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Identification of Insertion Site by RESDA-PCR in Chlamydomonas Mutants Generated by AphVIII Random Insertional Mutagenesis
在通过AphVIII随机插入诱变产生的衣藻突变体中采用RESDA-PCR鉴定插入位点   

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The Plant Journal
Apr 2017

Abstract

Chlamydomonas reinhardtii is frequently used as a model organism to study fundamental processes in photosynthesis, metabolism, and flagellar biology. Versatile tool boxes have been developed for this alga (Fuhrmann et al., 1999; Schroda et al., 2000; Schroda, 2006). Among them, forward genetic approach has been intensively used, mostly because of the high efficiency in the generation of hundreds of thousands of mutants by random insertional mutagenesis and the haploid nature therefore phenotypic analysis can be done in the first generation (Cagnon et al., 2013; Tunçay et al., 2013). A major bottleneck in the application of high throughput methods in a forward genetic approach is the identification of the genetic lesion(s) responsible for the observed phenotype. In this protocol, we describe in detail an improved version of the restriction enzyme site-directed amplification PCR (RESDA-PCR) originally reported in (González-Ballester et al., 2005). The improvement includes optimization of primer combination, the choice of DNA polymerase, optimization of PCR cycle parameters, and application of direct sequencing of the PCR products. These modifications make it easier to get specific PCR products as well as speeding up subcloning steps to obtain sequencing data faster.

Keywords: Chlamydomonas reinhardtii (莱茵衣藻), Insertional mutagenesis (插入诱变), RESDA PCR (RESDA PCR), Forward genetic (正向遗传), Chlamydomonas mutant (衣藻突变体)

Background

In addition to the restriction enzyme site-directed amplification PCR (RESDA-PCR) (González-Ballester et al., 2005), several other molecular techniques have been developed to identify insertion sites within the nuclear genome, including Genome Walker (Stirnberg and Happe, 2004), thermal asymmetric interlaced PCR (TAIL-PCR) (Dent et al., 2005), 3’-rapid amplification of cDNA ends (3’RACE) (Meslet-Cladiere and Vallon, 2012), Mme1-based insertion site sequencing strategy (ChlaMmeSeq) (Zhang et al., 2014), or whole-genome resequencing (Goold et al., 2016). RESDA-PCR is based on the use of specific primers of the marker gene combined with the use of degenerate primers that anneal with sequences of restriction sites highly and randomly distributed in the nuclear genome. RESDA-PCR is one of the most commonly used, is not too expensive and has been found to give the highest possibility in identifying the flanking sequence in our hands.

Materials and Reagents

  1. Falcon conical centrifuge tubes, 15 ml (Corning, catalog number: 430055 )
  2. Eppendorf tube, 1.5 ml and 2.0 ml, Eppendorf QualityTM (Eppendorf, catalog numbers: 0030120086 and 0030120094 )
  3. Petri dishes, 90 mm in diameter (Thermo Fisher Scientific, SterilinTM, catalog number: 101R20 )
  4. Sterilized toothpick (Fujian Fuhua, FUHUA FANGYUANTM, catalog number: 855 )
  5. Chlamydomonas reinhardtii mutants generated by random insertional mutagenesis (Cagnon et al., 2013)
  6. One ShotTM TOP10 Chemically Competent cell (Thermo Fisher Scientific, catalog number: C404010 )
  7. Zero BluntTM TOPOTM PCR Cloning Kit (Thermo Fisher Scientific, catalog number: 450245 )
  8. Taq DNA polymerase (5,000 U ml-1), 10x Standard Taq Reaction Buffer, dNTPs (New England Biolabs, catalog number: M0273S )
  9. Dimethyl sulfoxide (DMSO) (Sigma-Aldrich, catalog number: 472301 )
  10. PCR-grade H2O (Sigma-Aldrich, catalog number: W1754 )
  11. Ethanol (Sigma-Aldrich, catalog number: 34852 )
  12. KOD Xtreme Hot Start DNA polymerase, dNTPs (2 mM each), 2x Xtreme buffer (Merck, Novagen®, catalog number: 71842 )
  13. Agarose (Sigma-Aldrich, catalog number: A9539 )
  14. ExactLadder® DNA PreMix 2 log (OZYME, catalog number: OZYC002-500 )
  15. SYBRTM Safe DNA gel stain (Thermo Fisher Scientific, catalog number: S33102 )
  16. UltraPureTM 10x TAE buffer (Thermo Fisher Scientific, catalog number: 15558026 )
  17. Luria broth (Sigma-Aldrich, catalog number: L1900 )
  18. Kanamycin sulfate (Thermo Fisher Scientific, catalog number: 11815024 )
  19. NucleoSpin® Gel and PCR Clean-up Kit (MACHEREY-NAGEL, catalog number: 740609.10 )
  20. NucleoSpin® Plasmid Miniprep Kit (MACHEREY-NAGEL, catalog number: 740588.10 )
  21. Sodium dodecyl sulfate (SDS) (Sigma-Aldrich, catalog number: L3771 )
  22. Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: S9888 )
  23. Potassium chloride (KCl) (Sigma-Aldrich, catalog number: P9541 )
  24. Ethylenediaminetetraacetic acid (EDTA) (Sigma-Aldrich, catalog number: E9884 )
  25. Tris base (Sigma-Aldrich, catalog number: T6066 )
  26. Hexadecyltrimethyl ammonium bromide (CTAB) (Sigma-Aldrich, catalog number: H9151 )
  27. Isopropanol (Sigma-Aldrich, catalog number: W292907 )
  28. Phenol:chloroform:isoamyl alcohol (25:24:1) (Sigma-Aldrich, catalog number: P3803 )
  29. Proteinase K (20.0 mg ml-1) (Sigma-Aldrich, catalog number: P6556 )
  30. Lysis buffer (see Recipes)
    1. 0.7 M NaCl
    2. 10% SDS
    3. 0.5 M Tris-HCl, pH 8.0
    4. 10 mM EDTA
  31. 5 M KCl (see Recipes)
  32. 10% CTAB (see Recipes)
  33. 10% DMSO (see Recipes)
  34. 1.0% agarose gel (see Recipes)
  35. 1x TAE (see Recipes)
  36. 70% ethanol (see Recipes)

