Architectonic principles of polyproline II helix bundle protein domains.

Glycine rich polyproline II helix assemblies are an emerging class of natural domains found in several proteins with different functions and diverse origins. The distinct properties of these domains relative to those composed of α-helices and ß-sheets could make glycine-rich polyproline II helix assemblies a useful building block for protein design. Whereas the high population of polyproline II conformers in disordered state ensembles could facilitate glycine-rich polyproline II helix folding, the architectonic bases of these structures are not well known. Here, we compare and analyze their structures to uncover common features. These protein domains are found to be highly tolerant of distinct flanking sequences. This speaks to the robustness of this fold and strongly suggests that glycine rich polyproline II assemblies could be grafted with other protein domains to engineer new structures and functions. These domains are also well packed with few or no cavities. Moreover, a significant trend towards antiparallel helix configuration is observed in all these domains and could provide stabilizing interactions among macrodipoles. Finally, extensive networks of Cα-H···OC hydrogen bonds are detected in these domains. Despite their diverse evolutionary origins and activities, glycine-rich polyproline II helix assemblies share architectonic features which could help design novel proteins.

T-DNA characterization of genetically modified 3-R-gene late blight-resistant potato events with a novel procedure utilizing the Samplix Xdrop® enrichment technology.

Before the commercialization of genetically modified crops, the events carrying the novel DNA must be thoroughly evaluated for agronomic, nutritional, and molecular characteristics. Over the years, polymerase chain reaction-based methods, Southern blot, and short-read sequencing techniques have been utilized for collecting molecular characterization data. Multiple genomic applications are necessary to determine the insert location, flanking sequence analysis, characterization of the inserted DNA, and determination of any interruption of native genes. These techniques are time-consuming and labor-intensive, making it difficult to characterize multiple events. Current advances in sequencing technologies are enabling whole-genomic sequencing of modified crops to obtain full molecular characterization. However, in polyploids, such as the tetraploid potato, it is a challenge to obtain whole-genomic sequencing coverage that meets the regulatory approval of the genetic modification. Here we describe an alternative to labor-intensive applications with a novel procedure using Samplix Xdrop® enrichment technology and next-generation Nanopore sequencing technology to more efficiently characterize the T-DNA insertions of four genetically modified potato events developed by the Feed the Future Global Biotech Potato Partnership: DIA_MSU_UB015, DIA_MSU_UB255, GRA_MSU_UG234, and GRA_MSU_UG265 (derived from regionally important varieties Diamant and Granola). Using the Xdrop® /Nanopore technique, we obtained a very high sequence read coverage within the T-DNA and junction regions. In three of the four events, we were able to use the data to confirm single T-DNA insertions, identify insert locations, identify flanking sequences, and characterize the inserted T-DNA. We further used the characterization data to identify native gene interruption and confirm the stability of the T-DNA across clonal cycles. These results demonstrate the functionality of using the Xdrop® /Nanopore technique for T-DNA characterization. This research will contribute to meeting regulatory safety and regulatory approval requirements for commercialization with small shareholder farmers in target countries within our partnership.

Prediction and analysis of cis-regulatory elements in Dorsal and Ventral patterning genes of Tribolium castaneum and its comparison with Drosophila melanogaster.

