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Nov 2018
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Transient Expression Assay in Strawberry Fruits
草莓果实中的瞬时表达检测   

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Abstract

Strawberry, including the woodland strawberry Fragaria vesca (2x) and the cultivated strawberry (Fragaria × ananassa, 8x), has emerged as a model system for studying fruit development and ripening. Transient expression provides a quick assay for gene functions or gene interactions. In strawberry, virus-induced gene silencing (VIGS) and Agrobacterium tumefaciens-mediated transformation in fruit have been widely used as the transient expression approaches. Unlike VIGS, the latter one can be utilized not only for gene knock-down, but also for overexpression and knock-out. Here, we show the procedures of transiently expressing the 35S::FveMYB10 construct into fruit of the white-fruited F. vesca accession Yellow Wonder. As a master regulator of anthocyanin production, overexpressing FveMYB10 will cause fruit coloration, which was observed at one week post infiltration. We also exhibit the previous results of knocking down Reduced Anthocyanin in Petioles (RAP), encoding an anthocyanin transporter, by RNAi in fruit of the strawberry cultivar ‘Sweet Charlie’. Overall, Agrobacterium-mediated transient transformation in strawberry fruit is a quick and versatile approach for studying gene functions in fruit ripening.

Keywords: Transient expression (瞬时表达), FveMYB10 (FveMYB10), Overexpression (过表达), RAP (RAP), RNAi (RNA干扰), Agrobacterium (农杆菌), Strawberry (草莓)

Background

Cultivated strawberry (Fragaria × ananassa, 8x) is an economically important fruit crop grown worldwide with lovely appearance and rich nutrition. The wild diploid strawberry Fragaria vesca has emerged as a model plant for cultivated strawberry as well as other fleshy fruit species. Moreover, strawberry is a typical non-climacteric fruit, studies on the ripening of which lack a nice model system, like tomato as a model of the climacteric fruit. Therefore, strawberry is also frequently used for studying fruit ripening.

Stable transformation is doable in both woodland and cultivated strawberry; however, the entire process is labor-intensive and time-consuming. In contrast, the transient expression assay in fruit is much faster and more efficient. There are two transient gene expression approaches in strawberry, namely Virus-Induced Gene Silencing (VIGS) and Agrobacterium-mediated transformation (Spolaore et al., 2001). Some studies obtained nice results by using VIGS (Jia et al., 2011; Li et al., 2019). However, we utilize the Agrobacterium-mediated transformation more frequently as it can achieve more experimental aims, including gene knock-down (Hoffmann et al., 2006), overexpression (Huang et al., 2018), and knock-out (Tang et al., 2018).

The F. vesca fruit takes 25-30 days from being pollinated to ripen under our growth conditions. The entire process is divided into seven stages: flower/anthesis, small green, medium green, large green, white, turning, and red. Fruit receptacle (the juicy flesh) at the large green stage, when the firmness starts to decline rapidly (before the achenes turn red for the red strawberry fruit), is suitable for agrobacterium injection. Fruit receptacles at prior stages are recalcitrant to the injection owing to the texture. Thus, Agrobacterium-mediated transformation in strawberry fruit delimits functional studies on genes acting at late developmental stages.

The bright red color of strawberry fruit is caused by the accumulation of anthocyanin compounds, which is a visible phenotype to check. In strawberry, the R2R3-MYB transcription factor FveMYB10 is a master regulator of anthocyanin synthesis (Lin-Wang et al., 2014). The woodland strawberry includes both red-fruited and white-fruited varieties, owing to a natural SNP occurred in FveMYB10; overexpression of FveMYB10 results in accumulation of red pigments in fruit of the white-fruited accession Yellow wonder (YW) (Hawkins et al., 2016). Recently, we identified one anthocyanin transporter encoding gene Reduced Anthocyanin in Petioles (RAP) through chemical mutagenesis, knock-down of which greatly reduced fruit pigmentation (Luo et al., 2018).

In this study, as an example, we describe the procedures of transiently overexpressing FveMYB10 in fruit of YW. Moreover, we exhibit the results of knocking down RAP in fruit of the strawberry cultivar ‘Sweet Charlie’.