Equipment

  1. Shaker (Eppendorf, New BrunswickTM, model: Innova® 44 , catalog number: M1282-0002)
  2. AccumetTM pH meter (Fisher Scientific, model: 3-in-1 Set, catalog number: 13-636-AE153 )
  3. PCR Thermal Cyclers (Thermo Fisher Scientific, Applied BiosystemsTM, model: 2720 , catalog number: ED000651)
  4. Agarose electrophoresis tank (Bio-Rad Laboratories, model: Mini-Sub® Cell GT, catalog number: 1704401 )
  5. Gel Doc XR System (Bio-Rad Laboratories, model: Gel DocTM XR+ , catalog number: 5838)
  6. FisherbrandTM Common bench-top vortexer (Fisher Scientific, catalog number: 02-216-125 )
  7. Bench-top centrifuge (Beckman Coulter, model: Allegra® 64R , catalog number: 367586)
  8. Bench-top incubator (Eppendorf, New BrunswickTM, model: S41i , catalog number: S41I230011)
  9. NanoDrop 2000 (Thermo Fisher Scientific, model: NanoDropTM 2000 , catalog number: ND-2000)
  10. Multisizer 3 Coulter counter (Beckman Coulter, model: MultisizerTM 3 , catalog number: 6605697)

Software

  1. SPSS program (version 19.0)

Procedure

  1. DNA extraction and quantification
    1. Cultivate cells of Chlamydomonas reinhardtii (around 0.5 x 106 cells ml-1) in shake flasks at 25 °C under constant white fluorescent light (100 µmol photons m-2 m-1) in TAP liquid media (Harris, 2001) with gentle shaking. Normally it will reach exponential phase (around 5 x 106 cells ml-1) after 24 h.
    2. Isolate and purify the genomic DNA of the mutant with the CTAB method (Schroda et al., 2001). Briefly, after cells grow to logarithmic phase (around 5 x 106 cells ml-1), harvest a total of 5 x 107 cells by centrifugation and suspend in 500 µl lysis buffer (100 mM Tris-HCl, pH 8.0, 1.75 mM EDTA, 150 mM NaCl, 2% [w/v] SDS, and 1.0 mg ml-1 Proteinase K).
    3. Incubate the lysates for 2 h at 55 °C, and then add 80 µl of 5 M KCl and 70 µl of preheated 10% CTAB and incubate for 10 min at 65 °C.
    4. Extract the lysates twice with (500 µl) phenol:chloroform:isoamyl alcohol (25:24:1, v/v/v), and then add (500 µl) chloroform to eliminate the remaining phenol.
    5. The nucleic acids are precipitated for 20 min on ice by addition of 500 µl of isopropanol, and washed with 70% cold ethanol.
    6. Resuspend the air-dried pellets in 50 µl sterile water.
    7. Quantify the purified DNA using NanoDrop 2000, and add water to adjust the final concentration to be of 50 ng µl-1.
      Note: The concentration of the cells mentioned is measured with Multisizer 3 Coulter counter.