Insect embryonic development and morphology are characterized by their anterior-posterior and dorsal-ventral (DV) patterning. In Drosophila embryos, DV patterning is mediated by a dorsal protein gradient which activates twist and snail proteins, the important regulators of DV patterning. To activate or repress gene expression, some regulatory proteins bind in clusters to their target gene at sites known as cis-regulatory elements or enhancers. To understand how variations in gene expression in different lineages might lead to different phenotypes, it is necessary to understand enhancers and their evolution. Drosophila melanogaster has been widely studied to understand the interactions between transcription factors and the transcription factor binding sites. Tribolium castaneum is an upcoming model animal which is catching the interest of biologists and the research on the enhancer mechanisms in the insect's axes patterning is still in infancy. Therefore, the current study was designed to compare the enhancers of DV patterning in the two insect species. The sequences of ten proteins involved in DV patterning of D. melanogaster were obtained from Flybase. The protein sequences of T. castaneum orthologous to those obtained from D. melanogaster were acquired from NCBI BLAST, and these were then converted to DNA sequences which were modified by adding 20 kb sequences both upstream and downstream to the gene. These modified sequences were used for further analysis. Bioinformatics tools (Cluster-Buster and MCAST) were used to search for clusters of binding sites (enhancers) in the modified DV genes. The results obtained showed that the transcription factors in Drosophila melanogaster and Tribolium castaneum are nearly identical; however, the number of binding sites varies between the two species, indicating transcription factor binding site evolution, as predicted by two different computational tools. It was observed that dorsal, twist, snail, zelda, and Supressor of Hairless are the transcription factors responsible for the regulation of DV patterning in the two insect species.

DNA conformational dynamics in the context-dependent non-CG CHH methylation by plant methyltransferase DRM2.

DNA methylation provides an important epigenetic mechanism that critically regulates gene expression, genome imprinting, and retrotransposon silencing. In plants, DNA methylation is prevalent not only in a CG dinucleotide context but also in non-CG contexts, namely CHG and CHH (H = C, T, or A) methylation. It has been established that plant non-CG DNA methylation is highly context dependent, with the +1- and +2-flanking sequences enriched with A/T nucleotides. How DNA sequence, conformation, and dynamics influence non-CG methylation remains elusive. Here, we report structural and biochemical characterizations of the intrinsic substrate preference of DOMAINS REARRANGED METHYLTRANSFERASE 2 (DRM2), a plant DNA methyltransferase responsible for establishing all cytosine methylation and maintaining CHH methylation. Among nine CHH motifs, the DRM2 methyltransferase (MTase) domain shows marked substrate preference toward CWW (W = A or T) motifs, correlating well with their relative abundance in planta. Furthermore, we report the crystal structure of DRM2 MTase in complex with a DNA duplex containing a flexible TpA base step at the +1/+2-flanking sites of the target nucleotide. Comparative structural analysis of the DRM2-DNA complexes provides a mechanism by which flanking nucleotide composition impacts DRM2-mediated DNA methylation. Furthermore, the flexibility of the TpA step gives rise to two alternative DNA conformations, resulting in different interactions with DRM2 and consequently temperature-dependent shift of the substrate preference of DRM2. Together, this study provides insights into how the interplay between the conformational dynamics of DNA and temperature as an environmental factor contributes to the context-dependent CHH methylation by DRM2.

Application of transposon insertion site sequencing method in the exploration of gene function in microalgae.

Microalgae are a large group of organisms that can produce various useful substances through photosynthesis. Microalgae need to be genetically modified at the molecular level to become "Chassis Cells" for food, medicine, energy, and environmental protection and, consequently, obtain benefits from microalgae resources. Insertional mutagenesis of microalgae using transposons is a practical possibility for understanding the function of microalgae genes. Theoretical and technical support is provided in this manuscript for applying transposons to microalgae gene function by summarizing the sequencing method of transposon insertion sites.

Effect of polymorphisms in the 5'-flanking sequence of MC1R on feather color in Taihang chickens.

