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Virus-induced Gene Silencing (VIGS) in Phalaenopsis Orchids
蝴蝶兰的病毒诱导基因沉默(VIGS)   

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参见作者原研究论文

本实验方案简略版
New Phytologist
May 2014

Abstract

This is a protocol to produce stable silencing efficacy and efficiency for VIGS using CymMV as a silencing vector for floral functional genomics in Phalaenopsis orchids. This protocol is established based on a method created by Lu et al. (2007), and then modified by Hsieh et al. (2013a; 2013b), Lu et al. (2012) successfully engineered a cloning vector (pCymMV-Gateway) in that the target gene fragment is simple to insert and can be manipulated with high efficiency. The silencing vector is inoculated into plants by Agro-inoculation by using Agrobacterium tumefaciens (A. tumefaciens) strain EHA105. Agro-infiltration of leaves for use in VIGS study of orchid flowers is a time saver and produces less damage to flower buds.

Materials and Reagents

  1. Virus-free Phalaenopsis plants
  2. TOP10 Escherichia coli (E. coli) competent cells
  3. Agrobacterium tumefaciens strain EHA105
  4. Gel/PCR DNA fragments extraction kit (Geneaid Biotech, catalog number: DF100 )
  5. Gateway® BP-ClonaseTM II enzyme mix (Life Technologies, InvitrogenTM, catalog number: 11789-020 )
  6. Selective plate (LB plate with 50 µg/ml Kanamycin)
  7. High-speed plasmid mini kit (Geneaid Biotech, catalog number: PD100 )
  8. Acetosyringone (Sigma-Aldrich, catalog number: D134406 )
  9. Murashige and Skoog salt (Sigma-Aldrich, catalog number: M5524 )
  10. Tryptone (US Biological, catalog number: 12855 )
  11. Yeast extract (Affymetrix, catalog number: 23547 )
  12. NaCl (Sigma-Aldrich, catalog number: S1446 )
  13. Tris-HCl (US Biological, catalog number: 22676 )
  14. CaCl2 (Inter-County Mechanical Corp., catalog number: 03-11250 )
  15. Glycerol (J.T.Baker®, catalog number: 2136-01 )
  16. Luria Broth medium (LB medium) (see Recipes)
  17. Proteinase K solution (see Recipes)
  18. Murishige and Skoog medium (MS medium) salt (see Recipes)

Equipment

  1. GeneAmp PCR system 9700 (Life Technologies, Applied Biosystems®)
  2. Laminar flow cabinet
  3. BioChrom Libra S50 UV/Vis Spectrophotometer (Biochrom)
  4. Incubator shaker
  5. Electroporation machine (Kaneka Corporation, Eurogentec)
  6. Electroporation cuvettes (EquiBio, catalog number: ECU-102 )
  7. Centrifuges for MiniSpin Eppendorf and 50 ml conical centrifuge tubes
  8. 1-ml syringe with a needle
  9. Water bath

Procedure

  1. Construction of pCymMV-Gateway plasmid DNA
    1. DNA fragments for silenced target gene for insertion into pCymMV-Gateway vector (Figure 1A) is obtained by PCR amplification with oligonucleotide primers containing 29-nt attB recombination site in both ends (see Note 1). Purify attB-PCR products by using a gel extraction kit.
    2. Insert target gene fragment into pCymMV-Gateway vector by using BP recombination reaction (Figure 1B).
      1. In a 1.5 ml microcentrifuge tube at room temperature mix attB-PCR product (150 ng), pCymMV-Gateway vector (150 ng), and 4 µl of 5x BP ClonaseTM reaction buffer. Then add 1x TE buffer (pH 8.0) until a total volume of 16 µl is obtained.
      2. Remove the BP ClonaseTM enzyme mix from -80 °C storage and put in ice for 2 min to thaw and vortex briefly twice (2 sec each time).
      3. Add 4 µl of BP ClonaseTM enzyme mix to each reaction (step A2a, above). Mix well by vortexing twice and then microcentrifuge briefly. Store BP ClonaseTM enzyme mix at -80 °C immediately after usage.
      4. Incubate reactions at 25 °C for 60 min.
      5. Add 2 µl of the Proteinase K solution to each sample to terminate the reaction.
      6. Vortex briefly and incubate samples at 37 °C for 10 min.
    3. Transform competent E. coli with the pCymMV-Gateway vector for amplification (in the Laminar flow cabinet).
      1. Incubate TOP10 E. coli competent cells on ice for 10 min (see Note 2).
      2. Add 5 µl of each BP reaction (step A2) into 50-100 µl of TOP10 E. coli competent cells.
      3. Incubate on ice for 15 min.
      4. Heatshock cells by incubating at 42 °C water bath for 45 sec.
      5. Incubate on ice for 2 min.
      6. Add 200 µl of LB medium and incubate at 37 °C for 60 min while shaking at 200 rpm.
      7. Spread each transformation (305 µl) onto LB-agar medium plates containing 50 µg/ml of Kanamycin.
      8. Incubate at 37 °C for 13 h.
    4. Randomly select two to four colonies for each construct, and then subculture.
    5. Recover and purify the plasmids.
    6. Confirm each construct by PCR with the gene-specific forward primer and the    general    reverse    primer    (CymMV 5351, 5′-CTTCTGTACCATACACATAG-3′) on the gateway vector.

