搜索

Polyethylene Glycol-mediated Transformation of Drechmeria coniospora
聚乙二醇介导的圆锥掘氏梅里霉的转化   

下载 PDF 引用 收藏 提问与回复 分享您的反馈 Cited by

本文章节

Abstract

Drechmeria coniospora is a nematophagous fungus and potential biocontrol agent. It belongs to the Ascomycota. It is related to Hirsutella minnesotensis, another nematophagous fungus but, phylogenetically, it is currently closest to the truffle parasite Tolypocladium ophioglossoides. Together with its natural host, Caenorhabditis elegans, it is used to study host-pathogen interactions. Here, we report a polyethylene glycol-mediated transformation method (Turgeon et al., 2010; Ochman et al., 1988) for this fungus. The protocol can be used to generate both knock-in or knock-out strains (Lebrigand et al., 2016).

Keywords: Fungus(真菌), Genetic engineering(基因工程), Nematophagous(食线虫), Pathogen(病原体), Caenorhabditis elegans(秀丽隐杆线虫)

Background

D. coniospora has been developed as a model pathogen for the study of innate immunity in C. elegans (Lebrigand et al., 2016 and references therein). D. coniospora grows slowly on standard growth media, rendering in vitro study difficult and making it hard to develop transformation methods. We report here a culture method that allows the rapid production of large quantities of D. coniospora, opening the way to its genetic modification. Polyethylene glycol-mediated transformation is probably the simplest method that has been broadly applied to modify fungi. We found that it can be used with D. coniospora, thus providing the first approach to modify its genome deliberately.

Materials and Reagents

  1. Pasteur pipettes (VWR, catalog number: 6121701 )
  2. Eppendorf tube 1.5 ml (Sigma-Aldrich, catalog: T9661 )
  3. Petri dish (Greiner Bio one, catalog number: 633185 )
  4. 50 ml conical tube (Corning, Falcon®, catalog number: 352070 )
  5. 15 ml conical tube (Corning, Falcon®, catalog number: 352196 )
  6. 14 ml polystyrene round bottom tube (Corning, Falcon®, catalog number: 352051 )
  7. Miracloth (EMD Millipore, catalog number: 475855 )
  8. Syringe 25 ml (Terumo, catalog number: SS-20ES )
  9. Acrodisc® syringe filters (Pall, catalog number: 4187 )
  10. Aluminum foil (Fisher Scientific, catalog number: 01213102 )
  11. Escherichia coli strain OP50
  12. Caenorhabditis elegans strain N2
  13. Drechmeria coniospora recipient strain ATCC 96282
  14. pLH4237 plasmid DNA, contains a hygromycin B phosphotransferase::GFP chimeric gene driven by D. coniospora β-tubulin promoter (β-tubp::HPH::GFP, Lebrigand et al., 2016)
  15. Ampicillin sodium salt (Sigma-Aldrich, catalog number: A9518 )
  16. Gentamicin solution (Sigma-Aldrich, catalog number: G1272 )
  17. Hygromycin B (Thermo Fisher scientific, GibcoTM, catalog number: 10687010 )
  18. di-Potassium hydrogen orthophosphate, K2HPO4 (VWR, catalog number: 26931.263 )
  19. Potassium dihydrogen phosphate, KH2PO4 (VWR, catalog number: 0781-1KG )
  20. Bacto agar (BD, Bacto, catalog number: 214010 )
  21. Bacto peptone (BD, Bacto, catalog number: 211677 )
  22. Sodium chloride, NaCl (VWR, catalog number: 27810.295 )
  23. Cholesterol (Sigma-Aldrich, catalog number: C3045 )
  24. Ethanol (VWR, catalog number: 20821.321 )
  25. Magnesium sulfate, MgSO4 (Sigma-Aldrich, catalog number: M7506 )
  26. Calcium chloride, CaCl2 (Sigma-Aldrich, catalog number: C1016 )
  27. Yeast extract (Douchefa Biochemie, catalog number: Y1333 )
  28. D-sorbitol (Sigma-Aldrich, catalog number: S3889 )
  29. Aurintricarboxylic acid, ATA (EMD Millipore, catalog number: 189400 )
  30. Tris/HCl (Sigma-Aldrich, catalog number: T6666 )
  31. Polyethylene glycol (PEG 3350) (Sigma-Aldrich, catalog number: P4338 )
  32. Caylase C4 (CAYLA, catalog number: caseC4-5 ) Alternative can be lysing enzymes (Sigma-Aldrich, catalog number: L1412)
    Note: The hyperlink for caseC4-5 is not available anymore. Alternative could be lysing enzyme, and we are still testing its efficiency in our lab.
  33. Phosphate buffer (see Recipes)
  34. Medium (see Recipes)
    1. NGM-AG
    2. NGMY
    3. NGMY liquid
    4. NGMS
  35. 15 mM ATA solution (see Recipes)
  36. 50 mM NaCl solution (see Recipes)
  37. CaCl2 1700 (see Recipes)
  38. Lysis buffer (see Recipes)
  39. STC 1700 (see Recipes)
  40. PEG 1700 (see Recipes)

