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Virus-induced Gene Silencing (VIGS) in Barley Seedling Leaves
大麦幼苗叶的病毒诱导基因沉默(VIGS)   

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Abstract

Virus induced gene silencing (VIGS) is one of the most potent reverse genetics technologies for gene functional characterisation. This method exploits a dsRNA-mediated antiviral defence mechanism in plants. Using this method allows researchers to generate rapid phenotypic data in a relatively rapid time frame as compared to the generation of stable transformants. Here we describe a simple method for silencing a target gene in barley seedling leaves using vectors based on the Barley Stripe Mosaic Virus (BSMV).

Materials and Reagents

  1. Bacterial strain: Escherichia coli strain DH5 α
  2. Taq DNA polymerase (Life Technologies, InvitrogenTM, catalog number: 11304-011 )
  3. pGEM-T easy cloning kit (Promega Corporation, catalog number: A1380 )
  4. QIAGEN QIAprep Centrifuge Miniprep Kit (QIAGEN, catalog number: 27106 )
  5. Recombinant Taq DNA polymerase (Life Technologies, InvitrogenTM, catalog number: 10342-020 )
  6. Plasmid vectors: pα42 (pα), pβ42.sp1 (pβ), pSL038-1 (pγ), pSL038-PDS (pγ-PDS).
    Note: See Scofield and Brandt (2012) for the plasmid maps (provided by Prof. Steven R. Scofield, https://www.purdue.edu/gradschool/pulse/groups/faculty_cascade.cfm?alias=scofield).
  7. Luria-Bertani (LB) liquid broth (Sigma-Aldrich L3152) and solid media containing 1.2% (w/v) agar (OXOID, catalog number: LP0013 )
  8. Ampicillin (100 mg/l working concentration) (Sigma-Aldrich, catalog number: A0166-5G )
  9. Restriction enzymes:
    PacI (New England Biolabs, catalog number: R0547S )
    MluI (New England Biolabs, catalog number: R0198S )
    SpeI (New England Biolabs, catalog number: R0133S )
  10. Ethanol (Sigma-Aldrich, catalog number: 459844 )
  11. sodium acetate (Sigma-Aldrich, catalog number: S2889 )
  12. mMessage mMachine T7 in vitro transcription kit (Ambion Sigma-Aldrich, catalog number: AM1344 )
  13. John Innes compost No 2 (Westland Horticulture)
  14. Glycine (Sigma-Aldrich, catalog number: 410225 )
  15. K2HPO4 dibasic (Sigma-Aldrich, catalog number: P3786 )
  16. Sodium pyrophosphate decahydrate (Sigma-Aldrich, catalog number: 221368 )
  17. Bentonite (Aldrich, catalog number: 28,523-4 )
  18. Celite (Fluka, catalog number: 22141 )
  19. 5x GP buffer (see Recipes)
  20. FES buffer (see Recipes)

Equipment

  1. Plant growth chambers (24 °C, 16/8 photoperiod and 55% humidity) (CambridgeHOK containment glasshouse)
  2. Sterile culture tubes (Falcon, catalog number: 352057 )
  3. Centrifuge tubes (SARSTEDT AG, catalog number: 72.695.500 )
  4. Filter paper (Whatman, catalog number: 1001-090 )
  5. Cooled centrifuge (Eppendorf)
  6. Shaking incubators for cultures (New Brunswick Scientific)
  7. Gel apparatus (Helixx Mupid-exU) and image system (Fusion Fx vilber lourmat)
  8. Thermal cycler (MJ Research PTC200)
  9. Gel electrophoresis chamber (Helixx Mupid-exU)
  10. Autoclave (Priorclave)

Software

  1. Primer3 software (version 0.4.0; http://frodo.wi.mit.edu/primer3/)

Procedure

  1. Seed germination and plant growth
    1. Place barley seeds in a 9-cm diameter petri plate containing 2 pieces of filter paper (90 mm diameter) and 6ml of sterile water.
    2. Cover the petri plates with aluminum foil and stratify in the dark for 2 days at 4 °C.
    3. Transfer plates to 21 °C in dark for 2 days to germinate seeds.
    4. Transplant etiolated seedlings to 3-inch pots containing John Innes compost No 2 at a density of 2 seedlings/pot.
    5. Grow the plants at 22 °C under long day conditions (16 h/8 h), bottom watering every second day. Relative humidity was maintained at 70%.