  2. Transformation by electroporation after amplification of the recombined pCymMV-Gateway vector (steps B1-3 in the Laminar flow cabinet)
    1. Thaw the Agrobacterium tumefaciens (strain EHA105) competent cells at room temperature and immediately place on ice. Transfer 100 μl of the competent cells to a chilled electroporation cuvette on ice. Add 1-2 μl of recombined pCymMV-Gateway vector (150 ng of plasmid DNA can be used depending on the competence of the cells), mix gently and keep on ice.
    2. Perform the electroporation.
      1. Insert the cuvettes in the electroporation chamber and initiate the electroporation. The voltage and pulse time parameters are pre-set.
      2. Immediately add 200 μl of LB medium in the cuvettes and transfer the solution to a sterile Eppendorf tube.
      3. Incubate the mixture at 28 °C  for 30 min. while shaking at 200 rpm.
    3. Spread all the mixture on selected LB-agar medium plate containing 100 μM of Acetosyringone and 50 µg/ml of Kanamycin and incubate at 28 °C for 16 h.
    4. Confirm each colony by PCR with the gene-specific forward primer and the general    reverse    primer (CymMV 5351, 5′-CTTCTGTACCATACACATAG-3′) on the gateway vector for the presence of the modified constructs.


      Figure 1. (A) Schematic representation of pCymMV-Gateway vector. Green rectangles represent open reading frames encoded by Cymbidium mosaic virus (CymMV) genomic RNA. RNA-dependent RNA polymerase (RdRp); triple gene block 1, 2, and 3; coat protein (CP) and attB sites are indicated. (B) Amplification of target gene and construction of pCambia-CymMV vector.

  3. Agro-infiltration of Phalaenopsis plants
    1. Prepare electroporation competent Agrobacterium cells:
      1. Four days prior to infection, transfer a single colony of Agrobacterium tumefaciens containing recombinant plasmids into 5 ml of LB medium containing 100 μM of Acetosiringone and 50 µg/ml of kanamycin and culture at 28 °C for 16 h while shaking at 200 rpm.
      2. Subculture the bacterial cell in 50 ml of LB medium containing 100 μM of Acetosiringone and 50 µg/ml of kanamycin and incubate at 28 °C for 13-16 h while shaking at 200 rpm until OD600 reaches 0.8-1.0 (about 3-5 h).
      3. To harvest, transfer the culture to 50 ml centrifuge bottles and centrifuge for 10 min at 4 °C 3,000 x g. From this step the cells should be kept cold throughout the preparation.
      4. After centrifugation, remove as much of the supernatant as possible. Resuspend cell pellets by adding 300 µl of MS medium containing 100 µM of Acetosyringone. It is allowed to stand at room temperature for 0.5 hours without shaking before agro-infiltration.
    2. Infiltrate 100 µl of Agrobacterium tumefaciens containing recombined pCymMV Gateway vector in virus-free Phalaenopsis plants by use of a 1-ml syringe with a needle (see Note 3).
    3. Perform infiltration using either one of the two methods (Figures 2 and 3):
      1. For leaf injection, inject suspensions into the abaxial side of blade or laminar regions but excluding form tip, base, midrib, and margin parts of the leaf (usually the third youngest leaf for Phalaenopsis) right above the inflorescence emerge (Figure 2, left panel and Figure 3A).
      2. For inflorescence injection, inject suspensions into the floral stalk (Figure 2, right panel and Figure 3B) of the raceme with eight internodes and one visible floral bud (extruding out of its bract). The raceme stalk usually emerges from the stem between the third and the fourth leaves.
        After injection, the injection areas become dark green under the epidermal surface than the un-injected ones (Figure 3C-D).
    4. Plants after injected should be grown back in stable and regular growth condition in an insect-proof and thermal-controlled (20-25 °C) greenhouse.
    5. Agro-infiltrated inflorescences bloom with the first blooming flower appearing on 31-35 days post Agro-infiltration.
    6. Confirm Agrobacterium infiltration, virus movement and silencing of transgene expression by PCR (see Note 4).