Equipment

  1. Stereomicroscope (Leica Microsystems, model: MZ16 F )
  2. HERAsafe KS safety cabinet (Thermo Fisher Scientific, Thermo ScientificTM, model: HerasafeTM KS Class II , catalog number: 51022751)
  3. Centrifuge (Eppendorf, catalog number: 5810 R )
  4. Funnel
  5. Spatula
  6. Cloth-plugged 250 ml glass Erlenmeyer flask
  7. Rotary shaker for culture flasks (Eppendorf, model: New Brunswick Scientific Innova 4080 )
  8. Counting chambers (Burker, Tiefe 0.1000 m, 0.0025 mm2)
  9. Balancer
  10. Pipetman P1000
  11. autoclave

Procedure

  1. Mycelium preparation
    1. Thaw a frozen stock of fungal mycelia at room temperature and spread on NGM plates (with ampicillin and gentamycin). Culture at 25 °C for 1 week. Using a stereomicroscope, confirm appearance of spores. If no spores are visible, continue culture and check daily.
    2. Using a bent sterile glass Pasteur pipette, gently rub the fungal colonies to dislodge the spores. Wash the spores from the plates, using 1 ml of 50 mM NaCl (Figures 1A and 1B) and collect into an Eppendorf tube.
    3. Spread the spores (20-100 µl) on a 9 cm NGM-AG plate (Figure 1C) and allow to dry in a safety cabinet. In parallel, prepare a population of synchronized L4 stage worms (Stiernagle, 2006). Typically, from a single densely populated 9 cm plate, one can obtain 4,000-6,000 worms. Wash the worms from each plate with 5 ml 50 mM NaCl into a 15 ml Falcon tube and centrifuge at 721 x g for 1 min; carefully remove the supernatant, resuspend in the remaining solution and spread half the mix on the 9 cm NGM-AG plate. The worms will be infected by spores. Incubate the resulting plate at 25 °C for 10 days.
    4. Wash off spores as in step A2. Prepare four 9 cm NGM plates seeded with OP50 (Stiernagle, 2006). Spread the spores collected from one NGM-AG plate onto 4 NGM plates with OP50. If necessary, allow the solution to dry.


      Figure 1. Preparation of infection plates. A and B. Washing spores from NGM-AG plate; C. Spreading spore solution on an NGM OP50 plate; D. Spores from NGM-AG plate, photomicrograph using Nomarski optics.

    5. Transfer L4 or young adult N2 worm collected from one 9 cm plate as above (generally 4,000-6,000 worms) onto each spore-OP50 plate, and incubate for 24-30 h.
    6. Wash the infected worms from each plate with 1.5 ml 50 mM NaCl into a 15 ml Falcon tube and centrifuge at 721 x g for 1 min; carefully remove the supernatant, resuspend and divide the worm pellet between three 9 cm NGMY plates. Gently spread the pellet across the surface of the plate using a bent sterile glass Pasteur pipette. If necessary, allow to dry in a safety cabinet before incubating at 25 °C for 1 day. Most adult worms should be dead by this time (Figure 2). Before dying, they will have produced progeny and most of the worms of the next generation will be alive at this time.