  2. PCR amplification
    This procedure largely follows the protocol first published by González-Ballester et al. (2005). To identify the DNA sequence around the site of insertion, two subsequent sets of PCR reactions are required, named here as the 1st and 2nd amplification. The 1st PCR amplification is to amplify a fragment from genomic DNA of the mutant using one AphVIII gene-specific primer with one of the degenerate primers (Figure 1). To increase the chances of amplification, 4 degenerate primers can be tested, and each of them contains a sequence-specific part (Q0 at the 5’ end) and a degenerate part (3’ end). The PCR product will be used as a template for a second round of PCR amplification (the 2nd PCR amplification) with the use of two sequence-specific primers. Degenerate primers (DegTaqI, DegPstI, DegAluI and DegSacII, including Q0) and the marker gene (AphVIII) specific primers (RB1 and RB4) are shown in Table 1.


    Figure 1. Outline of the principals for RESDA PCR. This schematic is adapted from González-Ballester et al., 2005.

    Table 1. Primers and their sequences used in this protocol

    I, inosine, N, A + T + G + C; S, G + C; W, A + T.
    *Gene-specific primers (RB1 and RB4) can be designed based on the type of marker gene (i.e., AphVII, Ble and aadA) used for mutagenesis.

    1. The first amplification
      In the 1st amplification, the AphVIII gene-specific primer (RB1) and one of the degenerate primers are used to amplify a fragment flanking the antibiotic marker gene AphVIII from the genomic DNA purified from the mutant. Four independent reactions can be set up by using four different degenerate primers and RB1, respectively. The composition of the PCR reaction mixture and cycling program are shown in Table 2 and Table 3, respectively.

      Table 2. Composition of the PCR reaction mixture used for the 1st amplification

      *Use one of the degenerate primers first.

      Table 3. The PCR conditions used for the 1st amplification

      *Each cycle contains thirteen steps sequentially; and totally twenty cycles were set up.

    2. The second amplification
      To get more specific PCR products, the maker gene-specific primer (RB4) and the specific primer (Q0) at the 5’ end of degenerate primers are used to amplify using the PCR from the 1st amplification as a template (One µl diluted PCR products (1/1,000) from 1st amplification). The composition of the PCR reaction mixture and cycling program are shown in Table 4 and Table 5, respectively.

      Table 4. Composition of the PCR reaction mixture used for the 2nd amplification


      Table 5. The conditions used for the 2nd PCR amplification


  3. Agarose-gel electrophoresis and DNA fragments purification
    1. Separate the PCR products from the 2nd amplification on 1.0% agarose gel at 100 V for 30 min by electrophoresis, and then stain the gel with SYBR Safe (1/1,000) and take image with a GelDocXR System (Bio-Rad Laboratories). Some representative results are shown in Figure 2, and the examples of RESDA-PCR success rate after amplification by four independent degenerat primers and RB1, followed by further amplification with RB4 and Q0 primers are shown in Table 6.
    2. Cut–off the well-separated PCR products (marked with *) from the gel and purify using the NucleoSpin Gel and PCR Clean-up Kit (MACHEREY-NAGEL) according to manufacturer’s instructions.


      Figure 2. Examples of results of RESDA-PCR using the degenerate primer DegTaqI. The PCR products shown are the results of two rounds of PCR reactions: using DegTaqI and RB1, followed by further amplification with RB4 and Q0 primers.

      Note: M refers to DNA marker (Exact Ladder DNA PreMix 2 log); and each lane (1, 2, 3…) refers to the amplification from independent single mutant. The black asterisk indicates the PCR fragment that will be cloned before sequencing, and the red asterisk indicates the PCR fragment that can be directly sequenced.

      Table 6. Examples of RESDA-PCR success rate after amplification with four independent degenerat primers and RB1, followed by further amplification with RB4 and Q0 primers


  4. Subsequent cloning of DNA fragment
    Since the recovered PCR products generated by proofreading polymerases (KOD Xtreme Hot Start DNA polymerase) are blunt-end, in this protocol pCR II-Blunt-TOPO vector is used. Other Taq DNA polymerase can also be used in this step.
    1. Directly ligate the recovered fragments to pCR II-Blunt-TOPO vector. This step can be completed within 5 min without adding a single deoxyadenosine (A) to the 3’ ends of PCR products. Add the ligation reaction components into a tube as described in Table 7, mix gently and incubate for 5 min at 22 °C.
      Note: pCR-Blunt II-TOPO allows direct selection of recombinants via disruption of the lethal E. coli gene, ccdB (Bernard et al., 1994). Cells that contain a non-recombinant vector are killed upon plating.
    2. The ligated products are transformed into One Shot competent cells (Thermo Fisher Scientific) according to manufacturers’ instructions. Transformants are selected on LB agar plates containing 50 μg ml-1 kanamycin (37 °C for overnight).

      Table 7. Setup of the cloning reaction using the Zero BluntTM TOPOTM PCR cloning kit


  5. Sequencing
    1. For each PCR fragment, culture three independent colonies separately in LB medium and extract plasmids using NucleoSpin Plasmid (MACHEREY-NAGEL) following manufacturer’s instructions. 
    2. Sequence the purified plasmids using the T7 universal primer, and example of sequencing results for one representative mutant is shown in Figure 3.