  2. Virus-induced gene silencing
    Note: BSMV is a single-stranded RNA virus with three genome components termed α, β, and γ RNA. A transcribed sequence representing a fragment of the gene to be silenced is inserted immediately downstream of the termination codon of the γ open reading frame that is encoded within a DNA plasmid. RNA is synthesized from α, β, and γ encoded within DNA plasmids. Barley seedlings are infected with all three RNA fragments leads to synthesis of viral dsRNA, which activates the anti-viral RNA silencing pathway, resulting in silencing of the target barley gene. Here we showed VIGS of barley Brassinosteroid-Insensitive-1 (BRI1) gene using two constructs targeting non-overlapping gene fragments. A BSMV γ RNA construct containing a 185 bp-fragment of the barley phytoene desaturase (PDS) gene that protects chlorophyll from photo-bleaching was used as a positive control for the VIGS experiment.
    1. Two independent fragments of BRI1 gene were amplified from genomic DNA of barley cv. Akashinriki using the fragment-specific primers HvBri1A-F/R or HvBri1B-F/R (Table 1). PCR was undertaken in 10 µl volume consisting 1 µl 10X high fidelity PCR buffer, 0.4 µl 50 mM MgSO4, 0.2 µl 10 mM dNTP Mix, 0.2 µl each of 5 μM forward and reverse primer, 0.05 µl 5 U/μl platinum Taq DNA polymerase, 30 ng genomic DNA and sterile Milli-Q H2O to 10 µl. PCR reactions were conducted in a Peltier thermal cycler DNA engine and the PCR program consisted of an initial denaturation step at 94 °C for 2 min, 30 cycles of denaturation 94 °C for 30 sec, annealing at 60 °C for 30 sec, extension at 68 °C for 45 sec and a final extension step at 72 °C for 5 min

      Table 1. Primers used for VIGS of BRI1 gene
      VIGS primer name
      Forward primer (5΄- 3΄)
      Reverse primer (5΄- 3΄)
      HvBRI1:A
      CGATTAATTAAGCGGAGGCAGAAGAATGA
      CGACCCGGGGTCACCCTGGCCACTCAC
      HvBRI1:B
      CGATTAATTAAGTGAGTGGCCAGGGTGAC
      CGACCCGGGTGGATGATGTGCGGAATG
      HvBRi1:RT
      CAACGATGCTCAAGGTGATG
      CCGGTGGTCATCTTCCTAAT
      HvRNAH
      GCACAGGGAATCGTCAAAGT
      TCAAAACAACACAACATCGAAGT
      pGamma
      TGATGATTCTTCTTCCGTTGC
      TGGTTTCCAATTCAGGCATCG

      Primers were design using the Primer3 software (version 0.4.0; http://frodo.wi.mit.edu/primer3/).
      *Note: When designing the primers for gene silencing makes sure that the PCR products will not contain restriction sites for the enzymes used to linearize the plasmids at a later stage.
    2. The amplified gene fragments were cloned into the pGEM-T vector using the pGEM-T Easy cloning kit and were transformed in to E. Coli DH5α via electroporation.
    3. The recombinant pGEM-T vectors carrying the silencing fragments were then digested with PacI and SmaI. The inserts were purified by gel extraction and then cloned into PacI and SmaI -digested γ RNA vector pSL038-1.
    4. The resulting recombinant pSL038-1-BRI1A (pγ-BRI1A) and pSL038-1-BRI1B (pγ-BRI1B) plasmids harbouring the BRI1 gene fragments were transformed in to E. coli DH5α via electroporation and cloned products were subsequently sequenced by Macrogen Inc. (Korea) using the vector-specific primers pGamma-F/R (Table 1) to check the orientation of silencing fragment. Positive clones with correct orientations were stored at -80 °C as glycerol stocks.
    5. E. coli glycerol stocks carrying plasmid pα, pβ, pγ, pγ-PDS, pγ-BRI1A and pγ-BRI1B were streaked on LB-agar plates supplemented with 100 mg/L ampicillin and cultures grown overnight at 37 °C.
    6. A single colony from each plate was inoculated into 10 ml LB broth supplemented with 100 mg/L ampicillin and cultures were grown overnight at 37 °C.
    7. Plasmid extraction was carried out using the QIAGEN QIAprep Centrifuge Miniprep Kit as per the product protocol, but excluding RNaseA from buffer P1 (any residual RNaseA carried over in the plasmid DNA preparation will interfere with in vitro transcription at later stages).
    8. The plasmids pα, pγ, pγ-PDS, pγ-BRI1A and pγ-BRI1B were linearized with MluI and the plasmid pβ was linearized with SpeI. The linearized plasmids were precipitated overnight using ½ volume 5 M ammonium acetate and 2 volume absolute ethanol followed by centrifuging at 18,000 x g for 15 min. DNA pellet was washed with 70% ethanol, air-dried and resuspended in 50 µl RNase-free water. (For 50 seedlings, 1 µg linearized plasmids of each).
    9. Capped in vitro transcripts were prepared from the linearized plasmids pα, pβ, pγ, γ-PDS, pγ-BRI1A and pγ-BRI1B using the mMessage mMachine T7 in vitro transcription kit following the manufacturer’s protocol. The prepared capped transcripts were checked on a 1% agarose gel. Any smearing of the smaller band indicates degradation of the RNA transcript. There should be a faint band at approximately 10,000 bp and a bright band at approximately 3,000 bp.
    10. For each plant, VIGS inoculum was prepared by mixing 9 µl of FES Buffer with 0.35 µl each of the α, β and the relevant γ-based transcript [γ, γ-PDS (positive control), γ-BRI1A or γ-BRI1B]. FES buffer served as a mock treatment.
    11. The first leaf of 10-day-old seedlings was inoculated by rubbing with 10 µl of transcript mixtures or FES (mock treatment) in between thumb and index finger. Care was taken not to damage the leaf.
    12. After 14 days plants were assessed for visual symptoms of gene silencing. In the case of PDS-silenced plants, the leaves appeared photo bleached (Figure 1). The BRI1-silenced plants showed dwarfing symptoms, as expected (Figure 2).