      Figure 2. Infected worms on NGMY plate. Two dead adult worms with a number of live larvae.

    7. Add 1-2 ml of 50 mM NaCl to each plate, and using a bent sterile glass Pasteur pipette, rub to resuspend the dead worms. Collect the liquid from 3 plates in a 15 ml Falcon tube; transfer the solution into 250 ml NGMY liquid, culture on a shaking incubator at 170 rpm for 36-40 h.

  2. Protoplasting
    1. Pour the liquid culture into a funnel lined with Miracloth to filter the mycelia; wash with 100 ml CaCl2 1700 buffer, use a spatula to transfer the mycelia into a 50 ml Falcon tube (Figure 3). Keep the used funnel and Miracloth in a sterile environment for later use.


      Figure 3. Collecting mycelium with Miracloth

    2. Weigh the mycelia and resuspend with gentle vortexing in newly prepared lysis buffer; for every 1 g mycelia add 10 ml lysis buffer. If the volume exceeds 30 ml, use a sterile, cloth-plugged Erlenmeyer flask, otherwise a 50 ml Falcon tube.
    3. Incubate the mycelial solution at 30 °C with 60 rpm for 1-1.5 h. After 1 h, at regular intervals, remove a small aliquot and check the solution under a microscope. Mycelial fragments are quite resistant to hygromycin selection and can give rise to colonies; avoid fragmentation as much as possible. If short mycelial fragments appear (Figure 4), stop the incubation by moving to the next step.


      Figure 4. Mycelium solution before and after lysis buffer digestion. A. Colony before treatment; B. Colony after 1 h of treatment, with protoplasts (red arrows); C. Mycelial fragments after treatment (black arrows). Scale bars = 20 μm in A and B; Scale bar = 50 μm in C.

    4. Place the funnel together with the Miracloth used in protoplasting step 1 onto a 50 ml Falcon tube. Filter the mycelia and protoplasts in lysis buffer, retain the filtrate.
    5. Add an equal volume of ice-cold STC buffer to the protoplast solution, mix gently and leave on ice for 10 min.
    6. Centrifuge the mixture at 4 °C, 1,127 x g for 15 min.
    7. Carefully discard the supernatant; firstly, gently resuspend the pellet with 1 ml ice-cold STC buffer by pipetting, then add another 24 ml of cold STC buffer to the protoplast; centrifuge the mixture at 4 °C, 1,623 x g for 10 min.
    8. Repeat step B7 one more time.
    9. Discard the supernatant, add 300 µl ice-cold STC buffer to resuspend the protoplasts; remove a small aliquot and count the protoplasts using a haemocytometer (Figure 5). Dilute the protoplasts to a final concentration of 108 protoplasts/ml using ice-cold STC buffer. Then, stock the solution on ice or at 4 °C; it can be used for transformation for a maximum of 7 days.


      Figure 5. Protoplasts on a haemocytometer. Individual protoplasts are indicated with red arrows.

  3. Transformation
    1. Add 1 µl ATA onto the wall of a round-bottom 14 ml Falcon tube, mix with 10 µg or more of plasmid DNA (in a volume of around 10 µl), then mix with 150 µl of protoplast solution by gently pipetting; incubate the mixture for 40 min at room temperature.
    2. Using a Pipetman P1000, add 6 drops of PEG1700 buffer, mix by gently shaking; add another 850 µl PEG buffer, gently shake again, incubate for another 20 min.
    3. Add 10 ml of room temperature STC buffer; centrifuge for 10 min at 1,623 x g at room temperature.
    4. Discard the supernatant carefully; resuspend the protoplasts with 300 µl STC at room temperature.
    5. Add 150 µl of protoplast solution to a 9 cm plate (see Note 2), spread gently with sterile bent glass Pasteur pipette; culture at 25 °C for 3 weeks.
    6. Check any colonies for successful transformation, by PCR or under a fluorescent stereomicroscope when using a transformation construct that includes a fluorescent reporter gene.

