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Functional Evaluation of the Signal Peptides of Secreted Proteins
分泌蛋白信号肽功能评估   

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Xiaolin Chen Xiaolin Chen
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参见作者原研究论文

本实验方案简略版
Molecular Plant Microbe Interactions
Aug 2013

Abstract

Here, we describe a method that can be used to evaluate the function of predicted signal peptides. This method utilizes the yeast Saccharomyces cerevisiae YTK12 strain and pSUC2 vector in which the pSUC2 vector with fused predicted signal peptide is transformed into yeast. The function of the signal peptides can be evaluated by using different selective media and color reaction. In this protocol, we provide the detailed description of manipulation in order to implement easily.

Keywords: Secreted protein (分泌蛋白), Signal peptide (信号肽), Function (功能), Yeast (酵母菌), pSUC2 vector (pSUC2载体)

Background

Microbial eukaryotes, such as fungi and oomycetes, secret abundant of proteins to play a variety of functions. Currently, the most widely used method for prediction of signal peptide from amino acid sequences is to use software SignalP (Petersen et al., 2011). The yeast YTK12 strain is invertase negative and pSUC2 vector contains invertase gene but lack Methionine (Met) and signal peptide sequence, thus YTK12 strain and pSUC2 vector were widely used for biological evaluation of peptide secretion (Jacobs et al., 1997; Oh et al., 2009). In addition, the color reaction could be used to verify the result, because the invertase enzymatic activity can be detected by the reduction of 2,3,5-Triphenyltetrazolium Chloride (TTC) to insoluble red colored 1,3,5-Triphenylformazan (TPF). Although the method has been generally used to evaluate the function of signal peptides, there is no specific and detailed information about this method (Oh et al., 2009; Song et al., 2015). Here, we describe a modified high-efficiency yeast transformation method (Gietz and Schiestl, 2007) and detailed protocol to evaluate the function of signal peptides, which will be the primary part for the secretory proteins research.

Materials and Reagents

  1. Pipette tips
  2. 1.5 ml tubes
  3. 2 ml tubes
  4. 10 ml test tube
  5. Millipore filter units, 0.22 µm (Millex-GP, Merck, catalog number: SLGP033RB )
  6. Yeast YTK12 strain
  7. pSUC2 vector
  8. EcoRI restriction enzymes (Takara Bio, catalog number: 1040S )
  9. XhoI restriction enzymes (Takara Bio, catalog number: 1094S )
  10. Yeast extract (OXOID, catalog number: LP0021 )
  11. Peptone (BD, DifcoTM, catalog number: 211677 )
  12. Glucose (Sinopharm Chemical Reagent, catalog number: 10010518 )
  13. Agar A (Sangon Biotech, catalog number: A600010 )
  14. Adenine hemisulfate salt (Sigma-Aldrich, Vetec, catalog number: V900375 )
  15. Tris-HCl (Sigma-Aldrich, catalog number: V900312 )
  16. EDTA (Sinopharm Chemical Reagent, catalog number: 10009617 )
  17. NaOH (Sinopharm Chemical Reagent, catalog number: 10019718 )
  18. Single-stranded carrier DNA (salmon sperm DNA, Solarbio, catalog number: D8030 )
  19. LiAc (Sinopharm Chemical Reagent, catalog number: 30109760 )
  20. PEG (Polyethylene glycol, Sigma-Aldrich, catalog number: P3640 )
  21. DMSO (Dimethyl Sulfoxide, Sigma-Aldrich, catalog number: D5879 )
  22. YNB (yeast nitrogen base without amino acids, BD, DifcoTM, catalog number: 291920 )
  23. -Trp DO supplement (Clontech, catalog number: 630413 )
  24. Sucrose (Sinopharm Chemical Reagent, catalog number: 10021418 )
  25. Antimycin A (Sigma-Aldrich, catalog number: A8674 )
  26. Raffinose (D-(+)-Raffinose pentahydrate, Bomei, CAS Number: 17629-30-0)
  27. Sodium acetate (Sinopharm Chemical Reagent, catalog number: 10018718 )
  28. Acetic acid (Sinopharm Chemical Reagent, catalog number: 10000218 )
  29. KH2PO4 (Sinopharm Chemical Reagent, catalog number: 10017618 )
  30. Na2HPO4 (Sinopharm Chemical Reagent, catalog number: 10020318 )
  31. TTC (Tokyo Chemical Industry (TCI), CAS Number: 298-96-4)
  32. YPD medium (1 L) (see Recipes)
  33. 1x TE Buffer (100 ml) (see Recipes)
  34. Single-stranded carrier DNA (2 mg/ml) (see Recipes)
  35. 1.0 M LiAc (100 ml) (see Recipes)
  36. 50% (w/v) PEG (100 ml) (see Recipes)
  37. CMD-W medium (1 L) (see Recipes)
  38. Antimycin A stock solution (50 mg/ml) (see Recipes)
  39. YPRAA medium (1 L) (see Recipes)
  40. 10 mM acetic acid-sodium acetate buffer (100 ml, pH = 4.7) (see Recipes)
  41. Phosphate Buffer (150 ml) (see Recipes)
  42. TTC Stock solution (2%) (see Recipes)