      Figure 3. Example of sequencing results for one representative mutant
      Note: The partial sequences of AphVIII is underlined by red color and the sequences targeted to the genome of C. reinhardtii (v5.5) is underlined by black color, the sequences not underlined belong to the vector (pCR-Blunt II-TOPO) sequences.

Data analysis

The obtained sequences were firstly blasted against AphVIII expression cassette to confirm whether the RESDA-PRC fragment contains sequences of this cassette. If it is so, the obtained sequences were subsequently blasted against the genome of C. reinhardtii (v5.5 at Phytozome) (Merchant et al., 2007) to identify the flanking sequences around the cassette insertion site. The targeted genes of interest, i.e., maintenance-type DNA methyltransferase and acyl-CoA oxidase gene, were further studied by molecular genetics and biochemical analysis as we previously described (Kong et al., 2015 and 2017). All the experiments were performed in three biological replicates, and three plasmids of every single colony (containing PCR fragment) were sequenced, respectively. Statistical analysis was performed with SPSS program (version 19.0).

Notes

  1. This method is easily applicable to other insertional mutagenesis where marker genes other than AphVIII are used. Design of gene-specific primers is needed in this case.
  2. In 1st PCR amplification, it is essential to use Taq DNA polymerase (i.e., NEB) that has no proof-reading activity (lacking 3’ to 5’ DNase activity) to avoid the degradation of degenerate primers (containing inosine) hybridized to the genomic DNA. DMSO is used to reduce the hydrogen bonds in single-stranded DNA hairpin structures. We found out that the amplification with the DegPstI and DegTaqI degenerate primers had a higher probability of producing PCR products.
  3. In 2nd amplification, the diluted PCR product from the 1st PCR reaction at 1/1,000 was found to be the best situation to have higher chances of amplification. The PCR product often ranges from 0.8 kb to 1.5 kb. If the PCR product is specific with sufficient amount, it can be recovered and sequenced directly, i.e., without going through the step of cloning.
  4. pCR II-Blunt-TOPO vector is alternative for subsequent cloning of DNA fragment; it is also possible to use universal TA Cloning techniques for sub-cloning and DNA sequencing.

Recipes

  1. 0.7 M NaCl
    Add 40.95 g NaCl crystalline (Sigma-Aldrich) to 800 ml sterilized MilliQ water and stir until NaCl is dissolved, and make the final volume to 1 L
  2. 10% SDS
    Dilute SDS powder (Sigma-Aldrich) with sterilized MilliQ water (1:10, w/v) to make the working solution
  3. 0.5 M Tris-HCl, pH 8.0
    Add 60.55 g Tris base power (Sigma-Aldrich) to 800 ml sterilized MilliQ water and stir until Tris base is dissolved, adjust the pH to 8.0 with HCl using pH meter to monitor the pH change and make the final volume to 1 L
  4. 10 mM EDTA
    Add 2.92 g Tris base power (Sigma-Aldrich) to 800 ml sterilized MilliQ water and stir until EDTA is dissolved and make the final volume to 1 L
  5. Lysis buffer
    Dilute 0.5 M Tris-HCl, pH 8.0, zss10 mM EDTA, 0.7 M NaCl, 10% SDS and Proteinase K (20.0 mg ml-1) to a mixture of the final concentration (100 mM Tris-HCl, pH 8.0, 1.75 mM EDTA, 150 mM NaCl, 2% SDS, and 1.0 mg ml-1 Proteinase K)
  6. 5 M KCl
    Add 372.76 g KCl crystalline (Sigma-Aldrich) to 800 ml sterilized MilliQ water and stir until KCl is dissolved, and make the final volume to 1 L
  7. 10% CTAB
    Dissolve CTAB powder (Sigma-Aldrich) with 0.7 M NaCl (10:100, w/v)
  8. 10% DMSO
    Dilute the DMSO (Sigma-Aldrich) with PCR-grade H2O (1:10, v/v) to the working solution
  9. 1.0% agarose gel
    Add agarose (Sigma-Aldrich) to 1 x TAE (1:100, w/v)
  10. 1x TAE
    Dilute UltraPure 10x TAE buffer (Thermo Fisher Scientific, UltraPureTM) with sterilized MilliQ water (1:10, v/v) to make the working solution
  11. 70% ethanol
    Dilute Ethanol (Sigma-Aldrich) with sterilized MilliQ water (7:3, v/v) to make the working solution

Acknowledgments

The work was supported by the French Agence Nationale pour la Recherche (ANR-Diesalg and ANR-MUsCA). This protocol is based on the protocol published by González-Ballester et al. (2005). There are no conflicts of interest.