      Figure 2. Agrobacterium infiltration in virus-free Phalaenopsis plants. A. The Agrobacteriums with the recombined pCambia-CymMV vectors are injected into the leaf (left panel) or the floral stalk (right panel) with Agro liquid culture using a 1-milliliter syringe.


      Figure 3. Demonstration of performing infiltration in (A) abaxial side of leaf and (B) floral stalk. (C and D) The leaves before and after injection. The injection areas can be distinguishably observed (red arrows).

Notes

  1. To generate PCR products suitable for use as substrates in a Gateway® BP recombination reaction with a pCymMV-Gateway vector, the attB sites are needed to be incorporated into your PCR products:
    1. The forward PCR-primer must contain the attB1 site (5′ GGGGACAAGTTTGTACAAAAAAGCAGGCT-3′) at the 3′ end followed by at least 18-25 bp of template- or gene-specific sequences.
    2. The reverse PCR-primer must contain the attB2 site (5′-GGGGACCACTTTGTACAAGAAAGCTGGGT-3′) at the 3′ end followed by at least 18-25 bp of template- or gene-specific sequences.
    3. Remember that the gene-specific nucleotides need to be in frame with the attB sequence and that stop codons should be removed.
    4. The position and length of amplicon of the insert fragments used for silencing are different depending on individual genes and need to be optimized for each target genes. e.g. Both large (1,498 bp) and small (81 bp) sizes of insert fragments of PeUFGT3 showed significant silencing effects (Hsieh et al., 2013a). To avoid non-specific targeting, it is better to select gene-specific region for primer design.
    5. For insert fragment amplification, standard PCR reaction condition is used, but it is better to use DNA polymerase with proof reading ability. A higher agarose percentage enhances resolution of smaller bands to distinguish PCR products from the primers by agarose gel electrophoresis. Elute DNA in d.d.H2O or TE buffer after purify PCR products by using a gel extraction kit.
  2. The transformation efficiency of TOP10 E. coli competent cells should be equal to or exceed 1.0 x 109 colonies/ml.
  3. The optimal amount of Agrobacteirum mixture for infiltration depends on the growth stage, organ and variety of Phalaenopsis plants. We recommend infiltrating 100 µl of Agrobacteirum mixture in each mature Phalaenopsis plants to ensure that their development and growth are not affected.
  4. cDNA synthesis was performed with total RNAs extracted from leaf or floral tissues above injected regions. RT-PCR was performed with the gene-specific forward primer and the general reverse primer (CymMV 5351, 5′-CTTCTGTACCATACACATAG-3′) on the gateway vector.