Equipment

  1. Pipettes (Mettler-Toledo International, RAININ, model: XLS )
  2. Incubator (ZHICHENG, model: ZXDP-B2050 )
  3. Clean bench (AIRTECH, model: SW-CJ-2FD , catalog number: A11062689)
  4. Water bath (Shanghai Jinghong Laboratory Instrument, model: DK-8D )
  5. Vortex mixer (Kylin-Bell Lab Instruments, model: VORTEX-5 )
  6. Incubator shaker (Changzhou Zhiborui Instrument Manufacturing, model: THZ-D )
  7. Centrifuge (Eppendorf, model: 5424 )

Procedure

  1. pSUC2 vector construction
    1. DNA fragments that code the predicted signal peptides and the following additional two amino acids were amplified and introduced into pSUC2 using EcoRI and XhoI restriction sites (Figure 1). The DNA sequences around enzyme cutting site are as follows and the sequences of sites are underlined.
      EcoRI: 5’ TCCAAGCTCGGAATTTTAATTAAGAATTC 3’
      XhoI: 5’ CTCGAGGTTCTCCCTATAGTGAGTCGTAT 3’


      Figure 1. The vector information of pSUC2. This map was modified from Jacobs (Jacobs et al., 1997).

    2. Confirm the positive transformants by PCR with pSUC2 vector-specific primers as follows, which should be further confirmed by sequencing.
      pSUC2F: GGTGTGAAGTGGACCAAAGGTCTA
      pSUC2R: CCTCGTCATTGTTCTCGTTCCCTT
    3. In this study, we use the recombinant plasmid that contains Avr1b signal peptide from Phytophthora sojae that was demonstrated to be functional (Dou et al., 2008; Song et al., 2015).

  2. Transformation of plasmid DNA into yeast cells
    1. Streak an YPDA agar plate with the YTK12 yeast cells from a frozen yeast stock, incubate the plate upside down at 30 °C for 2-3 days until colonies appear.
      Note: YPD medium also can be used, although yeast can get a better situation on YPDA. If there are no special instructions, the following steps are done at the room temperature.
    2. Inoculate one colony (diameter 2-3 mm) into 3 ml YPDA liquid medium in a sterile test tube, incubate at 30 °C for 16-18 h with shaking at 220 rpm and measure the OD values at 600 nm to get suspension with OD value more than 0.6 (OD600 > 0.6).
    3. Pipette about 1.5 ml of cell suspension into a 2 ml tube and centrifuge the cells at 12,000 x g for 1 min at room temperature.
    4. Discard the supernatant and re-suspend the cells with 1.5 ml sterile distilled water or TE buffer.
    5. Centrifuge the cells at 12,000 x g for 1 min and discard the supernatant.
    6. For each transformation, add reagents in a 2 ml tube as following order and keep on ice: 34 μl plasmid DNA and sterile water (1 μg Plasmid DNA will be enough for transformation); 50 μl single-stranded carrier DNA (2.0 mg/ml) and mix well; 36 μl 1.0 M LiAc and mix well; 240 μl 50% (w/v) PEG 3350 and vortex mix vigorously or use a sterile pipette tip to break up the cell pellet to mix thoroughly.
    7. Incubate with shaking at 220 rpm for 30 min at 30 °C.
    8. Add 1/10 of the volume DMSO and mix gently.
    9. Incubate the cell suspension at 42 °C for 15 min and mix upside down for several times during this time.
    10. Incubate on ice for 2 min.
    11. Centrifuge at 12,000 x g for 1 min and discard the supernatant.
    12. Add 100 μl sterile distilled water or TE buffer and re-suspend the cells with a pipette carefully.
    13. Coat the cell suspension on selective media CMD-W plate using a sterile glass rod and incubate at 30 °C for 3 days.

