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Protocol for the Generation of a Transcription Factor Open Reading Frame Collection (TFome)
转录因子开放阅读框收集(Tfome)的生成方法   

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Abstract

The construction of a physical collection of open reading frames (ORFeomes) for genes of any model organism is a useful tool for the exploration of gene function, gene regulation, and protein-protein interaction. Here we describe in detail a protocol that has been used to develop the first collection of transcription factor (TF) and co-regulator (CR) open reading frames (TFome) in maize (Burdo et al., 2014). This TFome is being used to establish the architecture of gene regulatory networks (GRNs) responsible for the control of transcription of all genes in an organism. The protocol outlined here describes how to proceed when only an incomplete genome with partial annotation is available. TFome clones are made in a recombination-ready vector of the Gateway? system, allowing for the facile transfer of the ORFs to other Gateway?-compatible vectors, such as those suitable for expression in other host species. Although this protocol was developed for the maize TFome it can readily be applied to the generation of complete ORFeome collections in other eukaryotic species.

[Protocol overview] An important aspect of successful TFome generation is the initial effort spent to establish a reliable set of gene models so that they can be subsequently amplified or synthesized. An actual TFome construction protocol for a particular species will depend on available resources such as a full-length cDNA (flcDNA) collection and a reliable reference genome (Figure 1).
In the case of maize, a flcDNA collection and a draft genome was available, but the former provided only 30% of the needed clones, and the latter contained gaps and some erroneous gene models. In order to develop a near-complete set of target gene models for maize TFs, a bioinformatics pipeline was developed as described by Yilmaz et al. (2009). In brief, a two-pronged search process was developed. The first involved making a collection of protein sequences of TFs in other species and available from databases such as PlantTFDB, PlnTFDB and DBDTF. These sequences were then used to search gene models from the draft maize genome using BLASTP. The second process involved developing a collection of domains that define TF families and that are mostly annotated in the PFAM database (Finn et al., 2014). These domains were then used to search the draft maize genome using BLASTX. The number of TF families that exist and their naming is subject to change as new members are discovered and studied. Table 1 provides a list of known TF families with alternative names along with the respective PFAM domains whose presence or absence defines each TF family. HMM models for each domain can be obtained from the PFAM database (pfam.xfam.org). Following the BLAST search, redundant models are eliminated and then based on the TF motifs present in each gene model, gene models are assigned to a TF or Co-Regulator (CR) family according to the criteria specified in Table 1. Lastly, it is recommended to set up a database to store information on each TF family. The GRASSIUS (www.grassius.org) website was established to access the stored information on TF gene models for maize, sorghum, rice, Brachypodium, sugarcane and other grasses (Burdo et al., 2014). In the following section, an assumption is made that at least a draft genome or draft transcriptome is available and that a set of gene models is available that have been determined ab initio or with additional manual annotation. Familiarity with the use of PERL scripts is advantageous for the gene model assembly phase.



Figure 1. Flowchart for the generation of a TFome project. Flowchart outlining the general strategy for template identification, PCR amplification and cloning of transcription factor (TF) full length (FL) open reading frames (ORFs). (modified from Burdo et al., 2014)

Materials and Reagents

The main starting materials for embarking upon a large TFome project are the assembly of gene models and a collection of plasmid templates from existing cDNA collections. In this section, processes to develop these are outlined.

  1. Assembly of gene models and TF domain databases
    1. Assemble a collection of gene models from the available target genome. For plant genomes, the Phytozome database (phytozome.jgi.doe.gov) and EnsemblPlants (http://plants.ensembl.org/index.html) permit the downloading of all predicted protein models for a species as a single multi-sequence FASTA formatted file. For example, at the Phytozome website the BioMart tool permits one to select the genome dataset for a particular plant species. Once the genome is selected then attributes of the sequences to be downloaded can be specified such as peptide sequences or coding sequences only. By default all gene models are selected and then these can be downloaded as a single FASTA file that is the input file for subsequent steps below.
    2. Assuming the draft quality of the transcript annotation it would be desirable to eliminate redundant gene models in a multiseq FASTA file. The GRASSIUS website provides the custom perl script “IdentifySeqRedundancy.pl” (http://grassius.org/tools.html) for this purpose. This script also requires the perl module “Digest::MD5” which is available from the Comprehensive Perl Archive Network (CPAN) website (http://search.cpan.org/dist/Digest-MD5/MD5.pm). One can also eliminate redundant models within the species using BLAST searches. Proteins were arbitrarily considered duplicated if they are found in the same species, with a query coverage ≥ 90%, or have a query identity ≥ 90% and the query alignment starts less than 9 residues from the start codon. Alternatively, more complex criteria such as those of Gu et al., 2002, may be employed to identify duplicate proteins in a genome. If these conditions are satisfied, the longest protein is kept and the eliminated proteins were classified as identical or splicing variants. If there is access to RNA-Seq datasets they may be used to corroborate target TF gene models, but such an approach is not outlined here. Targeting the longest splice variant which is supported by EST or RNA-Seq data provides the maximum protein interaction space for identifying the protein-DNA and protein-protein interactions that gene regulatory networks are comprised of.
    3. Scanning the non-redundant multifasta file against a collection of protein domains as hidden Markov models (HMMs) such as provided by PFAM or Interpro provides a protein domain annotation of the putative proteome. For this particular step the software HMMER is required. In brief HMMER is a set of tools implementing the profile hidden Markov models to find similarity across protein sequences and is necessary to search the PFAM HMM models in a group of protein sequences. It is possible to call HMMER from a variety of scripting languages such as perl or python, using pre-build HMMER wrappers. PFAM provides one of those scripts written in perl (pfamscan.pl) with a set of PFAM pre-established parameters. Source code may be downloaded from the following website ftp://ftp.sanger.ac.uk/pub/databases/Pfam/Tools/PfamScan.tar.gz . Table 1 is a compilation of protein domains that have been used to define TF and CR families in plants (Buitrago-Florez et al., 2014; Burdo et al., 2014; Perez-Rodriguez et al., 2010; Yilmaz et al., 2009). This table describes 62 TF and 26 CR families that were used in defining gene models for inclusion in the maize TFome. Twelve of these families do not have ascribed PFAM domains but can be defined using “in house” HMMs as previously described (Buitrago-Florez et al., 2014; Perez-Rodriguez et al., 2010). The HMMs for the CCAAT-Hap2, 3, and 5 TF families can be obtained from the AGRIS database (http://arabidopsis.med.ohio-state.edu) (Yilmaz et al., 2011).
    4. Once the primary list of gene models are annotated with protein domains, they are assigned to TF or CR families using the criteria outlined in Table 1. The criteria include identifying the presence of one or more motifs and the absence of other (forbidden) motifs (Buitrago-Florez et al., 2014; Perez-Rodriguez et al., 2010; Yilmaz et al., 2009). The annotation involves a custom perl script “get_InterProScanDomains.pl” to sort proteins into TF families based on the interproscan output and is available at the GRASSIUS website (http://grassius.org/tools.html). The script keeps only significant hits with e-value ≤ 0.001 and verifies that the rules as defined in Table 1 are fulfilled. When the rules are only partially fulfilled then the protein is assigned into “Orphan” TFs. This can be the largest class of TFs for a species (~11.7% of the maize TFome), until Orphan members are assigned to families.
    5. Searches can be conducted with local computation resources or when not available using the iPlant Collaborative Discovery Environment public resource (http://www.iplantcollaborative.org).

  2. Screening of EST or flcDNA collections for plasmid templates
    1. Identify which cDNA resources are available for the target genome (see Note 1). Most genome projects for model species include generating an EST library, although many libraries are not publically available. For the maize TFome, extensive use of the maize flcDNA collection (http://www.maizecdna.org) was made (Soderlund et al., 2009). Individual cDNA clones for this library and many other plant species are available through the Arizona Genomics Institute (http://www.genome.arizona.edu).
    2. Using the coding sequence of a gene model as a query sequence, ESTs or flcDNAs were then identified using BLASTP or BLASTX, for which the amino terminus, including the start codon, was present and the sequence alignment was >99%. Alignments that are not 100% identical may occur because EST sequences are not high fidelity due to sequencing errors particularly at the 3’ end of sequences (Soderlund et al., 2009). Alternatively spliced isoforms not represented by a transcript model may also be targeted, as long as they maintain the reading frame of the original transcript.
    3. Once a likely flcDNA template is located, then the plasmid is acquired, isolated and sequenced from each end to confirm that: a) it is the correct template, and b) it is full length. Some cDNA libraries utilize enzymes during their construction that cut within the coding sequence leading to partial clones, which would be unsuitable for amplification of the entire coding sequence.
    4. Once a suitable target plasmid template is identified for a target gene, then a sample is diluted to 1 ng/µl and 1 ng is sufficient as template in a PCR reaction.

  3. Other reagents
    1. One Shot® TOP10 chemically competent cells (Life Technologies, catalog number: C404003 )
    2. PureLink® Plant RNA Reagent (Life Technologies, catalog number: 2322-012 )
    3. Turbo DNA-free™ kit (Life Technologies, Ambion®, catalog number: AM1907 )
    4. Thermo Scientific Maxima H minus 1st strand synthesis kit (Thermo Fisher Scientific, catalog number: K1652 )
    5. Ribolock® RNase inhibitor (Thermo Fisher Scientific, catalog number: EO0382 )
    6. EmeraldAmp® MAX PCR Master Mix (Takara Bio Company, catalog number: RR320A )
    7. Phusion® high fidelity polymerase (New England Biolabs, catalog number: M0530S )
    8. Gateway® pENTR™/D-TOPO® or pENTR™/SD/D-TOPO® vectors (Life Technologies, catalog numbers: K243520 and K242020 respectively)
    9. Genscript® Taq Polymerase (GenScript USA Inc., catalog number: E000071000 )
    10. EmeraldAmp® MAX PCR Master Mix (Takara Bio Company, catalog number: RR320A)
    11. 1 kb ladder molecular weight size standards (Thermo Fisher Scientific, catalog number: SM0311 )
    12. RNA extraction buffer I (see Recipes)
    13. RNA extraction buffer II stock solutions (see Recipes)
    14. RNA extraction buffer II (working solution) (see Recipes)
    15. Carlson lysis buffer (see Recipes)
    16. Freezing medium (see Recipes)

Equipment

Note: Most equipment listed here is standard molecular biology instrumentation present in most laboratories. A large TFome project would also benefit from the use of multichannel pippetters and 96 well plate formatted experimentation.