Recipes

  1. Luria Broth medium (LB medium)
    10 g/L tryptone
    5 g/L yeast extract
    10 g/L NaCl (pH 7.0)
  2. Proteinase K solution
    2 μg/μl Proteinase K in 10 mM Tris-HCl (pH 7.5)
    20 mM CaCl2
    50% glycerol
  3. Murishige and Skoog medium (MS medium) salt
    Major salts (macronutrients):
    1,650 mg/L ammonium nitrate (NH4NO3)
    440 mg/L calcium chloride (CaCl2.2H2O)
    370 mg/L magnesium sulphate (MgSO4.7H2O)
    170 mg/L potassium phosphate (KH2PO4)
    1,900 mg/L potassium nitrate (KNO3) Minor salts (micronutrients)
    6.2 mg/L boric acid (H3BO3)
    0.025 mg/L cobalt chloride (CoCl2.6H2O)
    0.025 mg/L cupric sulphate (CuSO4.5H2O)
    27.8 mg/L ferrous sulphate (FeSO4.7H2O)
    22.3 mg/L manganese sulphate (MnSO4.4H2O)
    0.83 mg/L potassium iodide (KI)
    0.25 mg/L codium molybdate (Na2MoO4.2H2O)
    8.6 mg/L zinc sulphate (ZnSO4.7H2O)
    37.2 mg/L Na2EDTA.2H2O
    pH 5.8

Acknowledgments

The authors would like to thank Dr. Lu, H. C. (Biotechnology Center in Southern Taiwan, Academia Sinica) for his assistance in viral vector construction.

References

  1. Hsieh, M. H., Lu, H. C., Pan, Z. J., Yeh, H. H., Wang, S. S., Chen, W. H. and Chen, H. H. (2013a). Optimizing virus-induced gene silencing efficiency with Cymbidium mosaic virus in Phalaenopsis flower. Plant Sci 201-202: 25-41.
  2. Hsieh, M. H., Pan, Z. J., Lai, P. H., Lu, H. C., Yeh, H. H., Hsu, C. C., Wu, W. L., Chung, M. C., Wang, S. S., Chen, W. H. and Chen, H. H. (2013b). Virus-induced gene silencing unravels multiple transcription factors involved in floral growth and development in Phalaenopsis orchids. J Exp Bot 64(12): 3869-3884.
  3. Lu, H. C., Chen, H. H., Tsai, W. C., Chen, W. H., Su, H. J., Chang, D. C. and Yeh, H. H. (2007). Strategies for functional validation of genes involved in reproductive stages of orchids. Plant Physiol 143(2): 558-569.
  4. Lu, H. C., Hsieh, M. H., Chen, C. E., Chen, H. H., Wang, H. I. and Yeh, H. H. (2012). A high-throughput virus-induced gene-silencing vector for screening transcription factors in virus-induced plant defense response in orchid. Mol Plant Microbe Interact 25(6): 738-746.

简介

这是使用CymMV作为蝴蝶兰花中的花功能基因组的沉默载体产生VIGS的稳定沉默效率和效率的方案。 该协议是基于由Lu等人(2007)创建的方法建立的,然后由Hsieh等人(2013a; 2013b)修改, (2012)成功地设计了克隆载体(pCymMV-Gateway),其中靶基因片段易于插入并且可以高效率操作。 通过使用根癌土壤杆菌(根瘤土壤杆菌)菌株EHA105通过农杆菌接种将沉默载体接种到植物中。 用于VIGS研究兰花花的叶的农杆菌浸润是一种节省时间并且对花芽产生较少的损害。

材料和试剂

  1. 无病毒蝴蝶兰植物
  2. TOP10大肠杆菌(大肠杆菌)感受态细胞
  3. 根癌农杆菌菌株EHA105
  4. Gel/PCR DNA片段提取试剂盒(Geneaid Biotech,目录号:DF100)
  5. (Life Technologies,Invitrogen TM ,目录号:11789-020)。
  6. 选择性板(含50μg/ml卡那霉素的LB平板)
  7. 高速质粒迷你试剂盒(Geneaid Biotech,目录号:PD100)
  8. Acetosyringone(Sigma-Aldrich,目录号:D134406)
  9. Murashige和Skoog盐(Sigma-Aldrich,目录号:M5524)
  10. 胰蛋白胨(US Biological,目录号:12855)
  11. 酵母提取物(Affymetrix,目录号:23547)
  12. NaCl(Sigma-Aldrich,目录号:S1446)
  13. Tris-HCl(US Biological,目录号:22676)
  14. CaCl 2(Inter-County Mechanical Corp.,目录号:03-11250)
  15. 甘油(J.T.Baker ,目录号:2136-01)
  16. Luria Broth培养基(LB培养基)(参见配方)
  17. 蛋白酶K溶液(见配方)
  18. Murishige和Skoog培养基(MS培养基)盐(参见配方)

