[an error occurred while processing this directive] [an error occurred while processing this directive] [an error occurred while processing this directive]
[an error occurred while processing this directive]

植物学报 >

2020 , Vol. 55 >Issue 2: 182 - 191

DOI: https://doi.org/10.11983/CBB19169

技术方法

双管基因枪介导的基因瞬时表达技术在拟南芥中的应用

展开
  • 1 西北农林科技大学旱区作物逆境生物学国家重点实验室, 杨凌 712100
    2 西北农林科技大学植物保护学院, 杨凌 712100

收稿日期: 2019-08-28

  录用日期: 2019-11-28

  网络出版日期: 2019-12-04

基金资助

国家自然科学基金(31500212);陕西省自然科学基础研究计划(No.2018JM3030)

收起

The Application of Double-barreled Particle Bombardment for Transient Gene Expression in Arabidopsis

Expand
  • 1 State Key Laboratory of Crop Stress Biology for Arid Areas, Northwest A&F University, Yangling 712100, China
    2 College of Plant Protection, Northwest A&F University, Yangling 712100, China

Received date: 2019-08-28

  Accepted date: 2019-11-28

  Online published: 2019-12-04

Fold

摘要

基因瞬时表达是植物中研究目标基因功能的常用手段。在模式植物拟南芥(Arabidopsis thaliana)中, 相比原生质体和农杆菌介导的基因异源表达技术, 利用粒子轰击进行基因瞬时表达一直鲜有报道。其主要原因是拟南芥叶型相对较小、基因枪操作相对烦琐以及基因表达效率差异较大。该研究通过优化双管基因枪系统, 在营养生长旺盛的拟南芥莲座叶中实现GFP和GUS基因高效表达。同时, 通过GUS报告基因明确了坏死诱导因子BAX、Avh238和ATR13/Rpp13激发拟南芥细胞坏死的表型。但在本氏烟(Nicotiana benthamiana)中明显诱导细胞坏死的Avrblb1/RB基因对, 在拟南芥中却丧失了诱导细胞坏死的活性。由于双管基因枪系统每次轰击时设置平行对照, 可有效降低转化实验中的样本变异度, 为拟南芥及其突变体研究中准确评价基因功能和高通量筛选目标基因提供新的技术参考。

关键词： 瞬时表达; 双管基因枪; 拟南芥; 效应基因; 抗病基因

本文引用格式

赵华,邵广达,高文鑫,顾彪 . 双管基因枪介导的基因瞬时表达技术在拟南芥中的应用[J]. 植物学报, 2020 , 55(2) : 182 -191 . DOI: 10.11983/CBB19169

Abstract

Transient gene expression is a favorite tool used for functional analysis of target genes in plant. Of three techniques applied for genetic transformation in the model plant Arabidopsis thaliana, biolistic delivery system was less used than protoplast- or Agrobacterium-mediated transformation. This is mainly due to the smaller leaf size of Arabidopsis, the complicated procedure of bombardment and the limited efficiency and consistency of gene expression. Here, we report applications of an optimized double-barreled particle bombardment system for transient transformation in Arabidopsis, which displayed high expression level of GFP and GUS reporter genes in leaf epidermal cells. By introducing the parallel control in the same shoot by co-bombardment, gene expression efficiency and consistency were dramatically improved, which allows quantitative analysis of target genes with several replicates. Furthermore, cell death inducers BAX, Avh238 or ATR13/Rpp13, were co-expressed with GUS in Arabidopsis rosette leaves and led to strong necrosis phenotypes visualized by significant reduction of number of GUS spots. On the contrary, Avrblb1/RB gene pair triggered strong cell death in Nicotiana benthaminana, but not in A. thaliana. Therefore, this time-saving protocol is an alternative to quantitatively evaluate biological functions of the gene of interest and high-throughput screening of immune suppressors in Arabidopsis and its mutants.