References

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  2. Cagnon, C., Mirabella, B., Nguyen, H. M., Beyly-Adriano, A., Bouvet, S., Cuine, S., Beisson, F., Peltier, G. and Li-Beisson, Y. (2013). Development of a forward genetic screen to isolate oil mutants in the green microalga Chlamydomonas reinhardtii. Biotechnol Biofuels 6(1): 178.
  3. Dent, R. M., Haglund, C. M., Chin, B. L., Kobayashi, M. C. and Niyogi, K. K. (2005). Functional genomics of eukaryotic photosynthesis using insertional mutagenesis of Chlamydomonas reinhardtii. Plant Physiol 137(2): 545-556.
  4. Fuhrmann, M., Oertel, W. and Hegemann, P. (1999). A synthetic gene coding for the green fluorescent protein (GFP) is a versatile reporter in Chlamydomonas reinhardtii. Plant J 19(3): 353-361.
  5. González-Ballester, D., de Montaigu, A., Higuera, J. J., Galvan, A. and Fernandez, E. (2005). Functional genomics of the regulation of the nitrate assimilation pathway in Chlamydomonas. Plant Physiol 137(2): 522-533.
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  7. Harris, E. (2001). Chlamydomonas as a model organism. Annu Rev Plan Physiol Plant Mol Biol 52: 363-406.
  8. Kong F., Liang Y., Légeret B., Beyly-Adriano A., Blangy S., Haslam R. P., Napier J. A., Beisson F., Peltier G. and Li-Beisson Y. (2017). Chlamydomonas carries out fatty acid β-oxidation in ancestral peroxisomes using a bona fide acyl-CoA oxidase. Plant J 90(2): 358-371.
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简介

莱茵衣藻(Chlamydomonas reinhardtii)是光合作用,代谢和鞭毛生物学的基础研究者。已经为这种藻类开发了多功能的工具箱(Fuhrmann等人,1999; Schroda等人,2000; Schroda,2006)。其中,正向遗传方法已经被广泛使用,主要是因为通过随机插入诱变产生了数十万个突变体的高效率,并且可以在第一代进行单倍体性质表型分析(Cagnon等人 2013,Tunçay et。 2013)。在正向遗传方法中应用高通量方法的主要瓶颈是鉴定与观察到的表型相关的遗传损伤。在该协议中,我们详细描述了最初在(González-Ballester等人,2005)中报道的限制性定点扩增PCR(RESDA-PCR)的改进版本。优化包括引物组合的优化,DNA聚合酶的选择,PCR循环参数的优化以及PCR产物直接测序的应用。这些修改使得获得特定的PCR产物变得更加容易,并且加速了亚克隆步骤以更快地获得测序数据。


【背景】除了限制酶位点定向扩增PCR(RESDA-PCR)(González-Ballester等人,2005)之外,还发现了其它几种分子技术。 包括Genome Walker(Stirnberg和Happe,2004),热不对称交错PCR(TAIL-PCR)(Dent等人,2005),cDNA末端的3'-快速扩增(3'RACE)( Meslet-Cladiere和Vallon,2012),基于Mme1的插入位点测序策略(ChlamEseq)(Zhang等,2014)或全基因组重测序(Goold等, 2016年)。 RESDA-PCR是基于使用特定引物的基因结合使用简并引物。 RESDA-PCR是最常用的之一,在我们手中没有被发现给出识别侧翼序列的最高可能性。