Materials and Reagents

  1. Tips
  2. Tag
  3. 1 ml syringe with needle (GEMTIER, catalog number: 0.45X16 RW LB)
  4. The white-fruited F. vesca accession Yellow Wonder (YW), and the strawberry cultivar ‘Sweet Charlie’
  5. Binary vectors: pK7WG2D for overexpression and pK7WIWG2D for RNAi. Both vectors contain a 35S::GFP cassette as a visual reporter
  6. Target genes: FveMYB10, FvH4_1g22020/gene31413; RAP, FvH4_1g27460/gene31672
  7. Yeast extract (OXOID, catalog number: LP0021)
  8. Tryptone (OXOID, catalog number: LP0042) 
  9. Agar (TSINGKE, catalog number: 1182GR500)
  10. MS (PhytoTechnology Laboratories, catalog number: M404-50L)
  11. GV3101 Chemically Competent Cell (Shanghai Weidi Biotechnology, catalog number: AC1001)
  12. NaCl (HUSHI, catalog number: 10019318, CAS: 7647-14-5)
  13. Sucrose (HUSHI, catalog number: 10021418, CAS: 57-50-1)
  14. Antibiotics, including Kanamycin (Kan), Gentamicin (Gent), Rifampicin (Rif), and Spectinomycin (Spe) (Biofroxx, catalog numbers: 1162GR005, 1463GR001, R3501, and S8040, respectively)
  15. Luria Broth (LB) medium (see Recipes)
  16. Injection buffer (see Recipes)
  17. Antibiotics (see Recipes)

Equipment

  1. Incubator used for the growth of Agrobacterium at 28 °C (JINGHONG, catalog number: DNP-9022)
  2. Double temperature controlled thermostat (MIULAB, catalog number: BTH-100)
  3. Centrifuge (Eppendorf, 5424)
  4. Clean workbench (AIRTECH, SW-CJ-1FD)
  5. Full temperature shaker (Peiying, catalog number: THZ-C-1)
  6. Ultra-low temperature refrigerator (ThermoFisher SCIENTIFIC, Thermo ScientificTM FormaTM 88000)
  7. Fluorescence dissecting stereomicroscope (Leica, catalog number: M205FA)
  8. Plant growth room: 25 ± 3 °C, 16 h light/8 h dark, light intensity at 100 μmol m-2 sec-1

Procedure

  1. Grow strawberry plants in the growth room for 3 to 4 months until the flowers open.
  2. Pollinate the flowers manually; wait until the fruits develop to the large green stage, at about 15-20 days post pollination when the receptacle starts to turn soft. 
  3. Transform the 35S::FveMYB10 construct (binary vector pK7WG2D) into the Agrobacterium tumefaciens strain GV3101, or use the Agrobacterium stock stored in the ultra-low temperature refrigerator. Pick a single positive colony and put into 2 ml of the liquid LB medium with the appropriate selective antibiotics, in this case, Gent + Rif + Spec. 
  4. Shake the culture overnight (~12-16 h) at 28 °C at the rate of 220 rpm. 
  5. (Optional) Amplify the fragments in the vector by regular PCR to make sure that the culture is correct. 
  6. Spin down the culture at 2,500 x g for 5 min, discard the supernatant. Suspend the culture with the injection buffer (MS salt + 2% sucrose) to reach a final OD600 of 0.8.
    Note: One milliliter cell suspension is enough for injecting 5-8 fruits. Two milliliters of culture usually makes 4 ml of cell suspension. The volume of the Agrobacterium culture could be increased if more injections are required.
  7. Use a 1 ml hypodermic syringe to do the injection immediately. Suck up the solution into the syringe, insert the needle tip into the center of the fruit from the apex, and gently press the syringe to release the solution. For a big fruit, such as that of the cultivated strawberry, more injections in other parts of the fruit are sometimes required to make the entire fruit soaked with the solution (Video 1).
    Note: Inject at least 10 fruits for each construct. Use fruits infiltrated only with the injection buffer as the negative control.