  1. Microcentrifuge
  2. High speed centrifuge
  3. Gradient thermocycler
  4. Gel electrophoresis equipment
  5. Water baths
  6. -80 °C freezer for the storage of stocks
  7. Aerosol pipette tips recommended for all DNA manipulation and cloning experiments
  8. Wizard® SV 96 Plasmid DNA Purification System (Promega Corporation, catalog number: A2255 )
  9. Wizard® Plus SV Minipreps DNA Purification System (Promega Corporation, catalog number: A1330 )
  10. Wizard® SV Gel and PCR Clean-Up System (Promega Corporation, catalog number: A9281 )
  11. Gene synthesis (Life Technologies)
  12. 2 ml 96 well culture dish (Thermo Fisher Scientific, catalog number: 12566121 )
  13. Breathable plate seal (Thermo Fisher Scientific, catalog number: AB0718 )
  14. 0.5 ml plates (USA Scientific, catalog number: 18965000 )
  15. 7 mm sized silicone seal that can be re-autoclaved (Thermo Fisher Scientific, catalog number: 0339649 )
  16. Matrix sample storage system (matrixtechcorp.com, Thermo Fisher Scientific, catalog numbers: 4111MAT ) (Capit-All® Capper/Decapper), 3740 (Barcoded screw cap tubes), and 4477 (Screw cap tray)
  17. (Optional) Vac-Man® 96 Vacuum Manifold (Promega Corporation, catalog number: A2291 ) for 96 well plasmid preparation
  18. (Optional) Vacuum pump capable of generating 38-51 cm of Hg or equivalent

Software

  1. OligoAnalyzer 3.1 software (www.idtdna.com)

Procedure

  1. Preparation of nucleic acid templates for PCR
    Here, three main sources of template for the amplification of TF coding sequences were used: 1) full length cDNA clones from representative cDNA libraries, 2) cDNA generated from RNA purified from a variety of host species tissues, and 3) genomic DNA from the host species which is suitable for the amplification of coding sequences from single exon genes. Methods for the preparation of nucleic acid templates are provided in this section.
    1. Plasmid templates for TF ORF amplification.
      Any traditional plasmid preparation will provide plasmid of sufficient quantity and purity to act as template for PCR amplification. For the maize TFome project, hundreds of plasmids were required and a 96-well format was desirable. The high throughput system that proved satisfactory was the Wizard® SV 96 Plasmid DNA Purification System. This system requires a vacuum pump capable of generating 38-51 cm of Hg or equivalent and the Vac-Man® 96 Vacuum Manifold. It is noted however that the final concentration of plasmid DNA from this method was usually less than the minimum required for automated Sanger dideoxy DNA sequencing which is approximately 50 ng/μl. If the plasmid template needs to be verified by DNA sequencing prior to amplification then a higher yielding protocol will be required (e. g. Wizard® Plus SV Minipreps DNA Purification System).
    2. cDNA template for TF ORF amplification by Reverse Transcription-PCR (RT-PCR).
      Most TF genes are restricted in their temporal and tissue specific gene expression pattern. When considering the use of RT-PCR for the generation of amplicons it is helpful to have some evidence for the tissue in which a particular TF is most highly expressed to maximize success in amplification. For maize and a few other plant species, the qTeller website (www.qteller.com) provides a searchable resource that displays the relative abundance of transcripts of genes from RNA-Seq data. For maize genes that were successfully amplified, it was found that they were always amplified from one of the top three ranked tissues where their expression was deemed highest using the qTeller resource. The Bio-Analytic Resource for Plant Biology (BAR) (www.bar.utoronto.ca) and The Plant Expression (PLEXDB) (http://www.plexdb.org) databases provide similar resources for a broader selection of plant species.
      For the collection of RNA samples, a large plant population (about 100 maize plants were employed) should be planted since multiple tissues will be required and the collection of certain tissues (e.g. developing ears) will entail harvesting the entire plant. For some genes, biotic and abiotic stress, or circadian rhythm, may cause transcript levels to increase significantly. In the case of the maize TFome, it was found that more than half of the clones derived by RT-PCR could be amplified from young seedling shoots and roots (Burdo et al., 2014).

      RNA isolation from plant tissues
      Total RNA was isolated from plant tissues by one of two methods. For non-starchy tissues PureLink® Plant RNA Reagent was employed according to the manufacturer's recommendations. For the isolation of RNA from starchy tissues, such as developing and mature seeds, the method described by Li and colleagues (Li and Trick, 2005) was utilized with slight modifications. For convenience, the protocol and solution recipes are detailed below. Typically, total RNA was isolated from 1 gram of tissue.
      1. Grind 1 gram of tissue in mortar and pestle with liquid nitrogen. For successful implementation, it is essential that tissues are immediately frozen in liquid nitrogen at harvest time and stored at -80 °C before grinding. The tissue should not be permitted to thaw at any time prior to the addition of extraction buffer.
      2. Transfer powder into RNase free centrifuge tube with 4 ml of extraction buffer I and immediately mix vigorously.
      3. Add 2.5 ml of phenol:chloroform mixture (1:1, pH 4.7) and mix well by inversion.
      4. Centrifuge at 13,000 x g for 15 in at 4 °C.
      5. Transfer upper aqueous phase (2.5 ml) to a new RNase free centrifuge tube containing 2.5 ml extraction buffer II. Samples are mixed by gentle inversion and incubated at room temperature for 10 min.
      6. Add 2 ml of chloroform-isoamyl alcohol (24:1), mix well and centrifuge the samples at 13,000 x g for 15 min at 4 °C.
      7. Recover supernatants (4.5 ml), and add 3 ml of isopropanol and 2.5 ml of 1.2 M NaCl. Samples are mixed by gentle inversion, incubated on ice for 15 min, and centrifuged at 13,000 x g for 15 min at 4 °C.
      8. Discard the supernatants and wash the pellets carefully with 400 µl of 70% ethanol.
      9. Dry the washed pellets at room temperature and resuspend in RNase free water (about 500 µl) prior to storing at -70 °C.
        Typically yields of total RNA were 2.2 ± 0.9 µg/µl with A260/A280 and A260/A230 ratios of 1.93 ± 0.06 and 2.32 ± 0.32 respectively (n = 30). Total RNA samples with ratios <1.7 and 2.0 were deemed unsuitable. RNA integrity must be assessed by separation on a denaturing gel. The appearance of acceptable quality total RNA is shown in Figure 2A. For most tissues, the large and small ribosomal bands should be sharp with the upper band being twice the intensity of the lower band. A faint smear extending above and below the ribosomal bands is indicative of mRNA. For germinating seeds, the ribosomal bands will have diminished intensity but the smear should still be visible. Some large molecular weight material is indicative of contaminating DNA, which is removed by DNase treatment (see below).

    Isolation of genomic DNA
    For TF genes whose coding sequence is contained within one exon (about 7% of TF genes in maize), then total genomic DNA is a suitable template for amplicon generation. The source material should match the reference genome that is being employed in the project. This will allow the user to discern if single nucleotide polymorphisms (SNPs) in the amplicons are due to errors during amplification and not due to naturally occurring SNPs in the germplasm. There are many suitable genomic DNA isolation protocols available and the one used in this project is summarized below.
    Plant DNA isolation protocol (modified from Carlson et al., 1991).
    1. Grind 1 g of leaf material in liquid nitrogen with a cold mortar and pestle.
    2. Transfer ground material into a sterile Oakridge tube containing 10 ml of Carlson Buffer preheated to 70 °C (by standing in water bath).
    3. Incubate for 20 min at 70 °C by standing in water bath - invert every few minutes to mix contents (use a shaking water bath if available).
    4. Cool samples to room temperature and add 5 ml of chloroform/isoamyl alcohol (24:1) and vortex well for 20 sec. Then centrifuge for 10 min at 4 °C at 5,000 x g.
    5. Transfer upper aqueous phase into a fresh (sterile) Oakridge tube. Add 50 µl of 10 mg/ml RNase A (preboiled and frozen at -20 °C) - incubate at 37 °C for 20 min to degrade RNA.
    6. Add an equal volume of isopropanol (2-propanol), vortex and incubate at room temperature for 20 min to allow the precipitation of DNA. Centrifuge for 20 min at 4 °C at 5,000 x g.
    7. Resuspend the pellet in 600 µl of TE (1 mM Tris, 0.1 mM EDTA, pH 8.0) and transfer into an Eppendorf tube.
    8. Add 5 µl of 10 mg/ml RNase A and incubate for 30 min at 37 °C.
    9. Extract the DNA with an equal volume of phenol/chloroform/isoamylalcohol (25:24:1) and centrifuge at 14,000 x g for 5 min at 4 °C.
    10. Transfer the top aqueous phase to a new Eppendorf tube. Extract with an equal volume (now about 0.5 ml) of chloroform/isoamylaclcohol (24:1) and centrifuge at 14,000 x g for 5 min in a benchtop microcentrifuge (4 °C).
    11. Precipitate DNA by adding 1/10 volume of 3 M NaOAc or 5 M NH4OAc and 2 volumes of 100% ethanol. Mix by gentle inversion and centrifuge at 14,000 x g for 5 min at 4 °C.
    12. Wash the DNA pellet with 70% ethanol once or twice.
    13. Remove the wash supernatant and let the pellet air dry for about 10 min but it is best not to let the DNA become completely dry. Resuspend the DNA in about 200 µl of TE buffer (1 mM Tris, 0.1 mM EDTA, pH 8.0) and determine the concentration by UV spectroscopy.
      DNA isolated by this method should have an A260/A280 ratio > 1.7 and an A260/A230 ratio > 2.0. For a complex genome such as maize 100 ng should be used as template in a PCR reaction.

  2. Generation of complementary DNA (cDNA) from total RNA
    1. Prior to the generation of cDNA it is important to remove any contaminating genomic DNA from the total RNA preparations. In this project the Turbo DNA-free™ kit was employed according to the manufacturer's recommendations. Four units of TURBO™ DNase are used to remove any contaminating DNA from 20 µg of total RNA. Following treatment at 37 °C for 30 min, the DNase deactivation matrix is added at room temperature and after 5 min incubation, separated by centrifugation at 10,000 x g. The supernatant is removed and used in a reverse transcription reaction.
    2. It is recommended that a high fidelity reverse transcriptase is employed to reduce errors in the cDNA that will act as template for target gene amplification. We employed Thermo Scientific Maxima H minus 1st strand synthesis kit according to the manufacturer's recommendations except that 40 units of Ribolock® RNase inhibitor was added in each reverse transcription reaction. Using this protocol, 12 µl of DNase treated RNA (approximately 5 µg total RNA) is annealed to the provided oligo dT primer in a 20 µl reaction. Between 0.5 to 1 µl of this reaction is sufficient for a single PCR amplification of most TF genes. Before proceeding with PCR, quality of the cDNA was assessed by amplifying the transcript of a housekeeping gene. For this purpose, low fidelity DNA polymerase such as the EmeraldAmp® MAX PCR Master Mix is adequate. For maize, a 1,001 bp portion of the ZmGAPDH (GRMZM2G046804) transcript was amplified using the primers ZmGAPDH_F (5’-ATGCAGGCAAGATTAAGATCGGAATCAAC-3’) and ZmGAPDH_R (5’-CATGTGGCGGATCAGGTCGAC-3’). The absence of a larger 2,817 bp PCR product confirmed the removal of genomic DNA (Figure 2B).