关键字:莱茵衣藻, 插入诱变, RESDA PCR, 正向遗传, 衣藻突变体

材料和试剂

  1. 猎鹰锥形离心管,15毫升(康宁,目录号:430055)
  2. (Eppendorf,产品目录号:0030120086和0030120094)。
  3. 直径90mm的培养皿(Thermo Fisher Scientific,Sterilin TM,目录号:101R20)
  4. 消毒牙签(福建福华,福华方圆,目录编号:855)
  5. 通过随机插入诱变产生的莱茵衣藻突变体(Cagnon et al。 ,2013)
  6. One Shot TM TOP10化学感受态细胞(Thermo Fisher Scientific,目录号:C404010)
  7. Zero Blunt TM TOPO TM PCR克隆试剂盒(Thermo Fisher Scientific,目录号:450245)
  8. Taq DNA聚合酶(5,000U ml -1),10x标准Taq反应缓冲液,dNTP(New England Biolabs,目录号:M0273S) br />
  9. 二甲亚砜(DMSO)(Sigma-Aldrich,目录号:472301)
  10. PCR级H 2 O(Sigma-Aldrich,目录号:W1754)
  11. 乙醇(Sigma-Aldrich,目录号:34852)
  12. KOD Xtreme热启动DNA聚合酶,dNTPs(各2mM),2x Xtreme缓冲液(Merck,Novagen,目录号:71842)。
  13. 琼脂糖(Sigma-Aldrich,目录号:A9539)
  14. ExactLadder®DNA PreMix 2 log(OZYME,目录号:OZYC002-500)
  15. SYBR TM Safe DNA凝胶染色(Thermo Fisher Scientific,目录号:S33102)
  16. UltraPure TM 10x TAE缓冲液(Thermo Fisher Scientific,目录号:15558026)
  17. Luria肉汤(Sigma-Aldrich,目录号:L1900)
  18. 硫酸卡那霉素(Thermo Fisher Scientific,目录号:11815024)
  19. NucleoSpin凝胶和PCR清洁试剂盒(MACHEREY-NAGEL,目录号:740609.10)
  20. NucleoSpin 质粒小量制备试剂盒(MACHEREY-NAGEL,目录号:740588.10)
  21. 十二烷基硫酸钠(SDS)(Sigma-Aldrich,目录号:L3771)
  22. 氯化钠(NaCl)(Sigma-Aldrich,目录号:S9888)
  23. 氯化钾(KCl)(Sigma-Aldrich,目录号:P9541)
  24. 乙二胺四乙酸(EDTA)(Sigma-Aldrich,目录号:E9884)
  25. Tris碱(Sigma-Aldrich,目录号:T6066)
  26. 十六烷基三甲基溴化铵(CTAB)(Sigma-Aldrich,目录号:H9151)
  27. 异丙醇(Sigma-Aldrich,目录号:W292907)
  28. 苯酚:氯仿:异戊醇(25:24:1)(Sigma-Aldrich,目录号:P3803)
  29. 蛋白酶K(20.0mg ml -1)(Sigma-Aldrich,目录号:P6556)
  30. 裂解缓冲液(见食谱)
    1. 0.7 M NaCl
    2. 10%SDS
    3. 0.5M Tris-HCl,pH8.0
    4. 10 mM EDTA
  31. 5 M KCl(见食谱)
  32. 10%CTAB(见食谱)
  33. 10%DMSO(见食谱)
  34. 1.0%琼脂糖凝胶(见食谱)
  35. 1倍TAE(见食谱)
  36. 70%乙醇(见食谱)

设备

  1. Shaker(Eppendorf,New Brunswick TM,型号:Innova 44,目录号:M1282-0002)
  2. Accumet TM pH计(Fisher Scientific,型号:3合1套装,产品目录号:13-636-AE153)
  3. PCR热循环仪(Thermo Fisher Scientific,Applied Biosystems TM,型号:2720,目录号:ED000651)
  4. 琼脂糖电泳槽(Bio-Rad Laboratories,型号:Mini-Sub Cell GT,产品目录号:1704401)
  5. Gel Doc XR系统(Bio-Rad Laboratories,型号:Gel Doc TM XR +,目录号:5838)
  6. Fisherbrand TM公共台式涡旋器(Fisher Scientific,目录号:02-216-125)
  7. 台式离心机(Beckman Coulter,型号:Allegra 64R,目录号:367586)
  8. 台式培养箱(Eppendorf,新不伦瑞克TM,型号:S41i,目录号:S41I230011)
  9. NanoDrop 2000(Thermo Fisher Scientific,型号:NanoDrop TM 2000,目录号:ND-2000)
  10. Multisizer 3 Coulter计数器(Beckman Coulter,型号:Multisizer TM 3,目录号:6605697)

软件

  1. SPSS程序(版本19.0)

程序

  1. DNA提取和定量
    1. 在摇瓶中于25℃恒定的白色荧光下(约0.5×10 6个细胞ml -1)培养莱茵衣藻(Chlamydomonas reinhardtii)细胞(约0.5×10 6个细胞/毫升)在TAP液体培养基(Harris,2001)中,用轻微摇动的100μmol光子m 2 / m -1。通常在24小时后它将达到指数期(约5×10 6个细胞ml -1)。
    2. 用CTAB方法分离和纯化突变体的基因组DNA(Schroda等人,2001)。简而言之,细胞生长至对数期后(约5×10 6个细胞ml -1),收获总共5×10 7个细胞通过离心并悬浮于500μl裂解缓冲液(100mM Tris-HCl,pH8.0,1.75mM EDTA,150mM NaCl,2%[w / v] SDS和1.0mg ml -1蛋白酶K)。
    3. 在55°C孵育裂解物2小时,然后加入80μL5MKCl和70μL预热的10%CTAB并在65°C孵育10分钟。
    4. 用(500μl)苯酚:氯仿:异戊醇(25:24:1,v / v / v)提取裂解物两次,然后加入(500μl)氯仿以除去剩余的苯酚。
    5. 在冰上通过加入500μl异丙醇将核酸沉淀20分钟,并用70%冷乙醇洗涤。
    6. 在50μl无菌水中重悬空气干燥的颗粒。
    7. 使用NanoDrop 2000量化纯化的DNA,加入水调节终浓度为50ngμl-1。
      注意:所提及细胞的浓度是用Multisizer 3 Coulter计数器测量的。