      Figure 1. Silencing of PDS gene in barley results in photobleaching and causes the leaves to turn white. A. FES treatment (negative control) B. BSMV:00 (mock treatment) C. BSMV:PDS (positive control).


      Figure 2. Silencing of HvBRI1 resulted in stunting of plant growth in barley plants. A. FES treatment (negative control) B. BSMV:00 (mock treatment) C. BSMV:HvBRI1.

    13. To confirm the silencing, the 3rd leaf was harvested and flash-frozen in liquid N2 and stored at -70 °C prior to RNA extraction. Total RNA was extracted from plants using the TriZolTM protocol as given by the manufacturer.
    14. Gene silencing was quantified by real-time RT-PCR using primers specific to BRI1 (HvBRi1:RT, Table 1) and relative to that of the RNA helicase housekeeping gene (HvRNAH, Table 1). Note: It is important to design the real-time primers outside the gene fragment that was cloned into the pγ plasmid.

Notes

  1. Precaution should be taken not to mix the γ–PDS with other γ samples.
  2. Gloves must be changed for every treatment
  3. When applying the transcripts to the leaf, be gentle by not damaging the leaf too much.

Recipes

  1. 5x GP buffer (500 ml)
    18.77 g glycine
    26.13 g K2HPO4 dibasic
    Bring to 500 ml with ddH2O and use immediately
  2. FES buffer (500 ml)
    100 ml GP buffer
    5 g sodium pyrophosphate decahydrate
    5 g bentonite
    5 g celite
    Bring to 500 ml with ddH2O
    Aliquot into 50 ml volumes and autoclave (121 °C for 20 min)
    Aliquots can be stored at room temperature under sterile condition

Acknowledgments

This work was supported by the Science Foundation Ireland research fund (IN10/IN.1/B3028) and Department of Agriculture Research Stimulus Grant RSF 07 513. Part of the procedures were adapted from a previously described methods by Ali et al. (2014), Holzberg et al. (2002) and Scofield et al. (2005).

References

  1. Ali, S. S., Gunupuru, L. R., Kumar, G. B., Khan, M., Scofield, S., Nicholson, P. and Doohan, F. M. (2014). Plant disease resistance is augmented in uzu barley lines modified in the brassinosteroid receptor BRI1. BMC Plant Biol 14: 227.
  2. Holzberg, S., Brosio, P., Gross, C. and Pogue, G. P. (2002). Barley stripe mosaic virus-induced gene silencing in a monocot plant. Plant J 30(3): 315-327.
  3. Scofield, S. R. and Brandt, A. S. (2012). Virus-induced gene silencing in hexaploid wheat using Barley stripe mosaic virus vectors. In: Watson, J. M. and Wang, M. B. (eds). Antiviral Resistance in Plants: Methods and protocols. Springer, 93-112.
  4. Scofield, S. R., Huang, L., Brandt, A. S. and Gill, B. S. (2005). Development of a virus-induced gene-silencing system for hexaploid wheat and its use in functional analysis of the Lr21-mediated leaf rust resistance pathway. Plant Physiol 138(4): 2165-2173.