  3. Screening the positive yeast colonies on selective media
    1. After transformation, spread the yeast cells on CMD-W media plates.
    2. Streak the colonies to fresh CMD-W plates and incubate at 30 °C for at least 2 days.
    3. Streak the colonies to new CMD-W plates and YPRAA plates (raffinose media) and incubate for another 2-3 days at 30 °C.

  4. Color observation to detect signal peptide secretion function
    1. The color change is used to detect signal peptide secretion function.
    2. Streak the single transformational colony into CMD-W liquid medium, the YTK12 yeast that does not contain any plasmid DNA was cultured in YPDA liquid medium as control.
    3. Incubate with shaking at 220 rpm for 36 h at 30 °C.
    4. Pipette about 1.5 ml of cell suspension into a 2 ml tube and centrifuge at 12,000 x g for 1 min at room temperature.
    5. Discard the supernatant, add 1.5 ml sterile distilled water and vortex shock to mix vigorously.
    6. Centrifuge at 12,000 x g for 1 min, discard the supernatant and add 1.5 ml sterile distilled water and vortex shock to mix well.
    7. Centrifuge at 12,000 x g for 1 min and discard the supernatant.
    8. Re-suspend with 750 μl sterile distilled water, add 250 μl 10 mM acetic acid–sodium acetate buffer (pH = 4.7), 500 μl 10 % sucrose solution (w/v), incubate at 37 °C for 10 min.
    9. Centrifuge at 12,000 x g for 1 min, take 100 μl of the supernatant, put into glass test tube and add 900 μl 0.1% TTC solution, incubate at room temperature for 5 min. Colorimetric change will be observed if the signal peptide is functional, while the red color will be not observed for YTK12 strain and YTK12 carrying the empty pSUC2 vector. 
      Note: The empty pSUC2 vector also contains a non-functional sequence.

Data analysis

If the signal peptide is functional, the yeast transformants can grow on CMD-W and YPRAA medium plates and the solution will turn red in the color test experiment (Figure 2).


Figure 2. Functional validation of the signal peptide of Avr1b.The yeast YTK12 strain carrying Avr1b signal peptide fragment fused in the pSUC2 vector are able to grow in both the CMD-W and YPRAA media, and also induce red color reaction, indicating the secretory function. The YTK12 strain and YTK12 carrying the empty pSUC2 vector served as controls.

Notes

Inoculate colony on CMD-W plates is to verify if the plasmid DNA is transformed into the yeast. We also observed that the yeast without any vector can get a small amount of growth on CMD-W and YPRAA plates under the condition of incubation for a long time (more than three days). To assay the invertase secretion, colonies were plated on YPRAA plates containing raffinose but lacking glucose for several times. The transformants containing pSUC2 vector can grow on YPRAA plate if incubated for more than three days. Thus, when the yeast colonies are streaked on CMD-W and YPRAA plates, it must be observed every day. The principle of color change is that the invertase enzymatic activity was detected by the reduction of 2,3,5-Triphenyltetrazolium Chloride (TTC) to insoluble red colored 1,3,5-Triphenylformazan (TPF).