MC1R plays an important role in the regulation of the formation, transfer, and deposition of melanin in animals and is important for determining coat color. Many studies have reported on single nucleotide polymorphisms (SNPs) in the coding sequence of MC1R. However, few studies have investigated the polymorphisms in the 5'-flanking sequence of MC1R. In this study, we sequenced 2000 bp of the 5'-flanking sequence of MC1R in 300 Taihang chickens with brown feathers (MTH) and 300 Taihang chickens with black feathers (HTH). The sequencing results showed that 4 SNPs (MC1R g.18838722 G > C, g.18838624 T > C, g.18838694 G > A, and g.18838624 C > T) were located in the 5'-flanking sequence of MC1R between the MTH and HTH groups. Association analysis showed that there was a significant correlation between the 4 SNPs and feather color in Taihang chickens. The correlation between MC1R g.18838624 T >C and feather color of Taihang chicken was 100%, of which the CC (E1) genotype is MTH and the TT (E2) genotype is HTH. Furthermore, there was a significant correlation between MC1R g.18838624 T > C and egg production at 302 d. E1 (184.14 ± 0.674) was significantly higher than that in E2 (181.75 ± 0.577) (P < 0.05). Luciferase reporter assays were used to detect the transcriptional activity of MC1R with different SNP genotypes. The results showed that the luciferase activity of E2 was significantly higher than that of E1 (P < 0.05). In addition, transcription factor-binding site predictions showed that E2 creates a new binding site for ZEB1. RT‒qPCR results revealed that the expression of MC1R in E2 was significantly lower than that in E1 (P < 0.05), and the expression of ZEB1 in E2 was significantly higher than that in E1 (P < 0.05). Overexpression and shRNA experiments demonstrated that ZEB1 regulates the expression of MC1R in DF1 cells. ZEB1 has a negative regulatory effect on the transcriptional activity of MC1R; it inhibits the expression of MC1R and affects the feather color of Taihang chickens. This study provides new insight into the molecular mechanism of feather color formation and the transcriptional regulation of MC1R in Taihang chickens.

Upstream Flanking Sequence Assists Folding of an RNA Thermometer.

Many heat shock genes in bacteria are regulated through a class of temperature-sensitive stem-loop (SL) RNAs called RNA thermometers (RNATs). One of the most widely studied RNATs is the Repression Of heat Shock Expression (ROSE) element associated with expression of heat shock proteins. Located in the 5'UTR, the RNAT contains one to three auxiliary hairpins upstream of it. Herein, we address roles of these upstream SLs in the folding and function of an RNAT. Bradyrhizobium japonicum is a nitrogen-fixing bacterium that experiences a wide range of temperatures in the soil and contains ROSE elements, each having multiple upstream SLs. The 5'UTR of the messenger (mRNA) for heat shock protein A (hspA) in B. japonicum has an intricate secondary structure containing three SLs upstream of the RNAT SL. While structure-function studies of the hspA RNAT itself have been reported, it has been unclear if these auxiliary SLs contribute to the temperature-sensing function of the ROSE elements. Herein, we show that the full length (FL) sequence has several melting transitions indicating that the ROSE element unfolds in a non-two-state manner. The upstream SLs are more stable than the RNAT itself, and a variant with disrupted base pairing in the SL immediately upstream of the RNAT has little influence on the melting of the RNAT. On the basis of these results and modeling of the co-transcriptional folding of the ROSE element, we propose that the upstream SLs function to stabilize the transcript and aid proper folding and dynamics of the RNAT.

Progress on methods for acquiring flanking genomic sequence.

Flanking genomic sequences refer to the DNA sequences flanking specific sites of known sequences in chromosome, which contain information such as candidate genes, transcriptional regulation, chromosome structure, and biosafety, and play an important role in genomics research. Flanking sequence acquisition technologies are mainly used in the cloning of regulatory sequences such as promoters and enhancers, identification of T-DNA or transposon insertion sites, chromosome walking, genome-wide gap filling, etc. It is an important means of structural genomics research and functional genomics research. It is applied in the identification of transgenic plants and animals and their safety management. With the development of molecular biology, many methods for obtaining flanking sequences have been established, including plasmid rescue, inverse PCR, ligation-mediated PCR, semi-random primer PCR, whole-genome resequencing etc. In this review, we summarize and compared different methods for acquiring flanking genomic sequence. The principles and research progress of each approach are discussed.

Efficient identification of genomic insertions and flanking regions through whole-genome sequencing in three transgenic soybean events.

