the main hormone responsible for the stimulation of gastric acid secretion;in addition,gastrin and its derivatives exert proliferative and antiapoptotic effects on several cell types.Gastrin synthesis and secretion are increased in certain situations,for example,when proton pump inhibitors are used.The impact of sustained hypergastrinemia is currently being investigated.In vitro experiments and animal models have shown that prolonged hypergastrinemia may be related with higher cancer rates;although,this relationship is less clear in human beings.Higher gastrin levels have been shown to cause hyperplasia of several cell types;yet,the risk for developing cancer seems to be the same in normo-and hypergastrinemic patients.Some tumors also produce their own gastrin,which can act in an autocrine manner promoting tumor growth.Certain cancers are extremely dependent on gastrin to proliferate.Initial research focused only on the effects of amidated gastrins,but there has been an interest in intermediates of gastrin in the last few decades.These intermediates aren't biologically inactive;in fact,they may exert greater effects on proliferation and apoptosis than the completely processed forms.In certain gastrin overproduction states,they are the most abundant gastrin peptides secreted.The purpose of this review is to examine the gastrin biosynthesis process and to summarize the results from different studies evaluating the production,levels,and effects of the main forms of gastrin in different overexpression states and their possible relationship with Barrett's and colorectal carcinogenesis.

Generation and Analysis of Pathogenicity-related Gene Mutants of Colletotrichum gloeosporioides Using a Novel Promoter Trapping System

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.

Transgenic japonica rice expressing the cry1C gene is resistant to striped stem borers in Northeast China

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.

Establishment of TaqMan Real-time Quantitative PCR Assay for Foreign Gene Copy Numbers in Transgenic 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.

Substitution of Nitrite Reductase of Thermosynechococcus elongatus BP-1 by the Homologous Gene of Phormidium laminosum

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.