设备

  1. GeneAmp PCR系统9700(Life Technologies,Applied Biosystems )
  2. 层流柜
  3. BioChrom Libra S50紫外/可见分光光度计(Biochrom)
  4. 培养箱振动器
  5. 电穿孔机(Kaneka Corporation,Eurogentec)
  6. 电穿孔杯(EquiBio,目录号:ECU-102)
  7. MiniSpin Eppendorf离心机和50 ml锥形离心管
  8. 带针头的1 ml注射器
  9. 水浴

程序

  1. pCymMV- Gateway质粒DNA的构建
    1. 用于沉默的靶基因的DNA片段,用于插入到pCymMV- Gateway中   载体(图1A)通过PCR扩增获得 在两者中含有29-nt attB重组位点的寡核苷酸引物   (见注1)。 通过使用凝胶提取纯化attB-PCR产物 kit。
    2. 通过使用BP重组反应将目标基因片段插入pCymMV-Gateway载体(图1B)
      1. 在室温下在1.5ml微量离心管中混合B-PCR 产物(150ng),pCymMV-Gateway载体(150ng)和4μl5×BP Clonase TM sup/TM反应缓冲液。 然后加入1×TE缓冲液(pH 8.0)直至总量 获得16μl的体积
      2. 去除BP Clonase TM 酶混合物   从-80℃储存并放在冰上2分钟解冻和短暂涡旋   两次(每次2秒)。
      3. 加入4μlBP Clonase TM sup /酶混合物 (步骤A2a,上述)。 通过涡旋混合两次,然后   微量离心机。 将BP Clonase TM 酶混合物储存在-80℃ 立即使用。
      4. 在25℃下孵育反应60分钟
      5. 向每个样品中加入2μl蛋白酶K溶液以终止反应
      6. 短暂涡旋并在37℃孵育样品10分钟。
    3. 转化能力。 大肠杆菌与用于扩增的pCymMV-Gateway载体(在层流箱中)。
      1. 孵育TOP10 E。 大肠杆菌感受态细胞在冰上10分钟(见注2)
      2. 加入5微升每个BP反应(步骤A2)到50-100微升TOP10 em。 大肠杆菌感受态细胞
      3. 在冰上孵育15分钟。
      4. 通过在42℃水浴中孵育45秒来杀死Heatshock细胞。
      5. 在冰上孵育2分钟。
      6. 加入200微升LB培养基,并在37℃孵育60分钟,同时以200转/分钟摇动
      7. 将每次转化(305μl)铺在含有50μg/ml卡那霉素的LB-琼脂培养基平板上
      8. 在37℃孵育13小时。
    4. 随机选择两个到四个菌落的每个构造,然后传代培养
    5. 回收和纯化质粒
    6. 使用基因特异性正向引物通过PCR确认每个构建体 和     general     reverse     primer    (CymMV 5351, 5'-CTTCTGTACCATACACATAG-3')。

  2. 在重组的pCymMV- Gateway载体(层流箱中的步骤B1-3)扩增后通过电穿孔转化
    1. 解冻根癌土壤杆菌(EHA105菌株)感受态细胞 室温下立即置于冰上。 转移100微升 感受态细胞至冰上的冷冻电穿孔杯。 加入1-2μl 的重组pCymMV- Gateway载体(可以使用150ng质粒DNA 取决于细胞的能力),轻轻混合并保持在冰上
    2. 执行电穿孔。
      1. 将比色杯插入电穿孔室并启动 电穿孔。 电压和脉冲时间参数已预先设置。
      2. 立即在比色杯中加入200μlLB培养基,并将溶液转移到无菌的Eppendorf管中
      3. 在28℃下孵育混合物 30分钟。 同时在200rpm下摇动。
    3. 将所有混合物铺在含有100的选定LB-琼脂培养基平板上   μM的乙酰丁香酮和50μg/ml的卡那霉素,并在28℃下孵育 保持16小时
    4. 通过PCR用基因特异性确认每个菌落 正向引物和一般     reverse    引物(CymMV 5351, 5'-CTTCTGTACCATACACATAG-3')在网关向量上存在 修改的结构
      (A)