Genomic insertions and flanking regions of transgenes in host genomes constitute a critical component of precise molecular characterization and event-specific detection, which are required in the development and assessment for regulatory approval of genetically modified (GM) crops. Previously, we reported three transgenic soybean events harboring the inverted repeats of the soybean mosaic virus NIb (nuclear inclusion b) gene, exhibiting significantly enhanced resistance to multiple Potyvirus strains. To facilitate safety assessment and event-specific detection, we identified the transgene insertion sites and flanking sequences of the events L120, L122, and L123 using whole-genome sequencing. More than 14.48 Gb sequence data (13 × coverage) were generated using the Illumina HiSeq Xten platform for each event. The sequence reads corresponding to boundaries of inserted T-DNA, and associated native flanking sequences were identified by bioinformatic comparison with the soybean reference genome (Wm82.a2.v1) and the transformation vector sequence. The results indicated that two T-DNA insertions occurred in L120, on Chr07 and Chr13, while L122 and L123 showed single insertions, on Chr02 and Chr06, respectively. Based on the flanking sequences of the inserted T-DNA, the event-specific detection for each event was established using specific PCR primers, and PCR amplification followed by sequencing of PCR products further confirmed the putative insertion loci and flanking regions in the transgenic lines. Our results demonstrate the efficacy and robustness of whole-genome sequencing in identifying the genomic insertions and flanking regions in GM crops. Moreover, the characterization of insertion loci and the establishment of event-specific detection will facilitate the application and development of broad-spectrum virus-resistant transgenic soybean cultivars.

Whole-Genome Sequencing: An Effective Strategy for Insertion Information Analysis of Foreign Genes in Transgenic Plants.

Molecular characterization is a key step in the risk assessment of genetically modified organisms (GMOs) for regulatory approval. Herein, we describe a method for analyzing copy number, insertion loci, and flanking sequences through whole-genome sequencing (WGS) and bioinformatics. Comprehensive molecular characterization of G2-6 transgenic rice was performed using this pipeline. The results showed that one copy of the foreign gene was inserted into rice chromosome 8. There was no vector backbone insertion but an unexpected insertion and DNA rearrangement at the 3' end of the T-DNA. We also obtained the 5' and 3' flanking sequences of the T-DNA. Our results suggested that the use of a combination of WGS and bioinformatics is an effective strategy for the molecular characterization of GMOs.

Insertional mutagenesis of Brachypodium distachyon using the Tnt1 retrotransposable element.

Brachypodium distachyon is an annual C3 grass used as a monocot model system in functional genomics research. Insertional mutagenesis is a powerful tool for both forward and reverse genetics studies. In this study, we explored the possibility of using the tobacco retrotransposon Tnt1 to create a transposon-based insertion mutant population in B. distachyon. We developed transgenic B. distachyon plants expressing Tnt1 (R0) and in the subsequent regenerants (R1) we observed that Tnt1 actively transposed during somatic embryogenesis, generating an average of 6.37 insertions per line in a population of 19 independent R1 regenerant plants analyzed. In seed-derived progeny of R1 plants, Tnt1 segregated in a Mendelian ratio of 3:1 and no new Tnt1 transposition was observed. A total of 126 flanking sequence tags (FSTs) were recovered from the analyzed R0 and R1 lines. Analysis of the FSTs showed a uniform pattern of insertion in all the chromosomes (1-5) without any preference for a particular chromosome region. Considering the average length of a gene transcript to be 3.37 kb, we estimated that 29 613 lines are required to achieve a 90% possibility of tagging a given gene in the B. distachyon genome using the Tnt1-based mutagenesis approach. Our results show the possibility of using Tnt1 to achieve near-saturation mutagenesis in B. distachyon, which will aid in functional genomics studies of other C3 grasses.

Polymerization retardation isothermal amplification strategy enables the sensitive and facile investigation of the flanking sequence preference of ten-eleven translocation 2 protein.