Notes

  1. Unless specified, temperature is always at 25 °C.
  2. Except OP50 NGM plates, all the other plates are with ampicillin and gentamicin. When transformation constructs include a hygromycin-resistance cassette, NGMS plates are supplemented with 25 µg/ml hygromycin.
  3. Autoclave the Miracloth (folded in half twice), in an aluminum foil bag with a cut corner, so that condensation does not accumulate and the cloth is dry after autoclaving (Figure 6).


    Figure 6. Miracloth preparation

Recipes

  1. 1 M phosphate buffer, pH 6.0
    108.3 g KH2PO4
    35.6 g K2HPO4
    Add ddH2O to 1 L
  2. Medium
    1. 1 L NGM
      20 g agar
      2.5 g peptone
      3 g NaCl
      5 mg/ml cholesterol in 1 ml ethanol
      Autoclave at 121 °C for 20 min, after autoclave, add:
      1 ml 1 M MgSO4 (sterilized)
      1 ml 1 M CaCl2 (sterilized)
      1 ml 1 M phosphate buffer pH 6.0 (sterilized)
    2. NGM-AG
      NGM with 100 µg/ml ampicillin and 50 µg/ml gentamicin
    3. NGMY
      NGM plus yeast extract 20 g/L
    4. NGMY liquid
      NGMY without agar
    5. NGMS
      NGM-AG plus D-sorbitol
  3. 15 mM ATA solution (autoclave)
    0.634 g aurintricarboxylic acid in 100 ml ddH2O and autoclave at 121 °C for 20 min
  4. 50 mM NaCl solution (autoclave)
    5.8 g NaCl in 2 L ddH2O and autoclave at 121 °C for 20 min
  5. CaCl2 1700 (500 ml, autoclave at 121 °C for 20 min)
    15.0 g CaCl2
    17.5 g NaCl
  6. Lysis buffer
    CaCl2 1700:C4 Caylase = 10 ml:100 mg (or CaCl2 1700:lysing enzyme = 10 ml:200 mg)
    Note: Prepare fresh every time, filter with syringe and film.
  7. STC 1700 (500 ml, autoclave)
    109.3 g sorbitol
    5 ml Tris/HCl
    22.8 g CaCl2
    1 g NaCl
  8. PEG 1700 (100 ml, autoclave)
    60 g PEG 4000
    1 ml Tris/HCl
    0.74 g CaCl2·2H2O
    Note: Tris/HCl solution stored at 1 M pH 7.5 , 60.6 g/500 ml, MW = 121.2.

Acknowledgments

We thank Dr. Eric Record for his help in establishing the protocol and Carole Couillault and Zhang Xing for their assistance. We also thank Dr. Nathalie Pujol for her input and suggestions. This work was supported by a program grant from the ANR (ANR-12-BSV3-0001-01) and institutional funding from INSERM, CNRS and AMU. Le He was supported by the Chinese Scholarship Council.

References

  1. Lebrigand, K., He le, D., Thakur, N., Arguel, M. J., Polanowska, J., Henrissat, B., Record, E., Magdelenat, G., Barbe, V., Raffaele, S., Barbry, P. and Ewbank, J. J. (2016). Comparative genomic analysis of Drechmeria coniospora reveals core and specific genetic requirements for fungal endoparasitism of nematodes. PLoS Genet 12(5): e1006017.
  2. Ochman, H., Gerber, A. S. and Hartl, D. L. (1988). Genetic applications of an inverse polymerase chain reaction. Genetics 120(3): 621-623.
  3. Turgeon, B. G., Condon, B., Liu, J. and Zhang, N. (2010). Protoplast transformation of filamentous fungi. In: Sharon, A. (Ed.). Molecular and cell biology methods for fungi. Humana pp: 3-19.
  4. Stiernagle, T. (2006). Maintenance of C. elegans. In: The C. elegans Research Community (Ed.). WormBook. WormBook.