  2. PCR扩增
    该程序主要遵循González-Ballester等人(2005)首次发表的方案。为了鉴定插入位点周围的DNA序列,需要两组后续的PCR反应,这里命名为第一和第二扩增。第一次PCR扩增是用一种简并引物(图1)使用一种AphVIII基因特异性引物从突变体的基因组DNA扩增片段。为了增加扩增的机会,可以检测4个简并引物,每个引物都含有序列特异性部分(5'端的Q0)和简并部分(3'端)。 PCR产物将用作第二轮PCR扩增(第二次PCR扩增)的模板,使用两个序列特异性引物。包括Q 0的简并引物(deg Taq I,Deg Pst I,Deg Alu I和Deg Sac II)和标记基因(emphVIII)特异性引物(RB1和RB4)显示在表1中。


    图1. RESDA PCR的主要部分概述此电路图改编自González-Ballester 等,2005年。

    表1.本协议中使用的引物及其序列

    肌苷,N,A + T + G + C; S,G + C; W,A + T.
    *基因特异性引物(RB1和RB4)可以根据标记基因的类型(即 , AphVII , > aadA )用于诱变。

    1. 第一次放大
      在第1次扩增中,使用AphVIII基因特异性引物(RB1)和一个简并引物扩增抗生素标记基因AphVIII侧翼的片段来自从突变体纯化的基因组DNA。四个独立的反应可以通过使用四个不同的简并引物和RB1分别建立。
      PCR反应混合物的组成和循环程序分别显示在表2和表3中
      表2.用于1强扩增的PCR反应混合物的组成 扩增

      *先使用一种简并引物。

      表3.用于1次扩增的PCR条件

      *每个循环依次包含十三个步骤;共设置了二十个循环。

    2. 第二次放大
      为了获得更多特异性PCR产物,使用简并引物5'末端的制造者基因特异性引物(RB4)和特异性引物(Q0)用来自第一次扩增的PCR扩增作为模板(1μl稀释的PCR产物(1 / 1,000),从1st扩增)。
      PCR反应混合物的组成和循环程序分别见表4和表5
      表4.用于扩增和/或扩增的PCR反应混合物的组成


      表5.用于第2次PCR扩增的条件


  3. 琼脂糖凝胶电泳和DNA片段纯化
    1. 将PCR产物从1.0%琼脂糖凝胶上进行2次扩增,在100V下电泳30分钟,然后用SYBR Safe(1 / 1,000)染色凝胶,并用GelDocXR系统(Bio-Rad Laboratories)。一些代表性的结果显示在图2中,并且通过四个独立的简并引物和RB1扩增后,用RB4和Q0引物进一步扩增的RESDA-PCR成功率的例子示于表6中。
    2. 从凝胶上切下充分分离的PCR产物(用*标记),用NucleoSpin Gel和PCR Clean-up Kit(MACHEREY-NAGEL)根据生产商的说明进行纯化。


      图2.使用简并引物DegQ TaqI的RESDA-PCR结果的实例所示的PCR产物是两轮PCR反应的结果:使用Deg > Taq I和RB1,然后用RB4和Q0引物进一步扩增。

      注:M指DNA标记(Exact Ladder DNA PreMix 2 log);每条泳道(1,2,3 ...)指独立单突变体的扩增。黑色星号表示测序前将被克隆的PCR片段,红色星号表示可直接测序的PCR片段。

      表6.用四个独立的简并引物和RB1扩增后,用RB4和Q0引物进一步扩增的RESDA-PCR成功率的实例


  4. 随后克隆DNA片段
    由于校正聚合酶(KOD Xtreme Hot Start DNA聚合酶)产生的回收的PCR产物是平端的,所以在该方案中使用pCR II-Blunt-TOPO载体。其他Taq DNA聚合酶也可用于此步骤。
    1. 将回收的片段直接连接到pCR II-Blunt-TOPO载体上。该步骤可以在5分钟内完成,而不向PCR产物的3'末端添加单个脱氧腺苷(A)。如表7中所述将连接反应组分加入管中,轻轻混合并在22℃孵育5分钟。
      注:pCR-Blunt II-TOPO允许通过破坏致死性大肠杆菌基因ccdB直接选择重组体(Bernard et al。,1994)。包含非重组载体的细胞在接种后被杀死。
    2. 根据制造商的说明将连接产物转化到One Shot感受态细胞(Thermo Fisher Scientific)中。在含有50μgml -1卡那霉素(37℃过夜)的LB琼脂平板上选择转化子。

      表7.使用Zero Blunt TM TOPO TM PCR克隆试剂盒设置克隆反应


  5. 测序
    1. 对于每个PCR片段,在LB培养基中分别培养三个独立的菌落,并使用NucleoSpin质粒(MACHEREY-NAGEL)按照生产商的说明提取质粒。 
    2. 使用T7通用引物对纯化的质粒进行测序,并且对于一个代表性突变体的测序结果的实例显示在图3中。