Active DNA demethylation process critically relies on the intrinsic properties of ten-eleven translocation proteins (Tets), particularly the flanking sequence preference. Challenged by the fact that the proximate bases to the 5-methylcytosine (5mC) are multitudinous and their influence on the Tets/DNA interplay is minute, the current methodologies are very limited in terms of cost, sensitivity and efficiency. Herein, we propose a polymerization retardation isothermal amplification (PRIA) strategy that enables sensitive and fast study of the flanking sequence preference of Tet. By arranging DNA polymerase to repetitively pass DNA strands through an isothermal replication-scission amplification reaction, the tiny difference in the Tet/DNA interplay can be consecutively accumulated and amplified. Low amount sample (80 ng) even multiple samples can be simultaneously analyzed within 10 h on an easily accessible laboratory real-time quantitative PCR instrument. For a proof-of-concept study, the binding preference (PB) of Tet2 for XmCGX, (X = C, G, T, A) was analyzed by PRIA and computational analysis, showing an order of AmCGT > TmCGA ≈ GmCGC > CmCGG. Furthermore, the binding and oxidation preference in Tet/DNA interplay process was individually considered. By comparative evaluation of the total flanking sequence preference (PT) and the PB, for the first time, we revealed that Tet2 has a lower oxidation preference (PO) to proximal flanking bases and the main contributor to PT of Tet2 is PB. The PRIA strategy, due to its reliable, cost-effective, high efficiency and low sample input, would hopefully advance epigenetic researches and other relative studies.

Comprehensive analysis of the molecular characterization of GM rice G6H1 using a paired-end sequencing approach.

Molecular characterization of exogenous DNA integrations in host genome is a key aspect in risk assessment of bioengineered crops. However, gaining a clear understanding of the molecular characters of a bioengineered crop using conventional techniques remains a challenging task. Herein, we report the full molecular characterization of one new transgenic rice event G6H1 via a paired-end sequencing approach and bioinformatics analysis pipelines. Also, the molecular characterization reported was validated using conventional PCR, Sanger sequencing, and digital PCR. The results showed there is only one copy of the exogenous DNA inserted, which is located within chromosome 7 of the G6H1 genome. There is no other unintended integration of sequences from the transformation plasmid. These results indicated that the paired-end sequencing approach, combined with bioinformatics pipeline developed, is well suited to elucidate the molecular characteristics of bioengineered crops, and is efficient, low cost, and comprehensive.

Highly active G-quadruplex/hemin DNAzyme for sensitive colorimetric determination of lead(II).

A UV-vis, CD, and differential pulse voltammetric study was performed on the deactivation of the activity of parallel G-quadruplex/hemin DNAzymes (G4 DNAzymes) by Pb(II). The G4 DNAzyme carries a d[TC] sequence at its 3' end and is stabilized by potassium(I). On addition of Pb(II), the K(I) ions in the parallel G4 are replaced by Pb(II) to keep the parallel topology. Intruded Pb(II) decrease the affinity between the topology and hemin, this leads to a decrease of DNAzyme activity for catalyzing the oxidation of 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) by hydrogen peroxide to form a green dye with an absorption maximum at 420 nm. The assay does not use any amplification, and has a linear response in the 0.01 to 10 µM Pb(II) concentration range and a 7.1 nM limit of detection. The method was successfully applied to the analysis of spiked water samples. Graphical abstractSchematic diagram of the colorimetric lead(II) assay based on the competition between K+ and Pb2+ stabilized G-quadruplex/hemin DNAzymes (G4 DNAzymes).

Biolistic Transformation of Haematococcus pluvialis With Constructs Based on the Flanking Sequences of Its Endogenous Alpha Tubulin Gene.

Haematococcus pluvialis has high commercial value, yet it displays low development of genetic transformation systems. In this research, the endogenous 5' and 3' flanking sequences of the constitutive alpha tubulin (tub) gene were cloned along with its encoding region in H. pluvialis, in which some putative promoter elements and polyadenylation signals were identified, respectively. Three selection markers of tub/aadA, tub/hyr and tub/ble with three different antibiotic-resistance genes fused between the endogenous tub promoter (Ptub) and terminator (Ttub) were constructed and utilized for biolistic transformation of H. pluvialis. Stable resistant colonies with introduced aadA genes were obtained after bombardments of either H. pluvialis NIES144 or SCCAP K0084 with the tub/aadA cassette, the efficiency of which could reach up to 3 × 10-5 per µg DNA through an established manipulation flow. Two key details, including the utilization of culture with motile flagellates dominant and controlled incubation of them on membrane filters during bombardments, were disclosed firstly. In obtained transformants, efficient integration and transcription of the foreign tub/aadA fragments could be identified through genome PCR examination and qPCR analysis, nonetheless with random style instead of homologous crossover in the H. pluvialis genome. The presented selection marker and optimized transforming procedures in this report would strengthen the platform for genetic manipulation and modification of H. pluvialis.