  3. Amplification of TF coding sequences
    1. Primers are synthesized to amplify the corresponding ORFs without the respective stop codons, and an additional 5'-CACC-3’ nucleotide tail added to the forward primer, for directional cloning into the pENTR™ vectors. For maize, 1,273 primer pairs were analyzed in regards to GC content and melting temperature (Tm). Tm values were estimated using the OligoAnalyzer 3.1 software (www.idtdna.com). The forward primers had an average length of 25 ± 4 bp including the 5’ CACC tail, which was similar to that of the reverse primers (23 ± 4 bp). However the GC content of the forward primers (62.8 ± 9.1) was about 12% higher than that of the reverse primer (50.2 ± 11.7). As a result the average Tm for the forward primer was about 3 °C higher (66 ± 5 °C) than that of the reverse primer (63 ± 5 °C). In addition the average GC content of the amplicons was 62.4 ± 8.8 % with a range from 78.9 to 38.2% (n = 1,411). Due to the relatively high GC content the buffer for GC rich templates was routinely employed for CDS amplification. When designing primers an effort should be made to have the difference in Tm (ΔTm) for primers in a pair to be as low as possible. In this project the average ΔTm was 3.2 ± 4.1 °C however amplicons were successfully obtained even when the ΔTm was as much as 19 °C.
    2. PCR products are next generated using a high-fidelity polymerase. For this project, the Phusion® high fidelity polymerase was chosen because it exhibits an error rate more than 50-fold lower than that of Taq DNA Polymerase. In addition, Phusion® polymerase has an enhanced processivity domain that requires an extension time of only about 15 sec per kb, and we found that an extension time of 1 min was sufficient for even the longest amplicons. The coding sequences of TFs in the maize genome were on average 1,024 ± 474 bp in length with a range from 90 to 3,705 bp. The recommended initial composition for PCR are listed in Table 2. It was determined that the use of GC buffer and 10% glycerol was the most successful initial condition (Table 2, setup A). If this initial condition was unsuccessful it is recommended to next adjust the annealing temperature (Note 2). It was found that the addition of DMSO or extra MgCl2 also improved the success rate for some templates (Table 2; Setups B and C). The cycling parameters for PCR are as follows: A single initial denaturation cycle of 98 °C for 1 min, 25- to 35 cycles of amplification of 98 °C denaturation for 20 sec, ~ 60 °C annealing for 40 sec, 72 °C extension for 15 sec per kb, 1 final extension step of 72 °C for 5 min. It is important that following completion of the final extension step that the PCR reactions be removed and processed or stored at -20 °C until processing (Note 3). The number of cycles depends on the template source. As few as 25 cycles may be employed to amplify from plasmid templates but usually 35 cycles are required when using a cDNA template. In general, a fewer number of cycles will reduce the chances of errors due to polymerase.

      Table 2. Recommended PCR composition


      Options

      Components (add in this order)
      A
      B
      C

      vol (µl)
      vol (µl)
      vol (µl)
      H2O
      11
      15.75 -15
      10 - 10.75
      GC Buffer (5x) contains 7.5 mM MgCl2
      5
      5
      5
      Forward Primer (20 µM stock)
      1
      1
      1
      Reverse Primer (20 µM stock)
      1
      1
      1
      dNTPs  (25 mM stock)
      1
      1
      1
      MgCl2 (50 mM stock)
      0
      0
      0.25 -1.0
      Glycerol (50% stock)
      5
      0
      5
      DMSO (100% stock)
      0
      0.25 - 1.0
      0
      Template* (plasmid, cDNA, or DNA)
      0.5
      0.5
      0.5
      Phusion® Polymerase* (5 U/µl)
      0.5
      0.5
      0.5
      Total Volume
      25 µl
      25 µl
      25 µl
      *Template amounts, Plasmid -1 ng, cDNA - 0.5 µl of RT reaction, genomic DNA-100 ng

    3. Once the PCR is complete the products are separated by gel electrophoresis to identify products of the expected size. When plasmid templates are employed it is usual to observe a single band, however when genomic DNA or cDNA is employed or when longer fragments are expected it is more usual to observe multiple bands (Figure 2C). The presence of a band of the correct size is not a guarantee that the correct coding sequence has been amplified however especially for genes that belong to a TF family for which multiple paralogs exist.

  4. Cloning and confirmation of PCR amplicons
    1. DNA bands of the correct size is identified by DNA gel electrophoresis are excised and gel purified. There are many suitable kits for purification for this step. For this project we used the Wizard® SV Gel and PCR Clean-Up System. Carefully quantify the purified product prior to ligation.
    2. Purified PCR products are then cloned into the Gateway® pENTR™/D-TOPO® or pENTR™/SD/D-TOPO® vectors. These vectors allow for the facile transfer of the clones into alternative expression vectors and greatly increase the utility of the TFome collection. Ligations were performed according to the manufacturers recommended protocol and used to transform One Shot® TOP 10 chemically competent cells. If desired half the reaction size can be performed using the recommended ratio of insert to vector.
    3.  Kanamycin-resistant colonies were then screened for inserts of the correct size and orientation by colony PCR or by restriction digestion of candidate plasmids. We noted considerable variation in the cloning efficiency using the Gateway® entry cloning kits. This difficulty was overcome by using an entire kit at one time to maximize efficiency. If cloning efficiency is high then plasmid DNA may be isolated from one or two colonies and these used for confirmation. It was found however that due to variation in cloning efficiencies, that colony PCR is often a more efficient way of identifying inserts.
      Colony PCR to identify positive clones
      First a master mix is prepared of a general purpose DNA polymerase such as Genscript® Taq Polymerase, in the manufacturer provided buffer (50 mM KCl, 10 mM Tris HCl, pH 9.0 at 25 °C), 1.5 mM MgCl2, 1% Triton X-100). The EmeraldAmp® MAX PCR Master Mix was also found suitable for colony PCR). Between 5-10% DMSO may also be added. To this mixture, the appropriate forward and reverse primers are added to a final concentration of 0.4 µM. Then 25 µl aliquots are added to the well of individual PCR tubes or a 96 well PCR plate. A sterile toothpick or pipette tip is then used to isolate a single reference colony, that is first mixed into an individual PCR reaction tube, and then to plate a replica of the colony being screened. The cycling parameters for PCR are as follows: A single initial denaturation cycle of 95 °C for 5 min, 35 cycles of amplification of 95 °C denaturation for 1 min, ~ 60 °C annealing for 1 min, 72 °C extension for 1 min per kb. Following PCR the products are separated by gel electrophoresis and examined for a product of the expected size (Figure 2D). Between 6-8 colonies are routinely screened for each clone due to the variation of cloning efficiency (Figure 2D). Initial PCR reactions are set up with vector specific forward and reverse primers, so that a product is amplified only if the gene was inserted in the correct orientation. In the case of no product being amplified, multiple combinations of vector and/or gene specific primers can be tested. Once clones with inserts are identified, then plasmid is prepared (see above) for confirmation by DNA sequencing.
    4. Once a colony produced a product of the expected size in colony PCR screening, the sequence of cloned inserts was confirmed by single-read paired-end Sanger dideoxy sequencing and by comparing the clone sequence to that of the reference genome. For clones longer than 1,200 bp, internal primers were designed to complete the sequence confirmation. The most common issue to arise during confirmation is the occurrence of SNPs that do not conform to the reference genome. If the SNP causes a silent mutation, or if the SNP was entered in the GRAMENE database (www.gramene.org) as a natural variant, then it was deemed acceptable for addition to the TFome collection.
    5. It is important to make duplicate permanent glycerol stocks of each verified clone immediately to avoid confusion in generating the TFome collection.

  5. Gene synthesis of rare TF transcripts.
    Chemical gene synthesis is an option for genes that prove recalcitrant to PCR amplification. This option offers the advantage that codon usage may be optimized for expression in different hosts, as is the desired downstream use of the TFome collection, and the production time is fast. However, costs are currently competitive for clones < 1 kb in length. Another disadvantage is that by not using a template derived from plant tissues, then there may be little or no direct experimental support for the existence of a particular transcript in vivo. Thus, it is recommended where possible, that RNA-Seq support be sought for a particular gene model prior to chemical synthesis.
    For the maize TFome project, approximately 30% of the collection was chemically synthesized (Burdo et al., 2014). Gene synthesis was performed using the GeneArt® technology. This technology employs a codon optimization process, which is required for any complex or GC-rich sequence, and to increase expression in maize. In the case of the maize TFome, a pilot test revealed that maize optimized constructs expressed at a lower but significant level in yeast compared to yeast optimized versions. No significant difference in expression was observed between different codon optimized versions in maize protoplasts (Burdo et al., 2014). Thus, in the case of the maize TFome the chemically synthesized clones were optimized for expression in maize where most downstream experimentation is expected to be performed.
    Once the fragments are synthesized they are resuspended in water and are cloned into the relevant Gateway® entry vector as described in section D above.