Recipes

  1. YPD medium (1 L)
    10 g yeast extract
    20 g peptone
    20 g glucose
    Add distilled water to 1 L, autoclave for 15 min at 121 °C
    Add 20 g Agar A for solid medium
    Supplement of adenine hemisulfate to the final concentration 30 mg/L in YPD could provide yeast a better growth condition
  2. 1x TE Buffer (100 ml)
    1 M Tris-HCl (pH 7.5) 10 ml
    500 mM EDTA (pH 8.0) 2 ml
    Adjust pH to 7.5 with NaOH
    Make up to 100 ml
    Autoclave for 15 min at 121 °C
  3. Single-stranded carrier DNA (2 mg/ml)
    1. Dissolve 100 mg single-stranded carrier DNA in 50 ml of sterile TE buffer by using magnetic stir at 4 °C until no visible DNA is seen
    2. Dispense 1.0 ml sample into 1.5 ml tubes and denature the carrier DNA in a boiling water bath for 5 min and chill immediately in an ice/water bath for several minutes, and store at -20 °C
    3. Make sure to gradually thaw the denatured carrier DNA on ice whenever it is used
    4. Denatured carrier DNA could be boiled, frozen and thawed many times
  4. 1.0 M LiAc (100 ml)
    10.2 g LiAc
    Add distilled water to the volume of 100 ml
    Sterilize by filtering Millipore filter units, 0.22 µm
  5. 50% (w/v) PEG (100 ml)
    50 g PEG
    Add 80 ml distilled water to fully dissolved and then make the volume up to 100 ml
    Autoclave for 15 min at 121 °C
  6. CMD-W medium (1 L)
    6.7 g YNB (yeast nitrogen base without amino acids)
    0.74 g -Trp DO supplement
    20 g sucrose
    1 g glucose
    20 g Agar A
    Add distilled water to 1 L, autoclave for 15 min at 121 °C
  7. Antimycin A stock solution (50 mg/ml)
    50 mg antimycin A dissolved in DMSO (dimethylsulphoxide)
  8. YPRAA medium (1 L)
    10 g yeast extract
    20 g peptone
    20 g raffinose
    20 g Agar A
    Add distilled water to 1 L, autoclave for 15 min at 121 °C
    Add antimycin A to a final concentration of 2 mg/ml just before pouring medium plates
  9. 10 mM acetic acid-sodium acetate buffer (100 ml, pH = 4.6)
    0.136 g sodium acetate
    Add 90 ml water
    Adjust pH to 7.5 with acetic acid
    Make up to 100 ml
  10. Phosphate Buffer (150 ml)
    100 ml 66.7 mM KH2PO4
    50 ml 68.6 mM
    Mix well to get phosphate buffer
  11. TTC Stock solution (2%)
    1. Dissolve 2 g TTC in 100 ml water, store in brown bottle or keep away from light
      Note: Because TTC in water cannot be kept for a long time, it should be dissolved in phosphate buffer for long-term preservation.
    2. Dilute with 1 M NaOH to 0.1% when it is used

Acknowledgments

This research was supported by the National Natural Science Foundation of China (Grant No. 31701736) and the Fundamental Research Funds for the Central Universities (Grant Nos. 2662015PY195 and 2662017JC003). The authors have declared that no conflicts of interest or competing interests exist.

References

  1. Dou, D., Kale, S. D., Wang, X., Jiang, R. H., Bruce, N. A., Arredondo, F. D., Zhang, X. and Tyler, B. M. (2008). RXLR-mediated entry of Phytophthora sojae effector Avr1b into soybean cells does not require pathogen-encoded machinery. Plant Cell 20(7): 1930-1947.
  2. Gietz, R. D. and Schiestl, R. H. (2007). Large-scale high-efficiency yeast transformation using the LiAc/SS carrier DNA/PEG method. Nat Protoc 2(1): 38-41.
  3. Jacobs, K. A., Collins-Racie, L. A., Colbert, M., Duckett, M., Golden-Fleet, M., Kelleher, K., Kriz, R., LaVallie, E. R., Merberg, D., Spaulding, V., Stover, J., Williamson, M. J. and McCoy, J. M. (1997). A genetic selection for isolating cDNAs encoding secreted proteins. Gene 198(1-2): 289-296.
  4. Oh, S. K., Young, C., Lee, M., Oliva, R., Bozkurt, T. O., Cano, L. M., Win, J., Bos, J. I., Liu, H. Y., van Damme, M., Morgan, W., Choi, D., Van der Vossen, E. A., Vleeshouwers, V. G. and Kamoun, S. (2009). In planta expression screens of Phytophthora infestans RXLR effectors reveal diverse phenotypes, including activation of the Solanum bulbocastanum disease resistance protein Rpi-blb2. Plant Cell 21(9): 2928-2947.
  5. Petersen, T. N., Brunak, S., von Heijne, G. and Nielsen, H. (2011). SignalP 4.0: discriminating signal peptides from transmembrane regions. Nat Methods 8(10): 785-786.
  6. Song, T., Ma, Z., Shen, D., Li, Q., Li, W., Su, L., Ye, T., Zhang, M., Wang, Y. and Dou, D. (2015). An oomycete CRN effector reprograms expression of plant HSP genes by targeting their promoters. PLoS Pathog 11(12): e1005348.