Identification of T-DNA Insertion Site and Flanking Sequence of a Genetically Modified Maize Event IE09S034 Using Next-Generation Sequencing Technology.

Molecular characteristics including information of insertion site, flanking sequence, and copy numbers are the base for the safety assessment and subsequent monitoring of genetically modified organisms (GMOs), which has to be revealed thoroughly in a case-by-case manner. Although both polymerase chain reaction (PCR)-based and next-generation sequencing (NGS)-based approaches are proven to be effective in the molecular characterization of most of GM events, they often fail to work with GM maize events, mainly due to the genome complexity. In this study, by using NGS, we successfully identified the 3' end T-DNA insertion site and flanking sequence of a GM maize event IE09S034, which were confirmed by PCR amplification and Sanger sequencing. Notably, insertions of unintended exogenous elements were revealed in this event although the single copy of target exogenous genes was also confirmed by digital PCR. The output of this study provides novel and important genetic evidence for the safety assessment and monitoring of GM maize event IE09S034.

OSTRFPD: Multifunctional Tool for Genome-Wide Short Tandem Repeat Analysis for DNA, Transcripts, and Amino Acid Sequences with Integrated Primer Designer.

Microsatellite mining is a common outcome of the in silico approach to genomic studies. The resulting short tandemly repeated DNA could be used as molecular markers for studying polymorphism, genotyping and forensics. The omni short tandem repeat finder and primer designer (OSTRFPD) is among the few versatile, platform-independent open-source tools written in Python that enables researchers to identify and analyse genome-wide short tandem repeats in both nucleic acids and protein sequences. OSTRFPD is designed to run either in a user-friendly fully featured graphical interface or in a command line interface mode for advanced users. OSTRFPD can detect both perfect and imperfect repeats of low complexity with customisable scores. Moreover, the software has built-in architecture to simultaneously filter selection of flanking regions in DNA and generate microsatellite-targeted primers implementing the Primer3 platform. The software has built-in motif-sequence generator engines and an additional option to use the dictionary mode for custom motif searches. The software generates search results including general statistics containing motif categorisation, repeat frequencies, densities, coverage, guanine-cytosine (GC) content, and simple text-based imperfect alignment visualisation. Thus, OSTRFPD presents users with a quick single-step solution package to assist development of microsatellite markers and categorise tandemly repeated amino acids in proteome databases. Practical implementation of OSTRFPD was demonstrated using publicly available whole-genome sequences of selected Plasmodium species. OSTRFPD is freely available and open-sourced for improvement and user-specific adaptation.

Genome-wide analysis of flanking sequences reveals that Tnt1 insertion is positively correlated with gene methylation in Medicago truncatula.