  6. Storage of TFome
    Given the utility of a TFome collection, careful consideration should be applied to how the TFome collection is stored and made available for long-term distribution. The maize TFome is made publicly available through the Arabidopsis Biological Resource Center (ABRC) (abrc.osu.edu). It is recommended that one or more backup TFome collections are created and stored in separate locations. Storage in a 96 well format allows for easier replication of the entire library but can make distribution of individual clones more difficult. Here we describe a method for the storage of a TFome collection in a 96 well format.
    Cryogenic Storage of TFome in 96 well format.
    1. Prepare sterilized freezing medium according to the recipe provided below which is adapted from Woo et al. (1994). This medium allows for the growth of bacterial cells and contains cryoprotectant to allow direct freezing of cultures following growth. In a laminar airflow cabinet, aliquot 1.8 ml of freezing medium containing the appropriate antibiotic into a sterile 2 ml 96 well culture dish.
    2. Using aseptic technique, inoculate 96 well plate with the individual bacterial stocks either from a plate or a previously made glycerol stock. When inoculating stocks, it can be helpful to have a map of the wells so as to avoid errors. Seal the plate with a breathable plate seal that is suitable for ventilating and storing bacterial/cell cultures. Place the sealed plate at 37 °C in a shaking incubator overnight with 220 rpm rotation for proper aeration.
    3. Since the cells are grown in freezing medium it is simply a matter of aseptically aliquoting 400 µl of the cultures into sterile 0.5 ml plates, and sealing them for storage at -80 °C. Multiple permanent plates can be made at this time. The plates may be stored at -80 °C with a plastic lid or a 7 mm sized silicone seal that can be re-autoclaved. Permanents may also be made in a 96-well format that permits automated decapping such as with the Matrix sample storage system.

Representative data



Figure 2. Representative data. Sample outcomes for plant RNA isolation and quality control, RT-PCR of long templates, and colony PCR. A. Appearance of total RNA prior to DNase treatment separated by gel electrophoresis. A non-degraded RNA sample will exhibit two major bands representing ribosomal RNAs (boxed bands in lane 1) and a background smear indicative of mRNA (arrow in lane 4). Note that diminished amounts of ribosomal RNA are seen in germinating seeds since the endosperm is not alive. A higher molecular weight band is indicative of genomic DNA that must be removed by DNase treatment. Std = Generuler™ 1 kb ladder molecular weight size standards. Samples are as follows:- 1: plant prop roots from L13 stage plant, 2: mature tassel from L13 stage plant 3: Developing seeds 2 weeks after pollination 4: entire ear 1 day after pollination, 5 seedling roots from 3 week old plant, 6, 7, and 8: germinating seeds 1, 2, and 7 days after germination. B. Amplification of GAPDH from cDNA generated from RNA isolated in panel A. A single band of 1.1 kb is indicative of the GAPDH transcript whereas a 4.3 kb band is expected if amplification occurs from genomic DNA. Std = Generuler™ 1kb ladder molecular weight size standards as in Figure 2A. cDNA samples were derived from the same samples as shown in Figure 2A. C. Amplification of TFs with long coding sequences by RT-PCR. Typically multiple bands are observed during RT-PCR from total cDNA samples. Asterisks indicate correct sized band that was excised and used for cloning. Samples and expected amplicon lengths are as follows:- 1: GRMZM2G069365, 2,127 bp, 2: GRMZM2G171600, 2,526 bp, 3: GRMZM2G028980, 2,742 bp, 4, 5: GRMZM2G160005, 3,159 bp. Std = Generuler™ 1 kb ladder molecular weight size standards as in Figure 2A. D. Sample Colony PCR reactions. Lanes with asterisks represent colonies with clones containing inserts of the expected size. High variability in the success rate of cloning different genes underscores the need for a rapid PCR based screening method. Std = Generuler™ 1 kb ladder molecular weight size standards as in Figure 2A. Lanes 1- 8: amplicons from GRMZM2G140156, expected size 2.6 kb. Lanes 9-16: amplicons from GRMZM2G009478 expected fragment size of 1.8 kb.

Notes

  1. The availability of a quality flcDNA library will be one of the factors that will most influence the cost of the TFome project. Amplifying rare and long transcripts from cDNA by RT-PCR can be challenging and the synthesis of long coding sequences can be prohibitively expensive.
  2. If no bands are observed following the initial PCR, then lowering the annealing temperature by 2 °C increments is recommended. Conversely, if multiple bands are seen then raising the annealing temperature by 2 °C increments is recommended.
  3. It is recommended not to allow the PCR reactions to dwell at 4 °C for any length of time at the end of the amplification cycles, as this will permit degradation of the amplicon ends and reduce cloning efficiency.

Recipes

  1. RNA extraction buffer I (Li and Trick, 2005)             per liter             per 100 ml
    100 mM Tris (pH 8.0) MW 121.14                          12.11g               1.211 g
    150 mM LiCl MW 42.39                                         6.34 g                0.63 g
    50 mM EDTA MW 372.24                                      18.6 g                1.86 g
    1.5 % 2-mercaptoethanol                                     15 ml                 1.5 ml
  2. RNA extraction buffer II stock solutions (Li and Trick, 2005)              per liter                per 100 ml
    Stock 0.75 M sodium citrate           MW 294.1                                 220.5 g                  22 g
    Stock 2 M sodium acetate (anhydrous) pH 4.0 MW 82.03                  164 g                    16.4 g
    Note: It needs to be warmed and use glacial acetic acid to adjust to pH 4.0.
    Stock 10% lauryl sarcosine             MW 293.39                                29.34 g                 2.93 g
    Note: Use a 10% stock solution, which needs to be heated to 68 °C to dissolve.
  3. RNA extraction buffer II (working solution) (Li and Trick, 2005)    per liter                        per 100 ml
    4.2 M guanidine isothiocyanate (w/v) MW 118.16                        496.27 g                      49.6 g
    0.5% Lauryl sarcosine Sigma (from 10% Stock)                           50 ml                          5 ml
    1 M Sodium acetate (from 2 M stock)                                         500 ml                         50 ml
    25mM Sodium citrate (from 0.75 M stock)                                   33 ml                           3.3 ml
  4. Carlson lysis buffer (Carlson et al., 1991)                                    per liter
    100 mM Tris-Cl (pH 9.5) MW 121.14                                           12.114 g
    2% CTAB (cetryl trimethyl ammonium bromide)                           20.0 g
    1.4 M NaCl MW 58.44                                                               81.82 g
    1% PEG 6000 or 8000                                                              10 g
    20 mM EDTA MW 372.24 (>5 M stock = 18.61 g/100 ml)  40 ml of 0.5 M stock
    Need to heat to dissolve CTAB. After autoclaving the solution will appear slightly opaque (Cloudy).
    Note: Add beta-mercaptoethanol to tubes just prior to use (100 µl BM per 10 ml).
  5. Freezing medium (Woo et al., 1994)                                            per liter
    Luria-Bertani broth (LB) powder or granules                                 25 g
    36 mM K2HPO4 MW 174.2                                                          6.28 g
    13 mM KH2PO4 MW 136.09                                                        1.8 g
    1.9 mM Na3C6H5O7∙2H2O (sodium citrate) MW 258.06                  0.5 g
    6.8 mM (NH4)2SO4 (ammonium sulfate) MW 132.14                      0.9 g
    4.4% C3H8O3 (glycerol) MW 92.09                                              44 ml
    Bring to 1,000 ml using deionized distilled water and autoclave in large media bottles.
    Separately autoclave                                                                  per 100 ml
    1 M MgSO4∙7 H2O (Magnesium sulfate) MW 246.475                     24.6 g
    Immediately prior to use, aseptically add 0.4 ml of 1M magnesium sulfate stock per liter of freezing medium and swirl to mix. The appropriate antibiotic should be also added at this time.

Acknowledgments

We appreciate the willingness of the Arabidopsis Biological Resource Center (ABRC), for accepting the TFome collection for storage, propagation and distribution. We thank Diego Mauricio Riaño-Pachón for his assistance with curation of the gene families. We thank the contributions of more than 300 University of Toledo undergraduate students who participated in the FIRE (Fostering the Integration of Research with Educational laboratory classes) program, as well as Azam Abdollahzadeh, Andrew Reed, Erik Mukundi, Evans Kataka, Narmer Fernando Galeano Vanegas, Flavia Santos, Hai-Dong Yu, Jeffrey Campbell, Tina Agarwal, Jennifer Carstens, Katja Machemer-Noonan, Kelly Scarberry, Kengo Morohashi, Kristen Belesky, Maria Tobias, Noor Zayed, Thais Andrade and Tomoe Kusayanagi and SiGuE (Success in Graduate Education, http:// www.sigue-caps.org/) fellows Miriam Mills and Gilbert Kayanja, for their outstanding contributions in team-cloning. Michael dos Santos Brito thanks FAPESP (Sao Paulo Research Foundation) for postdoctoral fellowship BEPE 2012/20486-2. Support for this project was provided by NSF IOS-1125620 to JG, AID and EG.

References

  1. Buitrago-Florez, F. J., Restrepo, S. and Riano-Pachon, D. M. (2014). Identification of transcription factor genes and their correlation with the high diversity of stramenopiles. PLoS One 9(11): e111841.
  2. Burdo, B., Gray, J., Goetting-Minesky, M. P., Wittler, B., Hunt, M., Li, T., Velliquette, D., Thomas, J., Gentzel, I., dos Santos Brito, M., Mejia-Guerra, M. K., Connolly, L. N., Qaisi, D., Li, W., Casas, M. I., Doseff, A. I. and Grotewold, E. (2014). The Maize TFome--development of a transcription factor open reading frame collection for functional genomics. Plant J 80(2): 356-366.
  3. Carlson, J. E., Tulsieram, L. K., Glaubitz, J. C., Luk, V. W., Kauffeldt, C. and Rutledge, R. (1991). Segregation of random amplified DNA markers in F1 progeny of conifers. Theor Appl Genet 83(2): 194-200.
  4. Finn, R. D., Bateman, A., Clements, J., Coggill, P., Eberhardt, R. Y., Eddy, S. R., Heger, A., Hetherington, K., Holm, L., Mistry, J., Sonnhammer, E. L., Tate, J. and Punta, M. (2014). Pfam: the protein families database. Nucleic Acids Res 42(Database issue): D222-230.
  5. Gu, Z., Cavalcanti, A., Chen, F. C., Bouman, P. and Li, W. H. (2002). Extent of gene duplication in the genomes of Drosophila, nematode, and yeast. Mol Biol Evol 19(3): 256-262.
  6. Li, Z. and Trick, H. N. (2005). Rapid method for high-quality RNA isolation from seed endosperm containing high levels of starch. Biotechniques 38(6): 872, 874, 876.
  7. Perez-Rodriguez, P., Riano-Pachon, D. M., Correa, L. G., Rensing, S. A., Kersten, B. and Mueller-Roeber, B. (2010). PlnTFDB: updated content and new features of the plant transcription factor database. Nucleic Acids Res 38(Database issue): D822-827.
  8. Soderlund, C., Descour, A., Kudrna, D., Bomhoff, M., Boyd, L., Currie, J., Angelova, A., Collura, K., Wissotski, M., Ashley, E., Morrow, D., Fernandes, J., Walbot, V. and Yu, Y. (2009). Sequencing, mapping, and analysis of 27,455 maize full-length cDNAs. PLoS Genet 5(11): e1000740.
  9. Woo, S. S., Jiang, J., Gill, B. S., Paterson, A. H. and Wing, R. A. (1994). Construction and characterization of a bacterial artificial chromosome library of Sorghum bicolor. Nucleic Acids Res 22(23): 4922-4931.
  10. Yilmaz, A., Mejia-Guerra, M. K., Kurz, K., Liang, X., Welch, L. and Grotewold, E. (2011). AGRIS: the Arabidopsis Gene Regulatory Information Server, an update. Nucleic Acids Res 39(Database issue): D1118-1122.
  11. Yilmaz, A., Nishiyama, M. Y., Jr., Fuentes, B. G., Souza, G. M., Janies, D., Gray, J. and Grotewold, E. (2009). GRASSIUS: a platform for comparative regulatory genomics across the grasses. Plant Physiol 149(1): 171-180.