    Video 1. Procedure for the fruit injection as stated in Step 7

  8. Write down the construct name and the date on the tag, and tie the tag around the fruit stem. Put the plants back to the growth room, and let the fruit grow for about one week.
    Note: Fruit coloration may start to show at about 3 days post injection.
  9. Examine the fruit color and the GFP signal (if necessary) using the fluorescence dissecting stereomicroscope. Take pictures or collect tissues for downstream analysis.
    Note: Cut the fruit in order to observe the internal phenotypes. If necessary, the GFP fluorescence can be used as a guide for collecting tissues for downstream analysis.

Data analysis

Fruits of the F. vesca accession YW at the large green stage (15-20 days post pollination) were used for transiently overexpressing FveMYB10 (Figure 1A). We can see that the injected fruits are full of water under the receptacle skin (Figure 1B). One week later, fruit coloration has been fully developed (Figure 1C). Frequently, some area of the receptacle turns red, while the rest remains white. When the fruit is cut into two halves, tissues close to the skin turn red, while the inner part remains white. The intensity of the GFP fluorescence correlates well with the fruit coloration (data not shown). In contrast, fruits injected with only the injection buffer (negative control) stay white (Figure 1D). Anthocyanin synthesis or ripening genes are often transiently modified in cultivated strawberry. In order to illustrate that this approach is also suitable for gene knock-down, we exhibit the cultivated strawberry fruit transiently transformed with the RAP-RNAi construct (binary vector pK7WIWG2D) (Figure 1E) (Luo et al., 2018). Of note, the parts with GFP fluorescence overlap with the parts possessing color change, although GFP and RAP are independently driven by the 35S constitutive promoter in one construct.


Figure 1. Phenotypes of the transiently transformed strawberry fruits. A. One fruit of the F. vesca accession YW at the large green stage. B. The same fruit from A that was just injected. C. One YW fruit showing red color after one week of injecting the FveMYB10-ox construct. D. The control YW fruit injected only with the injection buffer. E. One fruit of the strawberry cultivar ‘Sweet Charlie’ showing reduced anthocyanin accumulation after one week of injecting the RAP-RNAi construct. Right images showing the GFP signal taken by the fluorescence microscope. Scale bars = 1 cm.

Notes

  1. Due to the limitation of fruit developmental stages used for injection, Agrobacterium-mediated transformation is more suitable for studying genes modulating late developmental processes, such as fruit ripening and the formation of fruit quality (coloration, formation of aroma and taste, etc.).
  2. The transformation efficiency varies greatly among individual fruit, even within one fruit. We suggest using vectors carrying a visible reporter to facilitate phenotype observation and tissue collection. GFP or other fluorescent proteins are better options than the β-glucuronidase (GUS).
  3. Grow the plants at about 25 °C. Lower or higher temperatures (30 °C) will greatly reduce the efficiency or even lead to a complete failure.
  4. We succeeded in transforming a mixture of agrobacteria harboring two constructs, respectively, in a ratio of 1:1. This provides more potentials of examining the interactions between two or more genes. 
  5. Besides F. vesca and F. × ananassa, this approach should be applicable to other Fragaria species as well.
  6. In this transient strawberry transformation, no acetosyringone is used. In the strawberry stable transformation, acetosyringone is added into the incubation buffer, and a few hours’ incubation of the cell suspension is required. This optimization might be tested in the future.

Recipes

  1. Luria Broth (LB) medium
    10 g L-1 Tryptone
    5 g L-1 Yeast extract
    10 g L-1 NaCl
    pH 7.0
    15 g L-1 Agar (for the solid LB medium)
  2. Injection buffer
    0.44 g MS powder
    2 g Sucrose
    100 ml ddH2O
    pH 5.8
  3. Antibiotics
    50 mg L-1 Gent
    50 mg L-1 Rif
    50 mg L-1 Spe

Acknowledgments

This work in Kang’s lab was supported by the National Natural Science Foundation of China (31572098, 31772274, and 31822044). This protocol was modified from our previously published work (Luo et al., 2018).

Competing interests

All the authors declare that they have no competing interests.