简介

病毒诱导的基因沉默(VIGS)是基因功能表征的最有效的反向遗传学技术之一。 这种方法利用dsRNA介导的抗病毒防御机制在植物中。 与稳定转化体的产生相比,使用该方法允许研究人员在相对快的时间框架中产生快速表型数据。 在这里我们介绍了一种简单的方法,使用基于大麦条纹花叶病毒(BSMV)的载体,在大麦幼苗叶片中沉默目标基因。

材料和试剂

  1. 细菌菌株:大肠杆菌菌株DH5α
  2. DNA聚合酶(Life Technologies,Invitrogen TM ,目录号:11304-011)。
  3. pGEM-T易克隆试剂盒(Promega Corporation,目录号:A1380)
  4. QIAGEN QIAprep离心机小量制备试剂盒(QIAGEN,目录号:27106)
  5. 重组Taq DNA聚合酶(Life Technologies,Invitrogen TM ,目录号:10342-020)
  6. 质粒载体:pα42(pα),pβ42.sp1(pβ),pSL038-1(pγ),pSL038-PDS(pγ-PDS)。
    注意:对于质粒图谱(由Steven R.Scofield教授提供,参见Scofield和Brandt(2012)), https://www.purdue.edu/gradschool/pulse/groups/faculty_cascade.cfm?alias=scofield < em>)。
  7. Luria-Bertani(LB)液体肉汤(Sigma-Aldrich L3152)和含有1.2%(w/v)琼脂(OXOID,目录号:LP0013)的固体培养基
  8. 氨苄青霉素(100mg/l工作浓度)(Sigma-Aldrich,目录号:A0166-5G)
  9. 限制酶:
    PacI(New England Biolabs,目录号:R0547S) MluI(New England Biolabs,目录号:R0198S) SpeI(New England Biolabs,目录号:R0133S)
  10. 乙醇(Sigma-Aldrich,目录号:459844)
  11. 乙酸钠(Sigma-Aldrich,目录号:S2889)
  12. mMessage mMachine T7体外转录试剂盒(Ambion Sigma-Aldrich,目录号:AM1344)
  13. John Innes堆肥No 2(Westland园艺)
  14. 甘氨酸(Sigma-Aldrich,目录号:410225)
  15. (Sigma-Aldrich,目录号:P3786)
    HPO
  16. 焦磷酸钠十水合物(Sigma-Aldrich,目录号:221368)
  17. 膨润土(Aldrich,目录号:28,523-4)
  18. Celite(Fluka,目录号:22141)
  19. 5x GP缓冲区(参见配方)
  20. FES缓冲区(请参阅配方)

设备

  1. 植物生长室(24℃,16/8光周期和55%湿度)(CambridgeHOK containment glasshouse)
  2. 无菌培养管(Falcon,目录号:352057)
  3. 离心管(SARSTEDT AG,目录号:72.695.500)
  4. 滤纸(Whatman,目录号:1001-090)
  5. 冷却离心机(Eppendorf)
  6. 摇动文化孵化器(New Brunswick Scientific)
  7. 凝胶装置(Helixx Mupid-exU)和图像系统(Fusion Fx vilber lourmat)
  8. 热循环仪(MJ Research PTC200)
  9. 凝胶电泳室(Helixx Mupid-exU)
  10. 高压灭菌器(Priorclave)

软件

  1. Primer3软件(版本0.4.0; http://frodo.wi.mit.edu/primer3/

程序

  1. 种子发芽和植物生长
    1. 将大麦种子放在含有2片滤纸(90mm直径)和6ml无菌水的9cm直径培养皿中。
    2. 用铝箔覆盖培养皿,在4℃下在黑暗中分层2天
    3. 转移板21°C在黑暗中2天,以发芽种子
    4. 将移植化的幼苗移植到含有John Innes堆肥No 2的3英寸盆中,密度为2个幼苗/盆。
    5. 在长日照条件下(16小时/8小时)在22℃下生长植物, 底部浇水每隔一天。相对湿度保持在 70%。