简介

任何模式生物的基因的开放阅读框(ORFeomes)的物理集合的构建是探索基因功能,基因调节和蛋白质 - 蛋白质相互作用的有用工具。在这里我们详细描述已经用于开发玉米中转录因子(TF)和共调节子(CR)开放阅读框(TFome)的第一集合的方案(Burdo等人, 2014)。该TFome用于建立基因调控网络(GRN)的架构,负责控制生物体中所有基因的转录。这里概述的协议描述如何进行时,只有一个不完整的基因组与部分注释可用。 TFome克隆在Gateway系统的重组准备好的载体中制备,允许ORF容易地转移到其它Gateway载体 - 例如那些合适的载体上用于在其他宿主物种中表达。虽然这个协议是为玉米TFome开发的,它可以很容易地应用于在其他真核物种中产生完整的ORFeome集合。

[Protocol overview] 成功的一个重要方面TFome代是用于建立可靠的一组基因模型的初始努力,使得它们随后可以被扩增或合成。用于特定物种的实际TFome构建方案将取决于可获得的资源,例如全长cDNA(flcDNA)收集和可靠的参考基因组(图1)。
在玉米的情况下,可获得flcDNA集合和基因组草图,但前者仅提供所需克隆的30%,后者含有缺口和一些错误的基因模型。为了开发用于玉米TF的接近完整的靶基因模型组,开发了如Yilmaz等人(2009)所述的生物信息学管道。简而言之,开发了一个双管齐下的搜索过程。第一个涉及在其他物种中制备TF的蛋白质序列的集合,并且可从数据库例如PlantTFDB,PlnTFDB和DBDTF获得。然后使用BLASTP将这些序列用于从玉米基因组草稿中搜索基因模型。第二个过程涉及开发定义TF家族并且在PFAM数据库中大多注释的域的集合(Finn等人,2014)。然后使用BLASTX将这些结构域用于搜索玉米基因组草图。存在的TF家族的数量及其命名可随着新成员被发现和研究而改变。表1提供了具有替代名称的已知TF家族的列表以及相应的PFAM域,其存在或不存在定义每个TF家族。可以从PFAM数据库( pfam.xfam.org )获取每个域的HMM模型。在BLAST搜索之后,消除冗余模型,然后基于每个基因模型中存在的TF基序,根据表1中指定的标准将基因模型分配给TF或共 - 调节子(CR)家族。最后,建议设置一个数据库来存储每个TF系列的信息。建立了GRASSIUS( www.grassius.org )网站,以访问存储的关于玉米,高粱的TF基因模型的信息,水稻,短柄草属(Brachypodium),甘蔗和其他草(Burdo等人,2014)。在下一部分中,假设至少一个基因组草图或草图转录组是可用的,并且一组基因模型是可用的,其已经从头确定或具有额外的手动注释。熟悉使用PERL脚本对于基因模型组装阶段是有利的。



图1.生成TFome项目的流程图。概述模板识别,PCR扩增和克隆转录因子的一般策略TF)全长(FL)开放阅读框(ORF)。 (修改自Burdo等人,2014)

  • Zudaire,E.,Gambardella,L.,Kurcz,C.and Vermeren,S。(2011)。 血管网络定量分析的计算工具 PLoS One < em> 6(11):e27385。
  • ... 下载可以指定如肽序列或编码 序列。默认情况下,选择所有基因模型,然后选择这些 可以作为输入文件的单个FASTA文件下载 后续步骤。
  • 假设草稿质量 转录本注释,需要消除冗余基因 模型在multiseq FASTA文件中。 GRASSIUS网站提供 自定义perl脚本"IdentifySeqRedundancy.pl" ( http://grassius.org/tools.html )。这个脚本也 需要的Perl模块"Digest :: MD5"可以从 综合Perl归档网络(CPAN)网站 ( http://search.cpan.org/dist/Digest-MD5/MD5。 pm )。一个也可以消除 使用BLAST搜索的物种内的冗余模型。蛋白质 如果它们在同一物种中发现,则任意地被认为是重复的,  查询覆盖率≥90%,或者查询身份≥90% 查询比对从起始密码子开始少于9个残基。 或者,可以使用更复杂的标准,例如Gu等人,2002, 可以用于鉴定基因组中的重复蛋白。如果这些 条件满足时,保留最长的蛋白质并消除  蛋白质被分类为相同或剪接变体。如果有  访问RNA-Seq数据集,他们可能用于证实目标TF 基因模型,但这种方法在这里没有概述。定位 最长剪接变体,其由EST或RNA-Seq数据支持 提供了最大的蛋白相互作用空间,用于鉴定 蛋白质-DNA和蛋白质 - 蛋白质相互作用 网络。
  • 扫描非冗余multifasta 文件作为隐藏的Markov模型的蛋白质结构域的集合 (HMM)(例如由PFAM或Interpro提供)提供蛋白质结构域 注释的推定蛋白质组。对于这个特定的步骤 需要软件HMMER。简而言之HMMER是一套工具 实现配置文件隐藏马尔可夫模型以找到相似性 蛋白质序列,并且是搜索PFAM HMM模型所必需的 蛋白质序列组。有可能从一个品种调用HMMER 的脚本语言,如perl或python,使用预构建HMMER 包装。 PFAM提供了以perl编写的那些脚本之一 (pfamscan.pl)与一组PFAM预先建立的参数。源代码  可从以下网站下载: ftp://ftp。 sanger.ac.uk/pub/databases/Pfam/Tools/PfamScan.tar.gz 。表格1  是已经用于定义TF的蛋白质结构域的汇编 和植物中的CR家族(Buitrago-Florez等人,2014; Burdo等人, 2014; Perez-Rodriguez等人,2010; Yilmaz等人,2009)。这个表 描述了用于定义基因的62个TF和26个CR家族 包括在玉米TFome中的模型。其中12个家庭没有  具有归属的PFAM域,但可以使用"内部"HMM来定义 (Buitrago-Florez et al。,2014; Perez-Rodriguez et al。 al。,2010)。 CCAAT-Hap2,3和5 TF家族的HMM可以是 从AGRIS数据库获取( http://arabidopsis.med.ohio-state.edu )  (Yilmaz等人,2011)。
  • 一旦基本模型的主要列表 注释与蛋白质域,他们分配到TF或CR家庭 使用表1中列出的标准。标准包括识别 一个或多个基序的存在和其他(禁止的)  图案(Buitrago-Florez等人,2014; Perez-Rodriguez等人,2010; Yilmaz等人,2009)。注释涉及自定义perl脚本 "get_InterProScanDomains.pl"将蛋白质分类为TF家族 interproscan输出,可在GRASSIUS网站上获得 ( http://grassius.org/tools.html )。脚本只保留重要的命中  e值≤0.001,并验证规则如表1所示 满足。当规则只是部分满足时 蛋白质被分配到"孤儿"TF。这可以是最大的类 TFs为一个物种(〜11.7%的玉米TFome),直到孤儿成员 分配给家庭。
  • 可以进行本地搜索 计算资源或当iPlant不可用时 协作发现环境公共资源 ( http://www.iplantcollaborative.org )。

  • 筛选质粒模板的EST或flcDNA集合
    1. 确定哪些cDNA资源可用于目标基因组 (见注1)。模型物种的大多数基因组项目包括产生 EST文库,虽然许多库不是公开的。 对于玉米TFome,广泛使用玉米flcDNA收集 ( http://www.maizecdna.org )(Soderlund 等人 ,2009)。个人  用于该文库和许多其它植物物种的cDNA克隆是可获得的  通过亚利桑那大学基因组学院( http://www.genome.arizona.edu )。
    2. 使用基因模型的编码序列作为查询序列, 然后使用BLASTP或BLASTX鉴定EST或flcDNA,其中 存在氨基末端,包括起始密码子 序列比对> 99%。不是100%相同的对齐 可能发生,因为EST序列由于测序而不是高保真 特别是在序列的3'末端的错误(Soderlund等人,2009)。   可选择的剪接同种型不由转录模型表示 也可以是目标,只要它们保持阅读框架 原始成绩单。
    3. 一旦找到可能的flcDNA模板, 然后从每个末端获得,分离和测序质粒 确认:a)它是正确的模板,b)它是全长。 一些cDNA文库在其构建期间利用酶进行切割 在导致部分克隆的编码序列内 不适合于整个编码序列的扩增。
    4. 一旦   鉴定靶基因的合适的靶质粒模板,然后a   样品稀释至1ng /μl,1ng作为模板在a中足够 PCR反应。

  • 其他试剂
    1. One Shot TOP10化学感受态细胞(Life Technologies,目录号:C404003)
    2. PureLink 植物RNA试剂(Life Technologies,目录号:2322-012)
    3. Turbo DNA-free TM 试剂盒(Life Technologies,Ambion ,目录号:AM1907)
    4. Thermo Scientific Maxima H minus 1 链合成试剂盒(Thermo Fisher Scientific,目录号:K1652)
    5. Ribolock?RNase抑制剂(Thermo Fisher Scientific,目录号:EO0382)
    6. EmeraldAmp MAX PCR Master Mix(Takara Bio Company,目录号:RR320A)
    7. Phusion 高保真聚合酶(New England Biolabs,目录号:M0530S)
    8. 或者pENTR TM/SD/D-TOPO载体(Life Technologies,目录号:K243520和/或pENTR TM/SD-D-TOPO) K242020)
    9. Genscript Taq聚合酶(GenScript USA Inc.,目录号:E000071000)
    10. EmeraldAmp MAX PCR Master Mix(Takara Bio Company,目录号:RR320A)
    11. 1kb梯度分子量大小标准品(Thermo Fisher Scientific,目录号:SM0311)
    12. RNA提取缓冲液I(参见配方)
    13. RNA提取缓冲液II储备溶液(参见配方)
    14. RNA提取缓冲液II(工作溶液)(参见配方)
    15. Carlson裂解缓冲液(参见配方)
    16. 冷冻介质(见配方)
  • 设备