References

  1. Hawkins, C., Caruana, J., Schiksnis, E. and Liu, Z. (2016). Genome-scale DNA variant analysis and functional validation of a SNP underlying yellow fruit color in wild strawberry. Sci Rep 6: 29017.
  2. Hoffmann, T., Kalinowski, G. and Schwab, W. (2006). RNAi-induced silencing of gene expression in strawberry fruit (Fragaria × ananassa) by agroinfiltration: a rapid assay for gene function analysis. Plant J 48(5): 818-826.
  3. Huang, D., Wang, X., Tang, Z., Yuan, Y., Xu, Y., He, J., Jiang, X., Peng, S. A., Li, L., Butelli, E., Deng, X. and Xu, Q. (2018). Subfunctionalization of the Ruby2-Ruby1 gene cluster during the domestication of citrus. Nat Plants 4(11): 930-941.
  4. Jia, H. F., Chai, Y. M., Li, C. L., Lu, D., Luo, J. J., Qin, L. and Shen, Y. Y. (2011). Abscisic acid plays an important role in the regulation of strawberry fruit ripening. Plant Physiol 157(1): 188-199.
  5. Li, C., Yamagishi, N., Kasajima, I. and Yoshikawa, N. (2019). Virus-induced gene silencing and virus-induced flowering in strawberry (Fragaria × ananassa) using apple latent spherical virus vectors. Horticulture research 6: 18-18.
  6. Lin-Wang, K., McGhie, T. K., Wang, M., Liu, Y., Warren, B., Storey, R., Espley, R. V. and Allan, A. C. (2014). Engineering the anthocyanin regulatory complex of strawberry (Fragaria vesca). Front Plant Sci 5: 651.
  7. Luo, H., Dai, C., Li, Y., Feng, J., Liu, Z. and Kang, C. (2018). Reduced Anthocyanins in Petioles codes for a GST anthocyanin transporter that is essential for the foliage and fruit coloration in strawberry. J Exp Bot 69(10): 2595-2608.
  8. Spolaore, S., Trainotti, L. and Casadoro, G. (2001). A simple protocol for transient gene expression in ripe fleshy fruit mediated by Agrobacterium. J Exp Bot 52(357): 845-850.
  9. Tang, T., Yu, X., Yang, H., Gao, Q., Ji, H., Wang, Y., Yan, G., Peng, Y., Luo, H., Liu, K., Li, X., Ma, C., Kang, C. and Dai, C. (2018). Development and validation of an effective CRISPR/Cas9 vector for efficiently isolating positive transformants and transgene-free mutants in a wide range of plant species. Front Plant Sci 9: 1533.

简介

草莓,包括林地草莓Fragaria vesca (2x)和栽培草莓(Fragaria×ananassa, 8x),已成为研究果实发育成熟的模型系统。瞬时表达为基因功能或基因相互作用提供了一种快速检测方法。在草莓中,病毒诱导的基因沉默(VIGS)和农杆菌介导的转化(tumefaciens-mediated transformation)在果实中的瞬时表达被广泛应用。与VIGS不同,VIGS不仅可以用于基因敲除,还可以用于过表达和敲除。在这里,我们展示了将35S::FveMYB10结构瞬时表达为白果F果实的过程。vesca加入黄色奇迹。过表达FveMYB10会引起果实着色,这是花青素生产的主调节因子,在浸润一周后观察到。我们还展示了RNAi在草莓品种‘Sweet Charlie’果实中敲除在叶柄中还原的花青素 (RAP),编码花青素转运体的结果。总的来说,农杆菌介导的草莓果实瞬时转化是研究果实成熟过程中基因功能的一种快速、多用途的方法。
【背景】栽培草莓(Fragaria×ananassa, 8x)是一种生长在世界各地的具有重要经济价值的水果作物,外观可爱,营养丰富。野生二倍体草莓Fragaria vesca已成为栽培草莓和其他肉质果实品种的模型植物。草莓是一种典型的非更年期水果,对其成熟过程的研究缺乏良好的模型体系,如番茄作为更年期水果的模型。因此,草莓也是研究果实成熟的常用材料。