  2. 病毒诱导的基因沉默
    注意:BSMV是具有称为α,β和γRNA的三种基因组组分的单链RNA病毒。代表待沉默的基因片段的转录序列直接插入在DNA质粒内编码的γ开放阅读框的终止密码子的下游。 RNA由在DNA质粒内编码的α,β和γ合成。大麦幼苗被所有三个RNA片段感染,导致病毒dsRNA的合成,其激活抗病毒RNA沉默途径,导致目标大麦基因沉默。在这里我们显示大麦油菜甾体 - 不敏感-1(BRI1)基因的VIGS使用两个构造目标非重叠基因片段。包含保护叶绿素免受光漂白的大麦八氢番茄红素去饱和酶(PDS)基因的185bp片段的BSMVγRNA构建体用作VIGS实验的阳性对照。
    1. 从基因组DNA扩增BRI1 基因的两个独立片段 的大麦cv。 Akashinriki使用片段特异性引物 HvBri1A-F/R或HvBri1B-F/R(表1)。在10μl体积中进行PCR  包含1μl10X高保真PCR缓冲液,0.4μl50mM MgSO 4,0.2 μl10mM dNTP Mix,0.2μl每种5μM正向和反向引物,0.05 μl5U /μl铂/Taq DNA聚合酶,30ng基因组DNA和无菌 Milli-Q H 2 O至10μl。 PCR反应在Peltier热进行 循环DNA发动机和PCR程序组成的初始 变性步骤在94℃2分钟,30个循环的变性94℃ 30秒,60℃退火30秒,68℃延伸45秒   和在72℃下5分钟的最终延伸步骤

      表1.用于BRI1 基因的VIGS的引物
      VIGS引物名称
      正向引物(5- 3)
      反向引物(5-3)
      HvBRI1:A
      CGATTAATTAAGCGGAGGCAGAAGAATGA
      CGACCCGGGGTCACCCTGGCCACTCAC
      HvBRI1:B
      CGATTAATTAAGTGAGTGGCCAGGGTGAC
      CGACCCGGGTGGATGATGTGCGGAATG
      HvBRi1:RT
      CAACGATGCTCAAGGTGATG
      CCGGTGGTCATCTTCCTAAT
      HvRNAH
      GCACAGGGAATCGTCAAAGT
      TCAAAACAACACAACATCGAAGT
      pGamma
      TGATGATTCTTCTTCCGTTGC
      TGGTTTCCAATTCAGGCATCG

      使用Primer3软件(版本0.4.0; http://frodo.wi.mit.edu)设计引物/primer3/)。
      *注意:当设计用于基因沉默的引物时,确保 PCR产物将不含有所用酶的限制性位点   以在稍后阶段线性化质粒。
    2. 扩增的基因 使用pGEM-T Easy将片段克隆到pGEM-T载体中 克隆试剂盒并转化到E中。 Coli DH5α。
    3. 携带沉默片段的重组pGEM-T载体   然后用PacI和SmaI消化。 通过凝胶纯化插入物 提取,然后克隆到PacI和SmaI消化的γRNA载体中 pSL038-1。
    4. 所得的重组pSL038-1-BRI1A(pγ-BRI1A)和   携带BRI1 基因片段的pSL038-1-BRI1B(pγ-BRI1B)质粒 转化到E。 大肠杆菌DH5α通过电穿孔并克隆 随后使用Macrogen Inc.(Korea)对产物进行测序 载体特异性引物pGamma-F/R(表1)检查方向   沉默片段。 具有正确方向的阳性克隆 储存在-80℃下作为甘油储备液。
    5. E。 大肠杆菌甘油原液 携带质粒pα,pβ,pγ,pγ-PDS,pγ-BRI1A和pγ-BRI1B进行条纹   在补充有100mg/L氨苄青霉素和培养物的LB-琼脂平板上 在37℃生长过夜
    6. 从每个平板的单个菌落 接种到补充有100mg/L氨苄青霉素的10ml LB肉汤中 培养物在37℃下生长过夜。
    7. 质粒提取 使用QIAGEN QIAprep离心机小量制备试剂盒进行 产物方案,但不包括RNaseA从缓冲液P1(任何残留 在质粒DNA制备中携带的RNA酶A将干扰后期阶段的体外转录。)
    8. 质粒pα,pγ, pγ-PDS,pγ-BRI1A和pγ-BRI1B用MluI和质粒线性化 pβ用SpeI线性化。 线性化质粒沉淀 使用1/2体积的5M乙酸铵和2体积绝对 乙醇,随后在18,000×g离心15分钟。 DNA沉淀 用70%乙醇洗涤,风干并重悬于50μl 无RNase水。 (对于50个幼苗,每个的1μg线性化质粒)。
    9. 加盖的体外转录物从线性化的 质粒pα,pβ,pγ,γ-PDS,pγ-BRI1A和pγ-BRI1B mMachine T7体外转录试剂盒 协议。 在1%琼脂糖上检查制备的加帽的转录物 凝胶。 任何较小条带的拖尾指示RNA的降解 成绩单。 应该有一个约10,000 bp的微弱带,   亮带约3,000bp。
    10. 对于每个植物,VIGS 通过将9μlFES缓冲液与0.35μl每种混合来制备接种物 α,β和相关的基于γ的转录物[γ,γ-PDS(阳性 对照),γ-BRI1A或γ-BRI1B]。 FES缓冲液用作模拟处理。
    11. 通过摩擦接种10日龄幼苗的第一叶 与10μl的转录混合物或FES(模拟处理)之间 拇指和食指。 注意不要损坏叶片。
    12. 14天后,评估植物的基因的视觉症状 沉默。 在PDS沉默的植物的情况下,叶子出现照片   漂白(图1)。 BRI1 沉默的植物表现出矮小的症状,   如图2所示。