    注意:这里列出的大多数设备是大多数实验室中存在的标准分子生物学仪器。一个大的TFome项目也将受益于使用多通道pippetters和96孔板格式化实验。

    1. 微量离心机
    2. 高速离心机
    3. 梯度热循环仪
    4. 凝胶电泳设备
    5. 水浴
    6. -80℃冰柜用于储存库存
    7. 建议用于所有DNA操作和克隆实验的气溶胶移液器吸头
    8. Wizard SV96质粒DNA纯化系统(Promega Corporation,目录号:A2255)
    9. 向导® Plus SV Minipreps DNA纯化系统(Promega公司,目录号:A1330)
    10. Wizard SV Gel and PCR Clean-Up System(Promega Corporation,目录号:A9281)。
    11. 基因合成(Life Technologies)
    12. 2ml 96孔培养皿(Thermo Fisher Scientific,目录号:12566121)
    13. 透气板密封(Thermo Fisher Scientific,目录号:AB0718)
    14. 0.5ml平板(USA Scientific,目录号:18965000)
    15. 7 mm大小的硅胶密封,可重新高压灭菌(Thermo Fisher Scientific,目录号:0339649)
    16. 矩阵样品储存系统(matrixtechcorp.com,Thermo Fisher Scientific,目录号:4111MAT)(Capit-All capper/Decapper),3740(条形码螺旋盖管)和4477(螺旋盖盘)
    17. (可选)用于96孔质粒制备的Vac-Man 96真空歧管(Promega公司,目录号:A2291)
    18. (可选)能够产生38-51厘米Hg或等效物的真空泵

    软件

    1. OligoAnalyzer 3.1软件( www.idtdna.com

    程序

    1. 用于PCR的核酸模板的制备
      这里,使用用于扩增TF编码序列的模板的三个主要来源:1)来自代表性cDNA文库的全长cDNA克隆,2)从从多种宿主物种组织纯化的RNA产生的cDNA,和3)来自宿主物种,其适合于从单个外显子基因扩增编码序列。本节提供了制备核酸模板的方法。
      1. 质粒模板用于TF ORF扩增。
        任何传统 质粒制备将提供足够量的质粒 纯度作为PCR扩增的模板。对于玉米TFome 项目中,需要数百个质粒并且96孔形式 合意。高通量系统证明是令人满意的 Wizard ® SV 96质粒DNA纯化系统。这个系统需要一个 真空泵能够产生38-51厘米汞柱或等效压力 Vac-Man ® 96真空歧管。然而,注意到最后 这种方法的质粒DNA浓度通常小于 自动Sanger双脱氧DNA测序所需的最低限度 约50ng /μl。如果质粒模板需要通过验证 DNA测序,然后进行更高产量的方案 将需要(例如


        Wizard ® Plus SV Minipreps DNA Purification 系统)。
      2. cDNA模板通过逆转录PCR(RT-PCR)进行TF ORF扩增。
        大多数TF基因在其时间和组织特异性基因中受到限制  表达模式。当考虑使用RT-PCR时 产生扩增子是有帮助的有一些证据 其中特定TF最高度表达以最大化的组织 成功扩增。对于玉米和一些其他植物物种, qTeller网站( www.qteller.com )提供了一个可搜索的资源, 显示来自RNA-Seq的基因转录物的相对丰度 数据。对于成功扩增的玉米基因,发现 他们总是从前三名的组织之一扩增  其中他们的表达被认为是最高使用qTeller资源。 植物生物学生物分析资源(BAR)( www.bar.utoronto.ca ) 和植物表达(PLEXDB)( http://www.plexdb.org )数据库 为更广泛的植物物种选择提供类似的资源。
        对于RNA样品的收集,大的植物群体(约100 玉米植物)应该从多个组织种植 将需要和收集某些组织(例如开发) 耳朵)将需要收获整个植物。对于一些基因,生物 和非生物胁迫,或昼夜节律,可能导致转录水平 显着增加。在玉米TFome的情况下,发现 可以扩增通过RT-PCR衍生的一半以上的克隆 从幼苗芽和根(Burdo等人,2014)。

        从植物组织中分离RNA
        通过两种方法之一从植物组织中分离总RNA。对于 非淀粉组织使用PureLink 植物RNA试剂 按照制造商的建议。用于从中分离RNA 淀粉组织,如发育和成熟种子,该方法 由Li和同事描述(Li和Trick,2005) 轻微修改。为了方便,协议和解决方案配方  详细如下。通常,从1克中分离总RNA 组织。
        1. 研磨1克组织在研钵和杵用液体 氮。为了成功实施,组织是必不可少的 立即在收获时在液氮中冷冻并储存 -80℃。不应允许组织解冻 在添加提取缓冲液之前的任何时间
        2. 将粉末转移到无RNA酶的离心管中,用4ml提取缓冲液I,立即剧烈混合
        3. 加入2.5ml苯酚:氯仿混合物(1:1,pH4.7),通过倒置充分混合
        4. 在4℃下以13,000×g离心15分钟。
        5. 转移上层水相(2.5毫升)到一个新的无RNA酶离心机  管中装有2.5ml提取缓冲液II。样品通过混合 温和倒置并在室温下温育10分钟
        6. 加入2ml氯仿 - 异戊醇(24:1),充分混合,并在4℃下将样品在13,000×g离心15分钟。
        7. 回收上清液(4.5ml),加入3ml异丙醇和2.5 ml的1.2M NaCl。通过温和倒置混合样品,温育 冰15分钟,并在4℃下以13,000×g离心15分钟。
        8. 弃去上清液,用400μl70%乙醇小心洗涤沉淀
        9. 在室温下干燥经洗涤的沉淀物,并在-70℃下储存之前重悬于不含RNase的水(约500μl)。
          通常,总RNA的产量为2.2±0.9μg/μl,其中A 260和A 280为 A n 260/a/230比率分别为1.93±0.06和2.32±0.32(n = 30)。 比率<1.7和2.0的总RNA样品被认为不适合。 RNA完整性必须通过在变性凝胶上分离来评估。的 可接受质量的总RNA的出现显示在图2A中。对于 大多数组织,大和小核糖体带应该是锋利的 上带是下带的强度的两倍。微弱 延伸在核糖体条带上方和下方的涂片是指示的 mRNA。对于发芽的种子,核糖体条带将减少 强度,但涂片应该仍然可见。一些大分子 重量物质指示被除去的污染DNA DNase处理(见下文)。

      基因组DNA的分离
      对于编码序列包含在一个外显子(约7%的玉米中的TF基因)内的TF基因,总基因组DNA是扩增子产生的合适模板。源材料应与项目中使用的参考基因组匹配。这将允许用户辨别扩增子中的单核苷酸多态性(SNP)是否是由于扩增过程中的错误,而不是由于种质中天然存在的SNP。有许多合适的基因组DNA分离方案可用,本项目中使用的方法总结如下 植物DNA分离方案(从Carlson等人修改,1991)。
      1. 用冷的研钵和杵研磨1g液体氮中的叶材料
      2. 将地面材料转移到含有10的无菌Oakridge管中 ml的预热至70℃的Carlson缓冲液(通过静置在水浴中)
      3. 在70℃下通过在水浴 - 倒置中孵育20分钟 每隔几分钟混合内容物(使用摇动水浴如果 可用)。
      4. 将样品冷却至室温,加入5毫升 氯仿/异戊醇(24:1)并涡旋振荡20秒。然后 在4℃下以5,000xg离心10分钟。
      5. 转移上 水相置入新鲜(无菌)Oakridge管中。加入50μl的10 mg/ml RNA酶A(预煮沸并在-20℃冷冻) - 在37℃孵育 20分钟以降解RNA
      6. 加入等体积的异丙醇 (2-丙醇),涡旋并在室温下孵育20分钟 允许DNA沉淀。在4℃,5,000x下离心20分钟  g 。
      7. 将沉淀重悬在600μlTE(1mM Tris,0.1mM EDTA,pH8.0)中,并转移到Eppendorf管中。
      8. 加入5微升的10毫克/毫升核糖核酸酶A,并在37℃下孵育30分钟
      9. 用等体积的DNA提取DNA 苯酚/氯仿/异戊醇(25:24:1)中,并在4℃下以14,000×g离心5分钟。
      10. 将顶部水相转移到新的Eppendorf管中。用等体积(现在约0.5ml)提取 氯仿/异戊醇(24:1)并在14,000×g离心5分钟  在台式微量离心机(4℃)中
      11. 通过加入沉淀DNA  1/10体积的3M NaOAc或5M NH 4 OAc和2体积的100%乙醇。 通过温和倒置混合并在4℃下以14,000×g离心5分钟。
      12. 用70%乙醇洗涤DNA沉淀一次或两次
      13. 取出洗涤上清液,让沉淀物空气干燥约10  min,但最好不要让DNA变得完全干燥。重悬 将DNA在约200μlTE缓冲液(1mM Tris,0.1mM EDTA,pH8.0) 并通过UV光谱法测定浓度。
        DNA分离  该方法应具有A <260> /A <280> /A <230>比>>。 2.0。对于复杂的基因组如玉米应该使用100 ng  作为PCR反应中的模板。

    2. 从总RNA中产生互补DNA(cDNA)
      1. 在产生cDNA之前,重要的是去除任何 污染来自总RNA制剂的基因组DNA。在这里 项目的Turbo DNA- free™工具包根据 制造商的建议。使用四单位的TURBO TM DNase 从20μg总RNA中去除任何污染的DNA。以下 在37℃处理30分钟,加入DNase失活基质  室温下孵育5分钟后,分离 10,000×g离心。除去上清液并用于a 逆转录反应。
      2. 建议高 保真逆转录酶用于减少cDNA中的错误 其将充当靶基因扩增的模板。我们就业 Thermo Scientific Maxima H minus 1 st 链合成试剂盒 制造商的建议,除了在每个逆转录反应中加入40单位的Ribolock RNase抑制剂。使用 该方案,12μlDNA酶处理的RNA(约5μg总RNA)  在20μl反应中与提供的寡聚dT引物退火。 对于单一PCR,0.5至1μl的该反应是足够的 大多数TF基因的扩增。在进行PCR之前,质量 通过扩增管家的转录物来评估cDNA 基因。为此目的,低保真DNA聚合酶, EmeraldAmp ® MAX PCR Master Mix就足够了。对于玉米,1,001 bp 部分的ZmGAPDH(GRMZM2G046804)转录物 引物ZmGAPDH_F(5'-ATGCAGGCAAGATTAAGATCGGAATCAAC-3')和 ZmGAPDH_R(5'-CATGTGGCGGATCAGGTCGAC-3')。缺少较大的2,817 bp PCR产物证实去除了基因组DNA(图2B)。