在林地和栽培草莓中均可实现稳定转化;然而,整个过程是劳动密集型和费时的。相比之下,瞬时表达法在果实中的检测速度更快、效率更高。草莓中存在两种瞬时基因表达途径,即病毒诱导的基因沉默(VIGS)和农杆菌介导的转化(Spolaore et al., 2001)。一些研究使用VIGS获得了很好的结果(Jia et al., 2011;李等。, 2019)。然而,我们更频繁地利用农杆菌介导的转化,因为它可以实现更多的实验目的,包括基因敲除(Hoffmann et al., 2006)、过表达(Huang et al., 2018)和敲除(Tang et al., 2018)。



F。在我们的生长条件下,vesca果实从授粉到成熟需要25-30天。整个过程分为七个阶段:花/花、小绿、中绿、大绿、白、转、红。果柄(多汁果肉)处于大绿期,硬度开始迅速下降(红草莓果实在瘦果变红之前),适宜农杆菌注射。由于质地的原因,早期的果座对注射有抵触情绪。因此,农杆菌介导的草莓果实转化,界定了对发育后期起作用的基因的功能研究。
草莓果实鲜红的颜色是由花青素化合物的积累引起的,这是一种可见的表型检查。在草莓中,R2R3-MYB转录因子FveMYB10 是花青素合成的主调控因子(Lin-Wang et al., 2014)。林地草莓既有红果品种,也有白果品种,其天然SNP发生在FveMYB10;过表达FveMYB10导致白果加入黄wonder (YW)果实中红色素的积累(Hawkins et al., 2016)。近年来,我们通过化学诱变鉴定出一种编码基因的花青素转运体,能够降低叶柄中的花青素含量 (RAP),其中的敲除作用显著降低了果实色素沉着(Luo et al., 2018)。
在本研究中,我们以YW果实为例,描述了瞬时过表达FveMYB10的过程。此外,我们还展示了在草莓品种“甜查理”果实中敲除RAP的结果。

关键字:瞬时表达, FveMYB10, 过表达, RAP, RNA干扰, 农杆菌, 草莓

材料和反应

  1. Tips
  2. 标签
  3. 1毫升带针的syringe (GEMTIER,目录编号:0.45x16 RW LB)
  4. 白色水果F。草莓品种“甜美的查理”
  5. 二进制向量:外泄用pK7WG2D, RNAi用pK7WIWG2D。两个向量contain a 35S: GFP视频记者
  6. 目标基因:FveMYB10, FvH4_1g22020/gene31413;说唱, FvH4_1g27460 / gene31672
  7. 最近的提取(OXOID,目录编号:LP0021)
  8. Tryptone (OXOID,目录编号:LP0042)
  9. Agar (TSINGKE,目录编号:1182GR500)
  10. MS(物理技术实验室,目录编号:M404-50L)
  11. GV3101化学能力细胞(上海生物技术,目录编号:AC1001)
  12. NaCl (HUSHI,目录编号:10019318,CAS: 7647-14-5)
  13. Sucrose (HUSHI,目录编号:10021418,CAS: 57-50-1)
  14. 抗生素,包括Kanamycin (Kan), Gentamicin (Gent), Rifampicin (Rif),和Spectinomycin (Spe) (Biofroxx,目录编号:1162GR005, 1463GR001, R3501,和S8040,尊重)
  15. 罗利亚肉质(LB)中
  16. 注入缓冲区(见模式)
  17. 抗生素(见处方)

设备

  1. 孵化器用于生长 28度C (JINGHONG,目录编号:DNP-9022)
  2. 双温控恒温器(MIULAB,目录号:BTH-100)
  3. 离心机(埃普多夫,5424)
  4. Clean workbench (AIRTECH, SW-CJ-1FD)
  5. 全温振动筛(培英,目录号:THZ-C-1)
  6. 超低温冰箱(ThermoFisher SCIENTIFIC, Thermo SCIENTIFIC TM Forma< /sup> TM 88000)
  7. 荧光解剖立体显微镜(徕卡,目录号:M205FA)
  8. 植物生长室:25±3°C, 16 h光/ 8 h黑暗,光强度在100μmol m <一口> 2 < /一口>秒1 <一口> < /一口> < br / >