简介

在这里,我们描述了一种可用于评估预测信号肽功能的方法。 该方法利用酵母酿酒酵母YTK12菌株和pSUC2载体,其中将具有融合的预测信号肽的pSUC2载体转化到酵母中。 信号肽的功能可以通过使用不同的选择性培养基和显色反应来评估。 在这个协议中,我们提供了操作的详细描述以便轻松实现。

【背景】微生物真核生物,如真菌和卵菌,可以分泌丰富的蛋白质来发挥各种功能。目前,从氨基酸序列预测信号肽的最广泛使用的方法是使用软件SignalP(Petersen等人,2011)。酵母YTK12菌株为转化酶阴性,pSUC2载体含有转化酶基因但缺少甲硫氨酸(Met)和信号肽序列,因此YTK12菌株和pSUC2载体广泛用于肽分泌的生物学评价(Jacobs等人 >,1997; Oh等人,2009)。此外,颜色反应可用于验证结果,因为转化酶的酶活性可以通过将2,3,5-三苯基氯化四唑(TTC)还原成不溶性红色1,3,5-三苯甲forma胺(TPF )。尽管该方法一般用于评估信号肽的功能,但没有关于该方法的具体和详细的信息(Oh等人,2009; Song等人 >,2015)。在这里,我们描述了一种改进的高效酵母转化方法(Gietz和Schiestl,2007)和详细的方案来评估信号肽的功能,这将是分泌蛋白质研究的主要部分。

关键字:分泌蛋白, 信号肽, 功能, 酵母菌, pSUC2载体

材料和试剂

  1. 移液器吸头
  2. 1.5毫升管
  3. 2毫升管
  4. 10毫升试管
  5. Millipore过滤器,0.22μm(Millex-GP,Merck,目录号:SLGP033RB)
  6. 酵母YTK12株
  7. pSUC2载体
  8. Eco RI限制性酶(Takara Bio,目录号:1040S)
  9. XhoI限制酶(Takara Bio,目录号:1094S)
  10. 10.酵母提取物(OXOID,目录号:LP0021)
  11. 蛋白胨(BD,Difco TM,产品目录号:211677)
  12. 葡萄糖(国药集团化学试剂,目录号:10010518)
  13. 琼脂A(Sangon Biotech,产品目录号:A600010)
  14. 腺嘌呤半硫酸盐(Sigma-Aldrich,Vetec,目录号:V900375)
  15. Tris-HCl(Sigma-Aldrich,目录号:V900312)
  16. EDTA(国药集团化学试剂,目录号:10009617)
  17. NaOH(国药集团化学试剂,目录号:10019718)
  18. 单链载体DNA(鲑鱼精子DNA,Solarbio,目录号:D8030)
  19. LiAc(国药集团化学试剂,目录号:30109760)
  20. PEG(聚乙二醇,Sigma-Aldrich,目录号:P3640)
  21. DMSO(二甲基亚砜,Sigma-Aldrich,目录号:D5879)
  22. YNB(不含氨基酸的酵母氮源,BD,Difco TM,目录号:291920)
  23. -Trp DO补充剂(Clontech,目录号:630413)
  24. 蔗糖(国药集团化学试剂,目录号:10021418)
  25. 抗霉素A(Sigma-Aldrich,目录号:A8674)
  26. 棉子糖(D - (+) - 棉子糖五水合物,Bomei,CAS编号:17629-30-0)
  27. 醋酸钠(国药集团化学试剂,目录号:10018718)
  28. 醋酸(国药集团化学试剂,目录号:10000218)
  29. KH 2 PO 4(国药集团化学试剂,目录号:10017618)
  30. Na 2 HPO 4(Sinopharm Chemical Reagent,目录号:10020318)
  31. TTC(东京化学工业(TCI),CAS编号:298-96-4)
  32. YPD培养基(1L)(见食谱)
  33. 1×TE缓冲液(100毫升)(见食谱)
  34. 单链载体DNA(2 mg / ml)(见食谱)
  35. 1.0 M LiAc(100毫升)(见食谱)
  36. 50%(w / v)PEG(100 ml)(见食谱)
  37. CMD-W培养基(1L)(见食谱)
  38. 抗霉素A储备液(50毫克/毫升)(见食谱)
  39. YPRAA培养基(1 L)(见食谱)
  40. 10 mM乙酸 - 醋酸钠缓冲液(100 ml,pH = 4.7)(见食谱)
  41. 磷酸盐缓冲液(150毫升)(见食谱)
  42. TTC库存解决方案(2%)(请参阅食谱)