      (B)

      图1。(A)的示意图 pCymMV- Gateway载体。 绿色矩形表示开放阅读框 由

  3. 对蝴蝶兰植物的农业渗透
    1. 准备电穿孔感受态农杆菌细胞:
      1. 四 天,将含有重组质粒的单个土壤杆菌属根瘤土壤杆菌转移到5ml LB培养基中 含有100μM乙酰共溶素和50μg/ml卡那霉素, 在28℃下培养16小时,同时以200rpm振荡。
      2. 亚文化   细菌细胞在50ml含有100μM的LB培养基中 Acetosiringone和50μg/ml卡那霉素,并在28℃孵育13-16   同时以200rpm振摇直至OD 600达到0.8-1.0(约3-5小时)。
      3. 收获,转移培养到50毫升离心瓶和 在4℃下离心10分钟3,000xg 。 从这一步细胞应该   在整个准备过程中保持冷
      4. 离心后,   去除尽可能多的上清液。 重悬细胞沉淀 通过加入300μl含有100μM乙酰丁香酮的MS培养基。 它是   在室温下放置0.5小时而不摇动 。
    2. 浸润100微升土壤杆菌 通过使用具有针的1ml注射器(参见注释3),在无病毒的蝴蝶兰植物中包含重组的pCymMV Gateway载体的根瘤土壤杆菌。
    3. 使用两种方法之一进行渗透(图2和图3):
      1. 对于叶片注射,将悬浮液注入叶片的背侧 或层状区域,但不包括尖端,基部,中心和边缘 部分叶子(通常是蝴蝶兰的第三最年轻的叶子) 正好在花序上方出现(图2,左图和图 (图3A)
      2. 对于花序注射,注射悬浮液 花茎(图2,右图和图3B) 八个节间和一个可见的花蕾(挤出它的 bract)。 外me茎通常从茎之间出现在第三者之间   和第四叶 注射后,注射区域在表皮表面下变成深绿色,比未注射的区域(图3C-D)。
    4. 植物注入后应该恢复稳定和规律 在防虫和热控制(20-25℃) 温室
    5. 农杆菌浸润的花序绽放,第一朵花在农杆菌浸润后31-35天出现。
    6. 通过PCR确认土壤杆菌渗入,病毒移动和转基因表达的沉默(参见注释4)。


      图   农杆菌 无病毒 蝴蝶兰 >植物。A.用重组的pCambia-CymMV载体注入土壤杆菌 进入具有农杆菌的叶(左图)或花秆(右图) 使用1毫升注射器进行液体培养

      图3.演示 在(A)叶的背轴侧和(B)花的进行渗透 茎。(C和D)注射前后的叶子。 注射 可以区分观察到的区域(红色箭头)。

笔记

  1. 为了产生适合在使用pCymMV-Gateway载体的Gateway BP重组反应中用作底物的PCR产物,需要将attB位点并入PCR产物中:
    1. 正向PCR引物必须含有attB1位点(5' GGGGACAAGTTTGTACAAAAAAGCAGGCT-3')在3'末端,随后至少 18-25bp的模板或基因特异性序列
    2. 反之 PCR引物必须包含attB2位点 (5'-GGGGACCACTTTGTACAAGAAAGCTGGGT-3')在3'末端,然后至少   18-25bp的模板或基因特异性序列
    3. 记住 基因特异性核苷酸需要与attB序列同框   并应删除终止密码子。
    4. 位置和长度 的用于沉默的插入片段的扩增子是不同的 这取决于个体基因并且需要针对每个靶标进行优化 基因。例如,大(1,498bp)和小(81bp)大小的插入物 片段PeUFGT3 显示显着的沉默效应(Hsieh et al。,   2013a)。 要避免非特定定位,最好选择 基因特异区域用于引物设计
    5. 对于插入片段 扩增,使用标准PCR反应条件,但是更好   使用具有校对阅读能力的DNA聚合酶。 较高的琼脂糖 百分比提高了较小条带的分辨率以区分PCR 产物从引物通过琼脂糖凝胶电泳。 洗脱DNA d.d.H sub 2 O或TE缓冲液中,通过使用凝胶提取纯化PCR产物   试剂盒。
  2. TOP10的转化效率。 大肠杆菌感受态细胞应等于或超过1.0×10 9个菌落/ml。
  3. 用于浸润的农杆菌混合物的最佳量取决于蝴蝶兰植物的生长阶段,器官和品种。 我们建议在每个成熟的蝴蝶兰植物中渗入100μl的农杆菌混合物,以确保它们的发育和生长不受影响。
  4. 使用从注射区域上方的叶或花组织提取的总RNA进行cDNA合成。 用网关载体上的基因特异性正向引物和一般反向引物(CymMV 5351,5'-CTTCTGTACCATACACATAG-3')进行RT-PCR。