过程

  1. 在生长室内种植草莓植物3到4个月,直到花朵开放。
  2. 人工授粉;等到果实发育到大的绿色阶段,授粉后约15-20天,花托开始变软。
  3. 将35S::FveMYB10 construct (binary vector pK7WG2D)转化为Agrobacterium tumefaciens strain GV3101,或者使用储存在超低温冰箱中的Agrobacterium stock。选择一个阳性菌落,放入2毫升液体LB培养基中,使用适当的选择性抗生素,本例中为Gent + Rif + Spec。
  4. 以220转/分的速度,在28°C下摇匀培养过夜(~12-16 h)。
  5. (可选)用常规PCR扩增载体片段,确保培养正确。
  6. 将培养物在2500 x g处旋转5分钟,弃上清。用注射缓冲液(MS盐+ 2%蔗糖)暂停培养,最终OD600,为0.8。< br / > 注:1毫升细胞悬液足以注射5-8个果实。两毫升的培养液通常可制成4毫升的细胞悬浮液。如果需要更多的注射,农杆菌培养物的体积可以增加。
  7. 立即使用1毫升皮下注射器进行注射。将溶液吸进注射器,将针尖从顶端插入果实中心,轻轻按压注射器释放溶液。对于一个大的水果,例如栽培的草莓,有时需要在水果的其他部分注射更多的针剂,使整个水果浸泡在溶液中(视频1)。< br / > 注:每个构形至少注入10个果实。使用仅用注射缓冲液浸润的水果作为阴性对照。 < br / > < br / > < br / > 视频1。步骤7 所述的水果注射程序 < br / >
  8. 在标签上写下结构名和日期,并将标签绑在水果茎上。把植物放回生长室,让水果生长一周左右。< br / > 注:注射后约3天,果实可能开始着色。
  9. 用荧光解剖立体显微镜检查果实颜色和GFP信号(如有必要)。拍照或收集组织进行下游分析。< br / > 注:切开果实观察内部表型。如有必要,GFP荧光可作为收集组织进行下游分析的指南。 < br / >

数据分析

果实F。采用大绿期(授粉后15-20天)vesca加入YW瞬时过表达FveMYB10(图1A)。我们可以看到注入的水果在容器表皮下充满了水(图1B)。一周后,果实着色完全完成(图1C)。通常情况下,花托的某些部分会变成红色,而其他部分则保持白色。当果实被切成两半时,靠近果皮的组织会变红,而内部仍然是白色的。GFP荧光强度与果实着色有很好的相关性(数据未显示)。相反,只注射缓冲液(阴性对照)的水果保持白色(图1D)。在栽培草莓中,花青素的合成或成熟基因常发生短暂的修饰。为了说明该方法也适用于基因敲除,我们展示了用RAP-RNAi构建物(二元载体pK7WIWG2D)对栽培草莓果实进行瞬时转化(图1E) (Luo et al., 2018)。值得注意的是,虽然GFP和RAP在一个结构中分别由35S本构子启动子驱动,但具有GFP荧光的部分与具有颜色变化的部分重叠。< br / > < br / > 图1 。短暂转化草莓果实的表型。 A.一个水果的F。在大的绿色阶段,vesca加入YW。B.和刚才注射的A的水果是一样的。C.注射FveMYB10< /em> -ox construct一周后,YW果实呈现红色。D.对照YW果实只注射了注射缓冲液。E.草莓品种‘Sweet Charlie’的一个果实在注射RAP-RNAi构形一周后,花青素积累减少。右图为荧光显微镜拍摄的GFP信号。标尺= 1厘米。