设备

  1. 移液器(梅特勒 - 托利多国际,RAININ,型号:XLS)
  2. 孵化器(ZHICHENG,型号:ZXDP-B2050)
  3. 洁净工作台(AIRTECH,型号:SW-CJ-2FD,产品目录号:A11062689)
  4. 水浴(上海景洪实验仪器,型号:DK-8D)
  5. 涡旋混合器(Kylin-Bell Lab Instruments,型号:VORTEX-5)
  6. 孵化器摇床(常州紫葆瑞仪器制造有限公司,型号:THZ-D)
  7. 离心机(Eppendorf,型号:5424)

程序

  1. pSUC2载体构建
    1. 将编码预测的信号肽和随后的另外两个氨基酸的DNA片段扩增并使用EcoRI和XhoI限制性位点(图1)引入pSUC2中。
      切酶位点周围的DNA序列如下,位点序列加下划线。
      Eco EcoRI:5'TCCAAGCTCGGAATTTTAATTAAGAATTC 3'
      Xho I:5'CTCGAGGTTCTCCCTATAGTGAGTCGTAT 3'


      图1. pSUC2的矢量信息该图由Jacobs(Jacobs et al。 ,1997>)进行了修改。

    2. 如下通过PCR用pSUC2载体特异性引物确认阳性转化体,其应通过测序进一步确认。
      pSUC2F:GGTGTGAAGTGGACCAAAGGTCTA
      pSUC2R:CCTCGTCATTGTTCTCGTTCCCTT
    3. 在本研究中,我们使用含有来自大豆疫霉(Phytophthora sojae)的Avr1b信号肽的重组质粒,该质粒被证明是功能性的(Dou等人,2008; Song等人,2015)。

  2. 质粒DNA转化酵母细胞
    1. 将YPDA琼脂平板与来自冷冻酵母原液的YTK12酵母细胞划线接种,在30℃下将平板颠倒孵育2-3天直至菌落出现。
      注意:YPD培养基也可以使用,尽管酵母可以在YPDA上获得更好的状态。如果没有特殊说明,则在室温下进行以下步骤。
    2. 在无菌试管中将一个菌落(直径2-3mm)接种到3ml YPDA液体培养基中,在30℃下以220rpm振荡孵育16-18小时,并在600nm处测量OD值以获得具有OD的悬浮液值大于0.6(OD <600> 0.6)。
    3. 吸取约1.5毫升细胞悬液到2毫升试管中,并在室温下以12,000×gg离心1分钟。
    4. 丢弃上清液并用1.5 ml无菌蒸馏水或TE缓冲液重新悬浮细胞。
    5. 将细胞以12,000×g离心1分钟并丢弃上清液。
    6. 对于每次转化,按如下顺序将试剂加入2ml试管中并保持在冰上:34μl质粒DNA和无菌水(1μg质粒DNA将足以转化); 50μl单链载体DNA(2.0mg / ml)并充分混合; 36μl1.0M LiAc并充分混合; 240μl50%(w / v)PEG 3350并涡旋混合,或使用无菌枪头打碎细胞沉淀以彻底混匀。

    7. 在30°C以220转/分的速度摇动30分钟
    8. 加入1/10体积DMSO并轻轻混合。
    9. 在42℃孵育细胞悬液15分钟,并在此期间颠倒混合数次。
    10. 在冰上孵育2分钟。
    11. 在12,000×gg下离心1分钟并丢弃上清液。
    12. 加入100μl无菌蒸馏水或TE缓冲液,并用移液管小心重新悬浮细胞。
    13. 使用无菌玻璃棒将细胞悬液涂布在选择性培养基CMD-W平板上,并在30℃孵育3天。