食谱

  1. Luria Broth培养基(LB培养基)
    10 g/L胰蛋白酶
    5g/L酵母提取物
    10g/L NaCl(pH 7.0)
  2. 蛋白酶K溶液
    2μg/μl在10mM Tris-HCl(pH7.5)中的蛋白酶K 20mM CaCl 2/
    50%甘油
  3. Murishige和Skoog介质(MS介质)盐
    主要盐(大量营养素):
    1,650mg/L硝酸铵(NH 4 NO 3)
    440mg/L氯化钙(CaCl 2)2/2H 2 O 2)。 370mg/L硫酸镁(MgSO 4)7H/7H 2 O)
    170mg/L磷酸钾(KH 2 PO 4)
    1,900mg/L硝酸钾(KNO 3)次要盐(微量营养素)
    6.2mg/L硼酸(H 3 BO 3)
    0.025mg/L氯化钴(CoCl 2)6/6H 2 O)。 0.025mg/L硫酸铜(CuSO 4钠)5 H 2 O 2)。 27.8mg/L硫酸亚铁(FeSO 4)7H/7H 2 O)
    22.3mg/L硫酸锰(MnSO 4·4H 2 O·4H 2 O)。 0.83mg/L碘化钾(KI)
    0.25mg/L的钼酸钠(Na 2 MoO 4+)。 2H 2 O 8.6mg/L硫酸锌(ZnSO 4,7H 2 O,7H 2 O)。 37.2mg/L Na 2 EDTA缓冲液。 2H 2 pH 5.8

致谢

作者要感谢Lu博士(台湾南部生物技术中心)在病毒载体构建中的帮助。

参考文献

  1. Hsieh,M. H.,Lu,H. C.,Pan,Z.J.,Yeh,H. H.,Wang,S. S.,Chen,W. H. and Chen,H. H.(2013a)。 使用花旗病毒在中优化病毒诱导的基因沉默效率 >蝴蝶兰花。 植物科学 201-202:25-41。
  2. Hsieh,M. H.,Pan,Z.J.,Lai,P. H.,Lu,H. C.,Yeh,H. H.,Hsu,C. C.,Wu,W. L.,Chung,M. C.,Wang,S. S.,Chen,W. H. and Chen,H. H.(2013b)。 病毒诱导的基因沉默解开了涉及蝴蝶兰花卉生长和发育的多种转录因子/em> orchids。 J Exp Bot 64(12):3869-3884。
  3. Lu,H.C.,Chen,H.H.,Tsai,W.C.,Chen,W.H.,Su,H.J.,Chang,D.C.and Yeh,H.H。(2007)。 涉及兰花繁殖阶段的基因的功能验证策略 植物Physiol 143(2):558-569。
  4. Lu,H.C.,Hsieh,M.H.,Chen,C.E.,Chen,H.H.,Wang,H.I。和Yeh,H.H。(2012)。 用于筛选病毒诱导的植物防御中的转录因子的高通量病毒诱导的基因沉默载体在兰花中的反应。 Mol Plant Microbe Interact 25(6):738-746。
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Copyright: © 2014 The Authors; exclusive licensee Bio-protocol LLC.
引用:Hsieh, M., Pan, Z., Yeh, H. and Chen, H. (2014). Virus-induced Gene Silencing (VIGS) in Phalaenopsis Orchids. Bio-protocol 4(24): e1359. DOI: 10.21769/BioProtoc.1359.
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