Key words： transient expression; double-barreled gene gun; Arabidopsis; effector gene; disease-resistance gene

[an error occurred while processing this directive]

参考文献

[1] Champouret N, Bouwmeester K, Rietman H, van der Lee T, Maliepaard C, Heupink A, van de Vondervoort PJI, Jacobsen E, Visser RGF, van der Vossen EAG, Govers F, Vleeshouwers VGAA ( 2009). Phytophthora infestans isolates lacking class I ipiO variants are virulent on Rpi-blb1 potato. Mol Plant Microbe Interact 22, 1535-1545.
[2] Coll NS, Vercammen D, Smidler A, Clover C, Van Breusegem F, Dangl JF, Epple P ( 2010). Arabidopsis type I metacaspases control cell death. Science 330, 1393-1397.
[3] Dong SM, Yin WX, Kong GH, Yang XY, Qutob D, Chen QH, Kale SD, Sui YY, Zhang ZG, Dou DL, Zheng XB, Gijzen M, Tyler BM, Wang YC ( 2011). Phytophthora sojae avirulence effector Avr3b is a secreted NADH and ADP-ribose pyrophosphorylase that modulates plant immunity. PLoS Pathog 7, e1002353.
[4] Dou DL, Kale SD, Wang XL, Chen YB, Wang QQ, Wang X, Jiang RHY, Arredondo FD, Anderson RG, Thakur PB, McDowell JM, Wang YC, Tyler BM ( 2008). Conserved C-terminal motifs required for avirulence and suppression of cell death by Phytophthora sojae effector Avr1b. Plant Cell 20, 1118-1133.
[5] Gu B, Kale SD, Wang QH, Wang DH, Pan QN, Cao H, Meng YL, Kang ZS, Tyler BM, Shan WX ( 2011). Rust secreted protein Ps87 is conserved in diverse fungal pathogens and contains a RXLR-like motif sufficient for translocation into plant cells. PLoS One 6, e27217.
[6] Kale SD, Tyler BM ( 2011). Assaying effector function in Planta using double-barreled particle bombardment. In: McDowell J, ed. Plant Immunity. Methods in Molecular Biology (Methods and Protocols), Vol. 712. New York: Humana Press. pp. 153-172.
[7] Koornneef M, Meinke D ( 2010). The development of Arabidopsis as a model plant. Plant J 61, 909-921.
[8] Mangano S, Gonzalez CD, Petruccelli S ( 2014). Agrobacterium tumefaciens-mediated transient transformation of Arabidopsis thaliana leaves. In: Sanchez-Serrano JJ, Salinas J, eds. Arabidopsis Protocols, Methods in Molecular Biology (Methods and Protocols), Vol. 1062. Totowa, NJ: Humana Press. pp. 165-173.
[9] Sohn KH, Lei R, Nemri A, Jones JDG ( 2007). The downy mildew effector proteins ATR1 and ATR13 promote disease susceptibility in Arabidopsis thaliana. Plant Cell 19, 4077-4090.
[10] Tsuda K, Qi YP, Nguyen LV, Bethke G, Tsuda Y, Glazebrook J, Katagiri F ( 2012). An efficient Agrobacterium-mediated transient transformation of Arabidopsis. Plant J 69, 713-719.
[11] Ueki S, Lacroix B, Krichevsky A, Lazarowitz SG, Citovsky V ( 2009). Functional transient genetic transformation of Arabidopsis leaves by biolistic bombardment. Nat Protoc 4, 71-77.
[12] Wang QQ, Han CZ, Ferreira AO, Yu XL, Ye WW, Tripathy S, Kale SD, Gu B, Sheng YT, Sui YY, Wang XL, Zhang ZG, Cheng BP, Dong SM, Shan WX, Zheng XB, Dou DL, Tyler BM, Wang YC ( 2011a). Transcriptional programming and functional interactions within the Phytophthora sojae RXLR effector repertoire. Plant Cell 23, 2064-2086.
[13] Wang XJ, Tang CL, Zhang HC, Xu JR, Liu B, Lv J, Han DJ, Huang LL, Kang ZS ( 2011b). TaDAD2, a negative regulator of programmed cell death, is important for the interaction between wheat and the stripe rust fungus. Mol Plant Microbe Interact 24, 79-90.
[14] Weigel D, Glazebrook J ( 2011). Arabidopsis: a Laboratory Manual. New York: Cold Spring Harbor Laboratory Press. pp. 119-140.
[15] Yang B, Wang QQ, Jing MF, Guo BD, Wu JW, Wang HN, Wang Y, Lin L, Wang Y, Ye WW, Dong SM, Wang YC ( 2017). Distinct regions of the Phytophthora essential effector Avh238 determine its function in cell death activation and plant immunity suppression. New Phytol 214, 361-375.
[16] Yoo SD, Cho YH, Sheen J ( 2007). Arabidopsis mesophyll protoplasts: a versatile cell system for transient gene expression analysis. Nat Protoc 2, 1565-1572.
Options
文章导航

/

〈 〉

[an error occurred while processing this directive]