  3. 在选择性培养基上筛选阳性酵母菌落
    1. 转化后,将酵母细胞铺在CMD-W培养基平板上。
    2. 将菌落划线到新鲜的CMD-W平板上,并在30°C孵育至少2天。
    3. 将菌落划线至新的CMD-W平板和YPRAA平板(棉子糖培养基)并在30℃孵育2-3天。

  4. 颜色观察来检测信号肽分泌功能
    1. 颜色变化用于检测信号肽分泌功能。
    2. 将单一转化菌落划入CMD-W液体培养基中,将不含任何质粒DNA的YTK12酵母培养于YPDA液体培养基中作为对照。

    3. 在220°C振荡培养36小时,30°C。
    4. 吸取约1.5毫升细胞悬液到2毫升试管中,并在室温下以12,000×gg离心1分钟。
    5. 丢弃上清液,加入1.5毫升无菌蒸馏水和涡流震荡混合剧烈。
    6. 在12,000×gg下离心1分钟,弃去上清液并加入1.5ml无菌蒸馏水并涡流震荡以充分混合。

    7. 12,000×g g离心1分钟,弃去上清液。
    8. 重悬于750μl无菌蒸馏水中,加入250μl10 mM乙酸 - 醋酸钠缓冲液(pH = 4.7),500μl10%蔗糖溶液(w / v),37°C孵育10 min。 />
    9. 在12,000×gg下离心1分钟,取100μl上清液,放入玻璃试管并加入900μl0.1%TTC溶液,在室温下孵育5分钟。如果信号肽具有功能,则会观察到比色变化,而YTK12菌株和携带空pSUC2载体的YTK12则不会观察到红色。&nbsp;
      注:空的pSUC2载体也包含一个非功能性序列。

数据分析

如果信号肽是功能性的,酵母转化体可以在CMD-W和YPRAA培养基平板上生长,并且在颜色测试实验中溶液会变成红色(图2)。


图2.Avr1b的信号肽的功能验证携带在pSUC2载体中融合的Avr1b信号肽片段的酵母YTK12菌株能够在CMD-W和YPRAA培养基中生长,并且还诱导红色反应,表明分泌功能。携带空pSUC2载体的YTK12菌株和YTK12用作对照。

笔记

在CMD-W平板上接种菌落是为了验证质粒DNA是否转化到酵母中。我们还观察到,在长时间孵育(超过三天)的条件下,没有任何载体的酵母可以在CMD-W和YPRAA平板上获得少量的生长。为了测定转化酶分泌,将菌落铺在含有棉子糖但缺少葡萄糖的YPRAA板上数次。如果孵育超过三天,含有pSUC2载体的转化体可以在YPRAA平板上生长。因此,当酵母菌落在CMD-W和YPRAA平板上划线时,必须每天观察。颜色变化的原理是通过将2,3,5-三苯基氯化四唑(TTC)还原为不溶性红色1,3,5-三苯甲forma(TPF)来检测转化酶的酶活性。

食谱

  1. YPD培养基(1升)
    10克酵母提取物
    20克蛋白胨
    20克葡萄糖
    将蒸馏水加入1L,在121℃高压灭菌15分钟。
    添加20克琼脂A固体培养基
    在YPD中补充30mg / L的腺嘌呤半硫酸盐可以为酵母提供更好的生长条件
  2. 1x TE缓冲液(100 ml)
    1M Tris-HCl(pH 7.5)10ml
    500 mM EDTA(pH 8.0)2 ml
    用NaOH调节pH至7.5
    弥补100毫升

    在121°C高压灭菌15分钟
  3. 单链载体DNA(2mg / ml)
    1. 通过在4℃下使用磁力搅拌将100mg单链载体DNA溶解于50ml无菌TE缓冲液中,直至看不到可见的DNA。
    2. 将1.0毫升样品分配到1.5毫升试管中,并将载体DNA在沸水浴中变性5分钟,立即在冰/水浴中冷却几分钟,并储存在-20℃。


    3. 每次使用时,请务必在冰上逐渐融化变性的载体DNA
    4. 变性的载体DNA可以煮沸,冷冻和解冻多次
  4. 1.0 M LiAc(100毫升)
    10.2克LiAc
    加蒸馏水至100毫升体积
    通过过滤Millipore过滤器单元进行消毒,0.22μm
  5. 50%(w / v)PEG(100 ml)
    50克PEG
    加入80毫升蒸馏水使其完全溶解,然后使体积达到100毫升