笔记

  1. 由于注射用的果实发育阶段的限制,农杆菌介导的转化更适合研究调控后期发育过程的基因,如果实成熟和果实品质的形成(着色、香气和味道的形成、等)。
  2. 在不同的水果之间,即使在一个水果内,转化效率也有很大的差异。我们建议使用携带可见报告基因的载体来促进表型观察和组织收集。GFP荧光蛋白质或其他更好的选择比β-glucuronidase(格斯)。
  3. 在25℃左右种植。较低或较高的温度(30℃)会大大降低效率,甚至导致完全失效。
  4. 我们成功地将含有两种结构的农杆菌混合物按1:1的比例进行转化。这为检验两个或多个基因之间的相互作用提供了更多的可能性。
  5. 除了 F。vesca 和 F。×ananassa,该方法同样适用于其他Fragaria种。
  6. 在这个短暂的草莓转化过程中,没有使用丙酮环酮。在草莓稳定转化过程中,将丙酮环酮加入孵育缓冲液中,需要孵育数小时的细胞悬液。这种优化可能在将来进行测试。< br / >

食谱

  1. 罗瑞亚肉汤(LB)培养基 10 g L-1 Tryptone 5 g L-1酵母提取物 10 g L-1 NaCl pH值7.0 < br / > 15 g L-1琼脂(固体LB培养基)
  2. 注入缓冲< br / > 0.44 g MS粉末 2克蔗糖 100ml ddH2O pH值5.8
  3. 抗生素< br / > 50 mg L-1 Gent 50 mg L-1 Rif 50 mg L-1 Spe

致谢

康的实验室工作得到了国家自然科学基金(31572098、31772274、31822044)的支持。本协议是从我们之前发表的工作中修改而来的(Luo et al., 2018)。

相互竞争的利益

所有的作者都说他们没有竞争优势。

参考

  1. 霍金斯,C。-卡鲁阿纳,J。希克斯尼斯,E.和刘,Z.(2016)。 Sci代表 6: 29017。
  2. 霍夫曼,T。, Kalinowski, G.和Schwab, W.(2006)。 rnair -基因表达在草莓水果中的迷迷性沉淀(Fragaria a href= https://www.ncbi.nlm.nih.gov/pubmed/30729008”“>×< / > ananassa )由agroinfiltration: a快速吸附法for吉恩function分析。植物 48(5): 818-826
  3. 黄,D。王,X。唐,Z。元,Y。徐,Y。他,J。江,X。彭,s.a.。李,L。布泰利,E。,邓,X,徐,Q.(2018)。 Nat Plants 4(11): 930-941。
  4. 贾,h.f。柴,ym。李,c.l.。路,D。罗,j.j.。(2011)Abscisic acid在草莓水果的监管中扮演重要角色。 植物物理 157(1): 188-199。
  5. 李,C。山石,N。, Kasajima, I.和Yoshikawa, N.(2019)。< a href = " https://www.ncbi.nlm.nih.gov/pubmed/30729008 " target = " _blank " > Virus-induced吉恩silencing and Virus-induced flowering草莓( Fragaria × ananassa ) using苹果latent spherical vectors病毒。 园艺研究 6: 18-18。
  6. Lin-Wang, K。麦克吉,t.k.。王,M。刘,Y。沃伦,B。斯托里,R。, Espley, r.v.艾伦,a.c.(2014)。工程草莓的anthocyanin regulatory complex (Fragaria vesca)”。 前植物滑雪板 5: 651。
  7. 罗,H。戴,C。李,Y。冯,J。刘,Z.和康,C.(2018)。J Exp Bot 69(10): 2595-2608。
  8. Spolaore, S。(2001)。是由农杆菌介导的成熟肉质果实瞬时基因表达的一种简单方法。jexp Bot 52(357): 845-850。
  9. 唐、T。Yu, X。杨,H。、高、Q。霁,H。王,Y。燕,G。彭,Y。罗,H。刘,K。李,X。,妈,C。,康,C.和戴,C.(2018)。载体的开发和验证,该载体可有效分离多种植物物种中的阳性转化子和无转基因突变体。 前端植物Sci 9: 1533。< br / >
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引用:Pi, M., Gao, Q. and Kang, C. (2019). Transient Expression Assay in Strawberry Fruits. Bio-protocol 9(11): e3249. DOI: 10.21769/BioProtoc.3249.
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