简介

Drechmeria coniospora 是一种无害真菌和潜在的生物防治剂。它属于子囊菌纲。它与另外一种没食子菌真菌Hirsutella minnesotensis有关,但在系统发育中,它目前最接近松露寄生虫Tolypocladium ophioglossoides 。与其天然宿主,秀丽隐杆线虫一起,它用于研究宿主 - 病原体相互作用。在这里,我们报告了这种真菌的聚乙二醇介导的转化方法(Turgeon等人,2010; Ochman等人,1988)。该方案可用于产生敲入或敲除菌株(Lebrigand等人,2016)。

背景 D。 coniospora 已被开发为用于研究先天免疫的模型病原体。 elegans (Lebrigand等人,2016和其中的参考文献)。 D。 coniospora 在标准生长培养基上缓慢增长,使得体外研究困难,难以开发转化方法。我们在这里报告一种允许快速生产大量的D的文化方法。 coniospora ,开辟了其遗传修饰的道路。聚乙二醇介导的转化可能是广泛应用于修饰真菌的最简单的方法。我们发现它可以与一起使用。 coniospora ,因此提供了故意修改其基因组的第一种方法。

关键字:真菌, 基因工程, 食线虫, 病原体, 秀丽隐杆线虫

材料和试剂

  1. 巴斯德移液器(VWR,目录号:6121701)
  2. Eppendorf管1.5ml(Sigma-Aldrich,目录号:T9661)
  3. Pertri菜(Greiner Bio一,目录号:633185)
  4. 50ml锥形管(Corning,Falcon ®,目录号:352070)
  5. 15ml锥形管(Corning,Falcon ®,目录号:352196)
  6. 14ml聚苯乙烯圆底管(Corning,Falcon ®,目录号:352051)
  7. Miracloth(EMD Millipore,目录号码:475855)
  8. 注射器25毫升(Terumo,目录号:SS-20ES)
  9. Acrodisc ®针筒过滤器(Pall,目录号:4187)
  10. 铝箔(Fisher Scientific,目录号:01213102)
  11. 大肠杆菌菌株OP50
  12. 秀丽隐杆线虫菌株N2
  13. 受精菌株ATCC 96282
    接种菌株D.rerechmeria coniospora
  14. pLH4237质粒DNA含有由D驱动的潮霉素B磷酸转移酶:: GFP嵌合基因。球孢子β微管蛋白启动子(β-tub):: HPH :: GFP,Lebrigand等人,2016)
  15. 氨苄青霉素钠盐(Sigma-Aldrich,目录号:A9518)
  16. 庆大霉素溶液(Sigma-Aldrich,目录号:G1272)
  17. 潮霉素B(Thermo Fisher Scientific,Gibco TM ,目录号:10687010)
  18. 二 - 正磷酸氢钾,K 2 HPO 4(VWR,目录号:26931.263)
  19. 磷酸二氢钾,KH 2 PO 4(VWR,目录号:0781-1KG)
  20. Bacto琼脂(BD,Bacto,目录号:214010)
  21. Bacto蛋白胨(BD,Bacto,目录号:211677)
  22. 氯化钠,NaCl(VWR,目录号:27810.295)
  23. 胆固醇(Sigma-Aldrich,目录号:C3045)
  24. 乙醇(VWR,目录号:20821.321)
  25. 硫酸镁,MgSO 4(Sigma-Aldrich,目录号:M7506)
  26. 氯化钙,CaCl 2(Sigma-Aldrich,目录号:C1016)
  27. 酵母提取物(Douchefa Biochemie,目录号:Y1333)
  28. D-山梨醇(Sigma-Aldrich,目录号:S3889)
  29. Aurintricarboxylic acid,ATA(EMD Millipore,目录号:189400)
  30. Tris/HCl(Sigma-Aldrich,目录号:T6666)
  31. 聚乙二醇(PEG 3350)(Sigma-Aldrich,目录号:P4338)
  32. Caylase C4(CAYLA,目录号:caseC4-5)替代可以是裂解酶(Sigma-Aldrich,目录号:L1412)
    注意:caseC4-5的超链接不再可用。替代方案可能是裂解酶,我们仍然在实验室中测试其效率。
  33. 磷酸盐缓冲液(见配方)
  34. 中等(见配方)
    1. NGM-AG
    2. NGMY
    3. NGMY液体
    4. NGMS
  35. 15 mM ATA溶液(见配方)
  36. 50 mM NaCl溶液(参见食谱)
  37. CaCl 2 <1700>(见配方)
  38. 裂解缓冲液(见配方)
  39. STC 1700(见食谱)
  40. PEG 1700(见食谱)