    在121°C高压灭菌15分钟
  6. CMD-W培养基(1L)
    6.7克YNB(不含氨基酸的酵母氮基)
    0.74克-Trp补充剂
    20克蔗糖
    1克葡萄糖
    20克琼脂A
    将蒸馏水加入1L,在121℃高压灭菌15分钟。
  7. 抗霉素A储备液(50毫克/毫升)
    将50mg抗霉素A溶于DMSO(二甲基亚砜)中
  8. YPRAA中(1升)
    10克酵母提取物
    20克蛋白胨
    20克棉子糖
    20克琼脂A
    将蒸馏水加入1L,在121℃高压灭菌15分钟。

    在即将浇注中型平板之前加入抗霉素A至最终浓度为2 mg / ml
  9. 10mM乙酸 - 乙酸钠缓冲液(100ml,pH = 4.6)
    0.136克醋酸钠
    加90毫升水

    用醋酸将pH调至7.5 弥补100毫升
  10. 磷酸盐缓冲液(150毫升)
    100毫升66.7毫摩尔KH 2 PO 4水溶液 50毫升68.6毫米
    充分混合即可得到磷酸盐缓冲液
  11. TTC库存解决方案(2%)
    1. 将2克TTC溶于100毫升水中,存放于棕色瓶中或远离光线
      注意:由于水中的TTC不能长期保存,因此应将其溶解在磷酸盐缓冲液中进行长期保存。

    2. 用1M NaOH稀释至0.1%

致谢

本研究得到国家自然科学基金(批准号:31701736)和中央高校基础研究基金(批准号:2662015PY195和2662017JC003)的资助。作者声明不存在利益冲突或利益冲突。

参考

  1. Dou,D.,Kale,S.D.,Wang,X.,Jiang,R.H.,Bruce,N.A.,Arredondo,F.D。,Zhang,X.and Tyler,B.M。(2008)。 RXLR介导的大豆疫霉菌效应子Avr1b进入大豆细胞不会需要病原体编码的机器。 Plant Cell 20(7):1930-1947。
  2. Gietz,R.D。和Schiestl,R.H。(2007)。 使用LiAc / SS载体DNA / PEG方法进行大规模高效酵母转化。 a> Protoc 2(1):38-41。
  3. Jacobs,KA,Collins-Racie,LA,Colbert,M.,Duckett,M.,Golden-Fleet,M.,Kelleher,K.,Kriz,R.,LaVallie,ER,Merberg,D.,Spaulding,V. ,Stover,J.,Williamson,MJ和McCoy,JM(1997)。 分离编码分泌蛋白的cDNA的遗传选择。 Gene > 198(1-2):289-296。
  4. Oh,SK,Young,C.,Lee,M.,Oliva,R.,Bozkurt,TO,Cano,LM,Win,J.,Bos,JI,Liu,HY,van Damme,M.,Morgan,W. ,Choi,D.,Van der Vossen,EA,Vleeshouwers,VG和Kamoun,S。(2009)。 在致病疫霉(Phytophthora infestans)的植物表达筛选中RXLR效应物揭示了不同的表型,包括激活Solanum bulbocastanum抗病蛋白Rpi-blb2。植物细胞21(9):2928-2947。
  5. Petersen,T.N.,Brunak,S.,von Heijne,G。和Nielsen,H。(2011)。 SignalP 4.0:从跨膜区域鉴别信号肽 Nat Methods em> 8(10):785-786。
  6. Song,T.,Ma,Z.,Shen,D.,Li,Q.,Li,W.,Su,L.,Ye,T.,Zhang,M.,Wang,Y.和Dou,D.( 2015年)。 卵菌CRN效应子通过靶向它们的启动子重新编程植物HSP基因的表达。 > PLoS Pathog 11(12):e1005348。
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引用:Yin, W., Wang, Y., Chen, T., Lin, Y. and Luo, C. (2018). Functional Evaluation of the Signal Peptides of Secreted Proteins. Bio-protocol 8(9): e2839. DOI: 10.21769/BioProtoc.2839.
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