From a single transgenic line harboring five Tnt1 transposon insertions, we generated a near-saturated insertion population in Medicago truncatula. Using thermal asymmetric interlaced-polymerase chain reaction followed by sequencing, we recovered 388 888 flanking sequence tags (FSTs) from 21 741 insertion lines in this population. FST recovery from 14 Tnt1 lines using the whole-genome sequencing (WGS) and/or Tnt1-capture sequencing approaches suggests an average of 80 insertions per line, which is more than the previous estimation of 25 insertions. Analysis of the distribution pattern and preference of Tnt1 insertions showed that Tnt1 is overall randomly distributed throughout the M. truncatula genome. At the chromosomal level, Tnt1 insertions occurred on both arms of all chromosomes, with insertion frequency negatively correlated with the GC content. Based on 174 546 filtered FSTs that show exact insertion locations in the M. truncatula genome version 4.0 (Mt4.0), 0.44 Tnt1 insertions occurred per kb, and 19 583 genes contained Tnt1 with an average of 3.43 insertions per gene. Pathway and gene ontology analyses revealed that Tnt1-inserted genes are significantly enriched in processes associated with 'stress', 'transport', 'signaling' and 'stimulus response'. Surprisingly, gene groups with higher methylation frequency were more frequently targeted for insertion. Analysis of 19 583 Tnt1-inserted genes revealed that 59% (1265) of 2144 transcription factors, 63% (765) of 1216 receptor kinases and 56% (343) of 616 nucleotide-binding site-leucine-rich repeat genes harbored at least one Tnt1 insertion, compared with the overall 38% of Tnt1-inserted genes out of 50 894 annotated genes in the genome.

[Identification and evaluation on methods with upstream flank sequences of CRISPR1, regarding Escherichia coli and Shigella].

Objective: To analyze the effect of the identification and evaluation of Escherichia (E.) coli and Shigella, based on the upstream flanking sequences of CRISPR1. Methods: Both CRISPR and cas sequences were obtained through the BLAST with repeating sequences against the publicly complete genome in GenBank that related to E. coli and Shigella. Clustal X was used to perform multi-sequences alignment of the flanking sequences. PCR method was used to amplify the upstream flanking sequences of CRISPR1 in order to appraise the effect of identification and evaluation of upstream flanking sequences on E. coli and Shigella, which were based on the upstream flanking sequences of CRISPR1. Results: The results showed that 73.4% of the strains containing the I-E CRISPR/Cas that belonged to the phylogroups A, B1, D while 8.4% strains carried the I-F CRISPR/Cas. Another 17.2% of the strains owned CRISPR3-4 (non-CRISPR/Cas) only belonged to the phylogroups B2. All the Shigella strains carried I-E CRISPR/Cas. More than 99% of similarity the CRISPR1 upstream-flanking sequences was seen in E. coli (except B2) and Shigella and E. coli (B2). Both sensitivity and specificity were greater than 91% after PCR amplification in the region to identify the E.coli and Shigella. Conclusion: The upstream of CRISPR1 could achieve a preliminary identification effect on E.coli and Shigella.

Functional Analysis of Maize Silk-Specific ZmbZIP25 Promoter.

ZmbZIP25 (Zea mays bZIP (basic leucine zipper) transcription factor 25) is a function-unknown protein that belongs to the D group of the bZIP transcription factor family. RNA-seq data showed that the expression of ZmbZIP25 was tissue-specific in maize silks, and this specificity was confirmed by RT-PCR (reverse transcription-polymerase chain reaction). In situ RNA hybridization showed that ZmbZIP25 was expressed exclusively in the xylem of maize silks. A 5' RACE (rapid amplification of cDNA ends) assay identified an adenine residue as the transcription start site of the ZmbZIP25 gene. To characterize this silk-specific promoter, we isolated and analyzed a 2450 bp (from -2083 to +367) and a 2600 bp sequence of ZmbZIP25 (from -2083 to +517, the transcription start site was denoted +1). Stable expression assays in Arabidopsis showed that the expression of the reporter gene GUS driven by the 2450 bp ZmbZIP25 5'-flanking fragment occurred exclusively in the papillae of Arabidopsis stigmas. Furthermore, transient expression assays in maize indicated that GUS and GFP expression driven by the 2450 bp ZmbZIP25 5'-flanking sequences occurred only in maize silks and not in other tissues. However, no GUS or GFP expression was driven by the 2600 bp ZmbZIP25 5'-flanking sequences in either stable or transient expression assays. A series of deletion analyses of the 2450 bp ZmbZIP25 5'-flanking sequence was performed in transgenic Arabidopsis plants, and probable elements prediction analysis revealed the possible presence of negative regulatory elements within the 161 bp region from -1117 to -957 that were responsible for the specificity of the ZmbZIP25 5'-flanking sequence.