      图3.一个代表性突变体的测序结果示例
      注:AphVIII的部分序列以红色标出,以莱茵衣藻基因组(v5.5)为靶标的序列以黑色标出,未加下划线的序列属于载体(pCR-Blunt II -TOPO)序列。

数据分析

首先将获得的序列轰击AphVIII表达盒以确认RESDA-PRC片段是否含有该盒的序列。如果是这样的话,所获得的序列随后被轰击到C的基因组中。 (在Phytozome的v5.5)(Merchant等,<2007年),以鉴定盒插入位点周围的侧翼序列。如前所述,通过分子遗传学和生物化学分析进一步研究了目的靶基因即维持型DNA甲基转移酶和酰基辅酶A氧化酶基因(Kong等人, 2015年和2017年)。所有的实验都进行了三个生物学重复,每个单个菌落(含有PCR片段)的三个质粒分别测序。统计分析采用SPSS程序(版本19.0)进行。

笔记

  1. 这种方法很容易适用于其他插入诱变,其中使用除了AphVIII以外的标记基因。在这种情况下需要设计基因特异性引物。
  2. 在第一次PCR扩增中,使用不具有校对活性的Taq DNA聚合酶(即,NEB)是必需的(缺少3个'至5'DNA酶活性)以避免与基因组DNA杂交的简并引物(含肌苷)的降解。 DMSO用于减少单链DNA发夹结构中的氢键。我们发现使用Deg Pst I和Deg TaqI I简并引物的扩增产生PCR产物的可能性更高。
  3. 在第二次扩增中,发现来自第一次PCR反应的1/1000稀释的PCR产物是具有较高扩增机会的最佳情况。 PCR产物通常在0.8kb至1.5kb的范围内。如果PCR产物具有足够的特异性,则可以直接回收和测序,即不经过克隆步骤。
  4. pCR II-Blunt-TOPO载体可用于随后克隆DNA片段;也可以使用通用TA克隆技术进行亚克隆和DNA测序。

食谱

  1. 0.7 M NaCl
    加入40.95克NaCl结晶(Sigma-Aldrich)到800毫升无菌MilliQ水中并搅拌直到NaCl溶解,并使最终体积达到1升。
  2. 10%SDS
    用灭菌的MilliQ水(1:10,w / v)稀释SDS粉末(Sigma-Aldrich)以制备工作溶液
  3. 0.5M Tris-HCl,pH8.0
    向800ml无菌MilliQ水中加入60.55g Tris基本能量(Sigma-Aldrich)并搅拌直至Tris碱溶解,用pH计用HCl调节pH至8.0以监测pH变化并使最终体积为1L />
  4. 4.10mM EDTA
    加入2.92克Tris基本能量(Sigma-Aldrich)至800毫升无菌MilliQ水中并搅拌至EDTA溶解并使最终体积达到1升。
  5. 裂解缓冲液
    将最终浓度(100mM Tris-HCl,pH 8.0,zss10mM EDTA,0.7M NaCl,10%SDS和蛋白酶K(20.0mg ml -1) HCl,pH8.0,1.75mM EDTA,150mM NaCl,2%SDS和1.0mg ml -1蛋白酶K)
  6. 5 M KCl
    加入372.76克氯化钾结晶(西格玛奥德里奇)到800毫升无菌MilliQ水,搅拌,直到氯化钾溶解,并使最终体积为1升
  7. 10%CTAB
    用0.7M NaCl(10:100,w / v)溶解CTAB粉末(Sigma-Aldrich)
  8. 10%DMSO
    用PCR级别H 2 O(1:10,v / v)稀释DMSO(Sigma-Aldrich)至工作溶液。
  9. 1.0%琼脂糖凝胶
    加入琼脂糖(Sigma-Aldrich)至1×TAE(1:100,w / v)
  10. 1x TAE
    用灭菌的MilliQ水(1:10,v / v)稀释UltraPure 10x TAE缓冲液(Thermo Fisher Scientific,UltraPure TM),制成工作溶液。
  11. 70%乙醇
    用无菌MilliQ水(7:3,v / v)稀释乙醇(Sigma-Aldrich)以制备工作溶液。

致谢

这项工作得到了法国国家法律研究所(ANR-Diesalg和ANR-MUsCA)的支持。该协议基于González-Ballester等人发表的协议(2005)。没有利益上的冲突。

参考

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Copyright: © 2018 The Authors; exclusive licensee Bio-protocol LLC.
引用:Kong, F. and Li-Beisson, Y. (2018). Identification of Insertion Site by RESDA-PCR in Chlamydomonas Mutants Generated by AphVIII Random Insertional Mutagenesis. Bio-protocol 8(3): e2718. DOI: 10.21769/BioProtoc.2718.
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