    3. 扩展TF编码序列
      1. 合成引物以扩增相应的ORF而没有  各个终止密码子和另外的5'-CACC-3'核苷酸尾 加入到正向引物中,用于定向克隆到pENTR TM中 载体。对于玉米,就GC分析了1,273个引物对 含量和熔融温度(Tm)。 Tm值使用  OligoAnalyzer 3.1软件( www.idtdna.com )。正向引物具有  平均长度为25±4bp,包括5'CACC尾,这是 类似于反向引物(23±4bp)。但是GC 正向引物(62.8±9.1)的含量约高12% 反向引物(50.2±11.7)。结果,平均Tm  正向引物比正向引物高约3℃(66±5℃) 反向引物(63±5℃)。此外,平均GC含量 扩增子为62.4±8.8%,范围为78.9-38.2%(n = 1,411)。 由于GC含量相对较高,缓冲区为GC丰富的模板 常规用于CDS扩增。当设计引物时  应该努力使引物在Tm中具有差异(ΔTm)  一对尽可能低。在这个项目中,平均ΔTm为 3.2±4.1℃,然而扩增子成功地获得,即使当 ΔTm高达19℃。
      2. 接下来使用a生成PCR产物  高保真聚合酶。对于这个项目,Phusion ®高保真 选择聚合酶,因为它表现出大于的错误率 比Taq DNA聚合酶低50倍。此外,Phusion 聚合酶具有增强的持续性结构域,其需要 延伸时间仅为约15秒/kb,我们发现a 延长时间为1分钟对于甚至最长的扩增子也是足够的。 玉米基因组中TF的编码序列平均为1,024± 长度为474bp,范围为90至3,705bp。推荐 PCR的初始组成列于表2中 使用GC缓冲液和10%甘油是最成功的 初始条件(表2,设置A)。如果这个初始条件是 不成功,建议接下来调整退火温度 (笔记2)。发现添加DMSO或额外的MgCl 2也是如此 提高了一些模板的成功率(表2;设置B和C)。 PCR的循环参数如下:单个初始 98℃变性1分钟,25-35个循环的扩增  98℃变性20秒,〜60℃退火40秒,72℃ 延伸15秒/kb,1个最后延伸步骤72℃5分钟。 重要的是在完成最终延伸步骤之后 取出PCR反应物并处理或储存在-20℃ 直到处理(注3)。循环数取决于模板 资源。少至25个循环可以用于从质粒扩增 模板,但是当使用cDNA模板时通常需要35个循环。  通常,较少的周期数将减少错误的机会 由于聚合酶
        表2.推荐的PCR组合


        选项

        组件(按此顺序添加)
        A
        B
        C

        vol(μl)
        vol(μl)
        vol(μl)
        H sub 2 O
        11
        15.75 -15
        10 - 10.75
        GC缓冲液(5x)含有7.5mM MgCl 2·h/v 5
        5
        5
        正向引物(20μM原液)
        1
        1
        1
        反向引物(20μM原液)
        1
        1
        1
        dNTPs (25mM储液)
        1
        1
        1
        MgCl 2(50mM储液)
        0
        0
        0.25 -1.0
        甘油(50%股票)
        5
        0
        5
        DMSO(100%储液)
        0
        0.25 - 1.0
        0
        模板*(质粒,cDNA或DNA)
        0.5
        0.5
        0.5
        Phusion 聚合酶*(5U /μl)
        0.5
        0.5
        0.5
        总量
        25μl
        25μl
        25μl
        *模板量,质粒-1ng,cDNA-0.5μlRT反应,基因组DNA-100ng

      3. 一旦PCR完成,产物通过凝胶分离 电泳鉴定产品的预期大小。当质粒  模板,但是通常观察单个频带 当使用基因组DNA或cDNA或当更长的片段时 预期更常见的是观察多个频带(图2C)。的 存在正确大小的带不是保证 正确的编码序列已被扩增,特别是对于基因 它们属于存在多个旁系同源物的TF家族。

    4. PCR扩增子的克隆和确认
      1. 通过DNA凝胶鉴定正确大小的DNA条带 切下电泳并凝胶纯化。有很多合适 用于该步骤的纯化试剂盒。对于这个项目,我们使用 Wizard ® SV凝胶和PCR清理系统。仔细定量纯化 结扎前的产品
      2. 然后克隆纯化的PCR产物 进入Gateway pENTR TM/D-TOPO 或pENTR TM/SD/D-TOPO 载体。这些 载体允许克隆轻易转移到备选中 表达载体并大大增加TFome的效用 采集。根据制造商进行连接 推荐的协议,并用于化学转化One Shot ® TOP 10 感受态细胞。如果需要,可以进行一半的反应尺寸 使用推荐的插入向量比例
      3. 然后筛选卡那霉素抗性菌落的插入片段 正确的大小和方向通过菌落PCR或限制性消化 的候选质粒。我们注意到克隆的相当大的变化 效率使用Gateway ®条目克隆试剂盒。这个困难是 通过一次使用整个套件克服,以最大化效率。如果 克隆效率高,则可以从一个或多个分离质粒DNA 两个殖民地和这些用于确认。然而,发现  由于克隆效率的变化,菌落PCR通常是a 更有效的识别插页的方法。
        菌落PCR鉴定阳性克隆
        首先,制备通用目的DNA聚合酶等的主混合物  作为Genscript Taq聚合酶,在制造商提供的缓冲液(50μl)中 mM KCl,10mM Tris HCl,pH 9.0,25℃),1.5mM MgCl 2,1%Triton X-100)。还发现EmeraldAmp MAX PCR主混合物适用于 菌落PCR)。也可以加入5-10%DMSO。向该混合物中 将合适的正向和反向引物加入最终 浓度为0.4μM。然后将25μl等分试样加入到孔中 单独的PCR管或96孔PCR板。无菌牙签或 移液管尖端然后用于隔离单个参考菌落,即 首先混合成单个PCR反应管,然后平板a 复制的菌落被筛选。 PCR的循环参数为  如下:95℃5分钟的单一初始变性循环,35 循环扩增95℃变性1分钟,〜60℃ 退火1分钟,72℃延伸1分钟/kb。 PCR后  产物通过凝胶电泳分离并检查a 产物的预期大小(图2D)。 6-8个殖民地之间 由于克隆的变化,对每个克隆进行常规筛选 效率(图2D)。用载体建立初始PCR反应 特异性正向和反向引物,从而产物被扩增 只有当基因以正确的方向插入时。如果是  没有产物被扩增,载体和/或基因的多种组合  可以测试特异性引物。一旦具有插入物的克隆 鉴定,然后制备质粒(见上文)以通过DNA确认  顺序。
      4. 一旦殖民地产生预期的产物 大小在菌落PCR筛选中,克隆插入片段的序列 通过单读配对末端Sanger双脱氧测序和 将克隆序列与参考基因组的序列进行比较。克隆  长于1,200 bp,设计内部引物以完成 序列确认。最常见的问题出现在 确认是不符合的SNP的发生 参考基因组。如果SNP导致沉默突变,或者如果SNP是  输入GRAMENE数据库( www.gramene.org )作为自然变体,  那么认为添加到TFome收集中是可接受的。
      5. 重要的是制备每种重复的永久甘油原液 立即验证克隆,以避免在生成TFome时出现混淆 采集。

    5. 罕见的TF转录物的基因合成 化学基因合成是证明难以进行PCR扩增的基因的选择。该选项提供的优点是,可以针对在不同宿主中的表达优化密码子使用,以及TFome收集的期望的下游使用,并且生产时间快。然而,目前, 1 kb长。另一个缺点是通过不使用源自植物组织的模板,那么对于特定转录物的存在在体内可能几乎没有或没有直接的实验支持。因此,建议在可能的情况下,在化学合成之前寻找特定基因模型的RNA-Seq支持。
      对于玉米TFome项目,约30%的收集是化学合成的(Burdo等人,2014)。使用GeneArt 技术进行基因合成。该技术采用密码子优化过程,其是任何复杂或富含GC的序列所需要的,并增加玉米中的表达。在玉米TFome的情况下,试验性测试显示,与酵母优化的形式相比,玉米优化的构建体在酵母中以较低但显着的水平表达。在玉米原生质体中不同密码子优化的形式之间没有观察到表达的显着差异(Burdo等人,2014)。因此,在玉米TFome的情况下,化学合成的克隆被优化用于在玉米中的表达,其中预期进行大多数下游实验。
      一旦合成片段,将它们重悬浮于水中,并克隆到上述D节中描述的相关Gateway 进入载体中。

    6. TFome的存储
      鉴于TFome收集的效用,应仔细考虑如何存储和提供TFome收集以供长期分发。玉米TFome通过拟南芥生物资源中心(ABRC)公开获得( abrc.osu.edu )。建议创建一个或多个备份TFome集合并存储在单独的位置。以96孔格式存储允许更容易地复制整个文库,但是可以使得个体克隆的分布更加困难。这里我们描述了一种用于存储96孔格式的TFome集合的方法 TFome的低温储存96孔格式
      1. 根据提供的配方准备灭菌的冷冻介质 其下面改编自Woo等人(1994)。这种介质允许 细菌细胞的生长和含有冷冻保护剂 生长后直接冷冻培养物。在层流气流中 内阁,等分1.8毫升含有适当冷冻介质 抗生素加入无菌的2ml 96孔培养皿中。
      2. 使用 无菌技术,用单个细菌接种96孔板  来自板或先前制备的甘油原液。什么时候 接种种群,可以有帮助的井的地图 以避免错误。用透气板密封板密封 适合通风和存储细菌/细胞培养物。放置 密封板在37℃下在振荡培养箱中以220rpm过夜 旋转适当曝气
      3. 由于细胞在其中生长 冷冻培养基,这只是一个无菌分装400μl的问题 的培养物加入无菌的0.5ml平板中,并将其密封以储存  -80℃。此时可以制作多个永久板。的 板可以储存在-80℃下,具有塑料盖或7mm大小 硅胶密封,可以重新高压灭菌。永久物也可以在a  96孔格式,允许自动去盖,如Matrix  样品存储系统。