设备

  1. 立体显微镜(Leica Microsystems,型号:MZ16 F)
  2. HERAsafe KS安全柜(Thermo Fisher Scientific,Thermo Scientific TM,型号:Herasafe TM KS/II类,目录号:51022751)
  3. 离心机(Eppendorf,目录号:5810 R)
  4. 漏斗
  5. Spatula
  6. 插入250毫升玻璃锥形瓶
  7. 用于培养瓶的旋转振荡器(Eppendorf,型号:New Brunswick Scientific Innova 4080)
  8. 计数室(Burker,Tiefe 0.1000m,0.0025mm 2
  9. 平衡器
  10. Pipetman P1000
  11. 高压釜

程序

  1. 菌丝体制备
    1. 在室温下解冻冷冻真菌菌丝体,并铺展在NGM平板(含氨苄青霉素和庆大霉素)上。在25°C培养1周。使用立体显微镜,确认孢子的外观。如果没有孢子可见,请继续培养并每天检查。
    2. 使用弯曲的无菌玻璃巴斯德吸管,轻轻擦拭真菌菌落以驱除孢子。使用1 ml的50 mM NaCl(图1A和1B)从板上清洗孢子,并收集到Eppendorf管中。
    3. 将孢子(20-100μl)撒在9cm NGM-AG板上(图1C),并在安全柜中干燥。同时,准备一批同步的L4阶段蠕虫(Stiernagle,2006)。通常,从一个人口稠密的9厘米平板,可以获得4,000-6,000个蠕虫。将每个板上的蠕虫用5ml 50mM NaCl洗涤到15ml Falcon管中,并以721×g离心1分钟;小心地去除上清液,重悬在剩余的溶液中,并将一半混合物铺在9cm NGM-AG板上。蠕虫将被孢子感染。将得到的板在25℃下孵育10天
    4. 像步骤A2一样冲洗孢子。准备用OP50种植的4个9cm NGM板(Stiernagle,2006)。将从一个NGM-AG板收集的孢子传播到具有OP50的4个NGM板上。如果需要,让溶液干燥。


      图1.感染板的制备 A和B.从NGM-AG板洗涤孢子; C.将孢子溶液撒在NGM OP50板上; D.来自NGM-AG板的孢子,使用Nomarski光学的显微照相。

    5. 将L4或年轻成虫N2蠕虫从上述一个9cm板(通常为4,000-6,000个蠕虫)收集到每个孢子-OP50板上,并孵育24-30小时。
    6. 将来自每个板的受感染的蠕虫用1.5ml 50mM NaCl洗涤到15ml Falcon管中,并以721×g离心1分钟;仔细取出上清液,将蠕虫颗粒重新悬浮并分成三个9cm NGMY板之间。使用弯曲的无菌玻璃巴斯德移液器轻轻地将颗粒散布在板的表面上。如果有必要,在25°C孵育1天后,在安全柜中干燥。这个时候大多数成虫都应该死了(图2)。在死亡之前,他们将产生后代,这时下一代的大部分蠕虫将会活着。


      图2. NGMY板上感染的蠕虫。两个死亡的成虫与多个活的幼虫。

    7. 向每个平板上加入1-2毫升50毫升的NaCl,并使用弯曲的无菌玻璃巴斯德吸管,擦去重悬死的蠕虫。从15毫升Falcon管中的3个板中收集液体;将溶液转移到250ml NGMY液中,在摇动培养箱中以170rpm培养36-40小时