      图1.大麦中PDS基因的沉默 导致光漂白并导致叶片变白。 A. FES 处理(阴性对照)B.BSMV:00(模拟处理)C.BSMV:PDS (正控制)。


      图2. Hv BRI1 的静音导致了 在大麦植物中植物生长的发育迟缓。 A。 FES治疗(阴性 控制)B.BSMV:00(模拟处理)C.BSMV:HvBRI1
    13. 至 确认沉默,收获3号叶并快速冷冻 液N 2并在RNA提取之前储存在-70℃。 总RNA为 提取自植物使用由TrizolTM提供的方案 制造商。
    14. 通过实时RT-PCR定量基因沉默   使用对BRI1(HvBR1:RT,表1)特异性的引物和相对于 RNA解旋酶管家基因(HvRNAH,表1)。 注意: 对于设计基因片段外的实时引物是重要的 将其克隆到pY质粒中。

笔记

  1. 应注意不要将γ-PDS与其他γ样品混合。
  2. 每次治疗都必须更换手套
  3. 当将成绩单应用于叶片时,请不要过度损坏叶片以使其温和。

食谱

  1. 5x GP缓冲液(500ml) 18.77g甘氨酸
    26.13g K 2 HPO 4二价
    用ddH 2 O转至500ml,立即使用
  2. FES缓冲液(500ml)
    100 ml GP缓冲液
    5克焦磷酸钠十水合物 5克膨润土
    5克celite
    用ddH 2 O 2加到500ml/dm 2 等分成50ml体积并高压灭菌(121℃20分钟) 等分试样可以在室温下在无菌条件下保存

致谢

这项工作得到了科学基金会爱尔兰研究基金(IN10/IN.1/B3028)和农业研究刺激剂研究部RSF 07 513的支持。部分程序改编自之前描述的方法,Ali等人 (2014),Holzberg等人(2002)和Scofield等人 (2005)。

参考文献

  1. Ali,S.S.,Gunupuru,L.R.,Kumar,G.B.,Khan,M.,Scofield,S.,Nicholson,P.and Doohan,F.M。 在油菜素类固醇受体BRI1中修饰的uzu大麦品系中植物病害抗性增强。 em> BMC Plant Biol 14:227.
  2. Holzberg,S.,Brosio,P.,Gross,C。和Pogue,G.P。(2002)。 在单子叶植物中大麦条纹花叶病毒诱导的基因沉默植物J 30(3):315-327。
  3. Scofield,S.R.and Brandt,A.S。(2012)。使用大麦条纹花叶病毒载体在六倍体小麦中病毒诱导的基因沉默。 In:Watson,J.M.and Wang,M.B。(eds)。 Antiviral Resistance in Plants:Methods and protocols。 Springer,93-112。
  4. Scofield,S.R.,Huang,L.,Brandt,A.S。和Gill,B.S。(2005)。 开发一种病毒诱导的六倍体小麦基因沉默系统及其在功能分析中的应用的 - 介导的叶锈病抗性途径。 植物生理学 138(4):2165-2173。
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Copyright: © 2015 The Authors; exclusive licensee Bio-protocol LLC.
引用:Gunupuru, L. R., Ali, S. S., Doohan, F. M. and Scofield, S. R. (2015). Virus-induced Gene Silencing (VIGS) in Barley Seedling Leaves. Bio-protocol 5(12): e1506. DOI: 10.21769/BioProtoc.1506.
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