    代表数据



    图2.代表性数据。植物RNA分离和质量控制,长模板的RT-PCR和菌落PCR的样品结果。 A.通过凝胶电泳分离的DNA酶处理前的总RNA的外观。未降解的RNA样品将显示代表核糖体RNA(泳道1中的带框带)的两条主带和指示mRNA的背景拖尾(泳道4中的箭头)。注意,在发芽的种子中看到减少的核糖体RNA的量,因为胚乳不存活。较高分子量条带指示必须通过DNA酶处理去除的基因组DNA。 Std = Generuler TM 1kb梯度分子量大小标准品。样品如下:-1:来自L13阶段植物的植物支持根,2:来自L13阶段植物的成熟雄花穗3:授粉后2周发育种子4:授粉后1天的整个穗,3周龄植物的5个秧苗根, 6,7和8:在萌发后1,2和7天发芽种子。 B.从在图A中分离的RNA产生的cDNA中扩增GAPDH。1.1kb的单条带指示GAPDH转录物,而如果扩增从基因组DNA发生,则预期4.3kb条带。 Std = Generuler TM 1kb梯度分子量大小标准品,如图2A所示。 cDNA样品来自如图2A所示的相同样品。 C.通过RT-PCR扩增具有长编码序列的TF。通常在RT-PCR期间从总cDNA样品观察到多条带。星号表示被切除并用于克隆的正确大小的条带。样品和预期的扩增子长度如下:-1:GRMZM2G069365,2,127bp,2:GRMZM2G171600,2,526bp,3:GRMZM2G028980,2,742bp,4,5:GRMZM2G160005,3159bp。 Std = Generuler TM 1kb梯度分子量大小标准品,如图2A所示。 D.样品菌落PCR反应。具有星号的泳道代表具有含有预期大小的插入物的克隆的集落。克隆不同基因的成功率的高可变性强调了对基于快速PCR的筛选方法的需要。 Std = Generuler TM 1kb梯度分子量大小标准品,如图2A所示。泳道1-8:来自GRMZM2G140156的扩增子,预期大小为2.6kb。泳道9-16:来自GRMZM2G009478的扩增子预期的片段大小为1.8kb。

    笔记

    1. 优质flcDNA文库的可用性将是影响TFome项目成本的最重要因素之一。通过RT-PCR扩增来自cDNA的罕见和长的转录物可能是具有挑战性的,长的编码序列的合成可能是非常昂贵的。
    2. 如果在初始PCR后没有观察到条带,则推荐将退火温度降低2℃。相反,如果看到多个条带,则建议将退火温度提高2℃
    3. 建议不要让PCR反应在扩增循环结束时在4℃下保持任何时间长度,因为这将允许扩增子末端降解并降低克隆效率。

    食谱

    1. RNA提取缓冲液I(Li and Trick,2005)            每升            每100 ml
      100 mM Tris(pH 8.0)MW 121.14                  ;     12.11g               1.211克
      150 mM LiCl MW 42.39                     ;                 6.34 g                0.63克
      50 mM EDTA MW 372.24                       ;                18.6 g                1.86克
      1.5%2-巯基乙醇                     ;              15毫升                 1.5 ml
    2. RNA提取缓冲液II储备溶液(Li and Trick,2005)           每升               每100 ml
      库存0.75 M柠檬酸钠          MW 294.1                                  220.5 g                   22克
      库存2 M乙酸钠(无水)pH 4.0 MW 82.03              164 g                    16.4克
      注意:需要加温,并使用冰醋酸调节至pH4.0。
      库存10%月桂基肌氨酸         MW 293.39         ;                     29.34 g                 2.93克
      注意:使用10%储备溶液,需要加热至68℃以溶解。
    3. RNA提取缓冲液II(工作溶液)(Li and Trick,2005)  每升                        每100 ml
      4.2 M异硫氰酸胍(w/v)MW 118.16                ;   496.27 g                       49.6克
      0.5%月桂基肌氨酸Sigma(10%股票)                  ;      50毫升                       5 ml
      1 M醋酸钠(2 M原液)                                       500 ml                      50 ml
      25mM柠檬酸钠(0.75M储备液)                                   33 ml                         3.3 ml
    4. Carlson裂解缓冲液(Carlson等人,1991)                                    每升
      100 mM Tris-Cl(pH 9.5)MW 121.14                 ;                      12.114克
      2%CTAB(十六烷基三甲基溴化铵)                  ;     20.0克
      1.4 M NaCl MW 58.44                    ;                          ;               81.82克
      1%PEG 6000或8000                                                            10克
      20mM EDTA,MW 372.24(> 5M储液= 18.61g/100ml) 40ml的0.5M原料
      需加热溶解CTAB。高压灭菌后,溶液将显得稍微不透明(多云)。
      注意:在使用前向管中加入β-巯基乙醇(每10ml中100μlBM)。
    5. 冷冻介质(Woo et al。,1994)                 ;                         ; 每升
      Luria-Bertani肉汤(LB)粉剂或颗粒剂                              25克
      36mM K 2 HPO 4 4 MW 174.2                                                         6.28克
      13mM KH sub 2 PO 4 sub MW 136.09                                                       1.8 g
      1.9mM Na 3 S 6 H 6 H 5 O 7(2H)2 H 2柠檬酸钠)分子量258.06                 0.5克
      6.8mM(NH 4)2 SO 4 SO 4(硫酸铵)MW 132.14                       0.9克
      4.4%C 3 H 8 O 3(甘油)MW 92.09                                             44 ml
      使用去离子蒸馏水和高压灭菌器在大型培养基瓶中加入1000ml 分别高压灭菌器                                                                 每100 ml
      1 M MgSO 4 4·H 2 O(硫酸镁)MW 246.475        ;         24.6克
      在即将使用前,无菌地每升冷冻介质中加入0.4ml 1M硫酸镁储备液并旋转混合。此时还应加入适当的抗生素。

    致谢

    我们赞赏拟南芥生物资源中心(ABRC)愿意接受储存,繁殖和分发的TFome收集品。我们感谢Diego MauricioRiaño-Pachón为基因家族的策划提供的帮助。我们感谢300多名托莱多大学本科生的贡献,他们参加了FIRE(促进研究与教育实验室课程的整合)计划,以及Azam Abdollahzadeh,Andrew Reed,Erik Mukundi,Evans Kataka,Narmer Fernando Galeano Vanegas ,Flavia Santos,Hai-Dong Yu,Jeffrey Campbell,Tina Agarwal,Jennifer Carstens,Katja Machemer-Noonan,Kelly Scarberry,Kango Morohashi,Kristen Belesky,Maria Tobias,Noor Zayed,Thais Andrade和Tomoe Kusayanagi和SiGuE ,http://www.sigue-caps.org/)研究员米里亚姆米尔斯和吉尔伯特Kayanja,他们在团队克隆的杰出贡献。 Michael dos Santos Brito感谢FAPESP(圣保罗研究基金会)博士后研究金BEPE 2012/20486-2。该项目的支持由NSF IOS-1125620提供给JG,AID和EG。

    参考文献

    1. Buitrago-Florez,F.J.,Restrepo,S.and Riano-Pachon,D.M。(2014)。 鉴定转录因子基因及其与原生藻菌素高度多样性的相关性 PLoS One 9(11):e111841。
    2. Burdo,B.,Gray,J.,Goetting-Minesky,MP,Wittler,B.,Hunt,M.,Li,T.,Velliquette,D.,Thomas,J.,Gentzel,I.,dos Santos Brito, M.,Mejia-Guerra,MK,Connolly,LN,Qaisi,D.,Li,W.,Casas,MI,Doseff,AI和Grotewold,E。(2014)。 玉米TFome - 为功能基因组学开发转录因子开放阅读框集合。 Plant J 80(2):356-366。
    3. Carlson,J.E.,Tulsieram,L.K.,Glaubitz,J.C.,Luk,V.W.,Kauffeldt,C。和Rutledge,R。(1991)。 在F1针叶树后代中随机扩增的DNA标记的分离。 Theor Appl genet 83(2):194-200。
    4. Finn,RD,Bateman,A.,Clements,J.,Coggill,P.,Eberhardt,RY,Eddy,SR,Heger,A.,Hetherington,K.,Holm,L.,Mistry,J.,Sonnhammer, ,Tate,J。和Punta,M。(2014)。 Pfam:蛋白质家族数据库 Nucleic Acids Res 42(数据库问题):D222-230。
    5. Gu,Z.,Cavalcanti,A.,Chen,F.C.,Bouman,P。和Li,W.H。(2002)。 果蝇,线虫和酵母基因组中的基因重复范围 Mol Biol Evol 19(3):256-262。
    6. Li,Z.和Trick,H.N。(2005)。 从含有高含量淀粉的种子胚乳中快速分离高质量RNA的方法。 Biotechniques 38(6):872,874,876.
    7. Perez-Rodriguez,P.,Riano-Pachon,D.M.,Correa,L.G.,Rensing,S.A.,Kersten,B.and Mueller-Roeber,B。 PlnTFDB:植物转录因子数据库的更新内容和新功能。 Nucleic Acids Res 38(数据库问题):D822-827
    8. Soderlund,C.,Descour,A.,Kudrna,D.,Bomhoff,M.,Boyd,L.,Currie,J.,Angelova,A.,Collura,K.,Wissotski,M.,Ashley, Morrow,D.,Fernandes,J.,Walbot,V.and Yu,Y。(2009)。 27,455玉米全长cDNA的测序,定位和分析 PLoS Genet 5(11):e1000740。
    9. Woo,S.S.,Jiang,J.,Gill,B.S.,Paterson,A.H.and Wing,R.A。(1994)。 <> Sorghum bicolor的细菌人工染色体文库的构建和表征。 Nucleic Acids Res 22(23):4922-4931。
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    Copyright: © 2015 The Authors; exclusive licensee Bio-protocol LLC.
    引用:Gray, J., Burdo, B., Goetting-Minesky, M. P., Wittler, B., Hunt, M., Li, T., Velliquette, D., Thomas, J., Agarwal, T., Key, K., Gentzel, I., Brito, M. d., Mejía-Guerra, M. K., Connolly, L. N., Qaisi, D., Li, W., Casas, M. I., Doseff, A. I. and Grotewold, E. (2015). Protocol for the Generation of a Transcription Factor Open Reading Frame Collection (TFome). Bio-protocol 5(15): e1547. DOI: 10.21769/BioProtoc.1547.
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