  2. 原创性
    1. 将液体培养液倒入带有Miracloth的漏斗中以过滤菌丝体;用100ml CaCl 2 1700缓冲液洗涤,使用刮刀将菌丝体转移到50ml Falcon管中(图3)。将二手漏斗和Miracloth放在无菌环境中供以后使用。


      图3.用Miracloth收集菌丝体

    2. 在新制备的裂解缓冲液中称重菌丝体并用温和的涡旋重悬;每1克菌丝体中加入10毫升裂解缓冲液。如果体积超过30ml,请使用无菌的布塞锥形烧瓶,否则使用50ml Falcon管。
    3. 将菌丝体溶液在30℃下以60rpm孵育1-1.5小时。 1小时后,定期取出小分,并在显微镜下检查溶液。菌丝体片段对潮霉素的选择具有相当的抗性,可以产生菌落;尽可能避免碎片化。如果出现短的菌丝体碎片(图4),请转到下一步骤停止孵化。


      图4.裂解缓冲液消化前后的菌丝体溶液。 A.处理前的菌落; B.殖民地1小时后处理,用原生质体(红色箭头); C.处理后的菌丝体碎片(黑色箭头)。 A和B中刻度棒=20μm;比例尺= 50μm
    4. 将漏斗与原生质体步骤1中使用的Miracloth一起放入50ml Falcon管中。在裂解缓冲液中过滤菌丝体和原生质体,保留滤液
    5. 将相同体积的冰冷STC缓冲液加入到原生质体溶液中,轻轻混合并在冰上离开10分钟。
    6. 将混合物在4℃,1,127×g离心15分钟
    7. 仔细丢弃上清液;首先用移液管用1ml冰冷的STC缓冲液缓缓沉淀,再加入24ml冷STC缓冲液至原生质体中;将混合物在4℃,1,623×g离心10分钟
    8. 再次重复步骤B7。
    9. 弃去上清液,加入300μl冰冷的STC缓冲液重悬原生质体;使用血细胞计数器去除小等分试样并计数原生质体(图5)。使用冰冷的STC缓冲液将原生质体稀释至最终浓度为108个原生质体/ml。然后,将溶液储存在冰上或4℃;它可以用于转换最多7天。


      图5.血细胞计数器上的原生质体。个体原生质体用红色箭头表示。

  3. 转型
    1. 加入1μlATA到圆底14ml Falcon管的壁上,与10μg或更多的质粒DNA(体积约10μl)混合,然后轻轻移液与150μl原生质体溶液混合;在室温下孵育混合物40分钟。
    2. 使用Pipetman P1000,加入6滴PEG1700缓冲液,轻轻混匀;再加入850μlPEG缓冲液,轻轻摇匀,再孵育20分钟
    3. 加入10ml室温STC缓冲液;在室温下以1,623×g离心10分钟。
    4. 仔细丢弃上清液;在室温下用300μlSTC重悬原生质体
    5. 加入150μl原生质体溶液至9 cm平板(见注2),用无菌弯曲玻璃巴斯德吸管轻轻涂抹;在25℃培养3周。
    6. 当使用包含荧光报告基因的转化构建体时,通过PCR或荧光立体显微镜检查任何菌落是否成功转化。

笔记

  1. 除非另有说明,温度始终为25°C
  2. 除了OP50 NGM板,所有其他板均为氨苄青霉素和庆大霉素。当转化构建体包含潮霉素抗性盒时,NGMS板补充有25μg/ml潮霉素。
  3. 高压灭菌后,将铝合金袋(折叠两次)放入带有切割角的铝箔袋中,以免冷凝不积聚,布料干燥(图6)。


    图6. Miracloth准备

食谱

  1. 1M磷酸盐缓冲液,pH 6.0 108.3g KH 2 PO 4
    35.6g K 2 HPO 4
    将ddH 2 O添加到1 L
  2. 中等//
    1. 1 L NGM
      20克琼脂
      2.5克蛋白胨
      3克NaCl
      5毫克/毫升胆固醇在1毫升乙醇中
      高压灭菌后,在121℃高压灭菌20分钟后,加入:
      1 ml 1 M MgSO 4(灭菌)
      1毫升1M CaCl 2(灭菌)
      1ml 1M磷酸盐缓冲液pH6.0(灭菌)
    2. NGM-AG
      NGM与100μg/ml氨苄青霉素和50μg/ml庆大霉素
    3. NGMY
      NGM加酵母提取物20 g/L
    4. NGMY液体
      NGMY无琼脂
    5. NGMS
      NGM-AG加D-山梨醇
  3. 15 mM ATA溶液(高压灭菌器)
    在100ml ddH 2 O中的0.634g三羟甲基胺在121℃下高压灭菌20分钟
  4. 50 mM NaCl溶液(高压釜)
    5.8g NaCl的2L ddH 2 O,并在121℃下高压灭菌20分钟
  5. CaCl 2 1700(500ml,121℃高压灭菌20分钟)
    15.0g CaCl 2
    17.5克NaCl
  6. 裂解缓冲液
    CaCl 2 1700:C4 Caylase = 10ml:(或CaCl 2 1700:裂解酶= 10ml:200mg)
    注意:每次准备新鲜,用注射器和胶片过滤。
  7. STC 1700(500ml,高压釜)
    109.3g山梨醇
    5ml Tris/HCl
    22.8g CaCl 2
    1克NaCl
  8. PEG 1700(100ml,高压釜)
    60克PEG 4000
    1 ml Tris/HCl
    0.74g CaCl 2·2H 2 O ○
    注意:以1M pH 7.5,60.6g/500ml,MW = 121.2储存的Tris/HCl溶液。

致谢

我们感谢Eric Record博士协助建立协议,Carole Couillault和Zhang Xing的协助。我们还感谢Nathalie Pujol博士的意见和建议。这项工作得到了ANR(ANR-12-BSV3-0001-01)的计划资助和INSERM,CNRS和AMU的机构资助。他由中国奖学金委员会支持。

参考文献

  1. Lebrigand,K.,Hele,D.,Thakur,N.,Arguel,MJ,Polanowska,J.,Henrissat,B.,Record,E.,Magdelenat,G.,Barbe,V.,Raffaele, Barbry,P.和Ewbank,JJ(2016)。 >>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>
  2. Ochman,H.,Gerber,AS和Hartl,DL(1988)。反向聚合酶链反应的遗传应用遗传学 120(3):621-623。
  3. Turgeon,BG,Condon,B.,Liu,J. and Zhang,N。(2010)。  丝状真菌的原生质体转化 In:Sharon,A.(Ed。)。真菌的分子和细胞生物学方法。 em a>>>>>::::::::::::
  4. Stiernagle,T。(2006)。维护 C。 elegans 在: C。线虫研究社区(Ed。)。 WormBook。 WormBook 。
  • English
  • 中文翻译
免责声明 × 为了向广大用户提供经翻译的内容,www.bio-protocol.org 采用人工翻译与计算机翻译结合的技术翻译了本文章。基于计算机的翻译质量再高,也不及 100% 的人工翻译的质量。为此,我们始终建议用户参考原始英文版本。 Bio-protocol., LLC对翻译版本的准确性不承担任何责任。
Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用:He, L. D. and Ewbank, J. J. (2017). Polyethylene Glycol-mediated Transformation of Drechmeria coniospora. Bio-protocol 7(5): e2157. DOI: 10.21769/BioProtoc.2157.
提问与回复

(提问前,请先登录)bio-protocol作为媒介平台,会将您的问题转发给作者,并将作者的回复发送至您的邮箱(在bio-protocol注册时所用的邮箱)。为了作者与用户间沟通流畅(作者能准确理解您所遇到的问题并给与正确的建议),我们鼓励用户用图片或者视频的形式来说明遇到的问题。由于本平台用Youtube储存、播放视频,作者需要google 账户来上传视频。

当遇到任务问题时,强烈推荐您提交相关数据(如截屏或视频)。由于Bio-protocol使用Youtube存储、播放视频,如需上传视频,您可能需要一个谷歌账号。