双管基因枪介导的基因瞬时表达技术在拟南芥中的应用

doi:10.11983/CBB19169

摘要/Abstract

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

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

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. 植物学报, 2020, 55(2): 182-191.

Hua Zhao,Guangda Shao,Wenxin Gao,Biao Gu. The Application of Double-barreled Particle Bombardment for Transient Gene Expression in Arabidopsis. Chinese Bulletin of Botany, 2020, 55(2): 182-191.

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链接本文: https://www.chinbullbotany.com/CN/10.11983/CBB19169

https://www.chinbullbotany.com/CN/Y2020/V55/I2/182

图/表 7

图1 利用双管基因枪在拟南芥和本氏烟叶片中瞬时表达GFP和GUS报告基因 (A) 用于基因瞬时表达的拟南芥植株, 红色箭头所示为选用的莲座叶(Bar=5 mm); (B) 拟南芥叶片中GFP报告基因荧光显微观察(Bar=50 μm); (C), (D) 分别为拟南芥和本氏烟叶片中GUS报告基因的表达情况。黑色数字代表统计的GUS蓝斑数量(Bars=5 mm)。

Figure 1 Transient expression of GFP and GUS reporter genes in Arabidopsis thaliana and Nicotiana benthamiana leaves via double-barreled particle bombardment (A) The rosette leaves (red arrow pointed) of A. thaliana were selected for transient gene expression (Bar=5 mm); (B) Observation of green fluorescent reporter gene (GFP) with fluorescent microscopy (Bar=50 μm); (C), (D) Transient expression of GUS in A. thaliana and N. benthamiana leaves, respectively. Numbers of GUS positive spots are indicated in black numbers (Bars=5 mm).

表1 PCR和qRT-PCR引物序列

Table 1 Primer sequences of PCR and qRT-PCR

Primer name	Forward primer (5'-3')	Reverse primer (5'-3')
GFP	CTAGCCCCGGGATGGTGAGCAAGGGCGAG	CTGAGGTACCTTACTTGTACAGCTCGTC
GUS	CTAGCCCCGGGATGGTAGATCTGAGGAACC	CTGAGGTACCTCACACGTGATGGTGATGG
BAX	CTAGCCCCGGGATGGACGGGTCCGGGGAG	GTGCAGGATCCCTGGAAGAAGATGGGCTGA
Avh238	CTAGCCCCGGGATGGAGGCGAAGCTCTTG	GTGCAGGATCCGTTGGCCAAGCCTGTGTAG
Avrblb1	CTAGCCCCGGGATGGTTTCATCCAATCTCAAC	GTGCAGGTACCCTAGCTAGGGCCAACGTTTTTATC
RB	CTAGCCCCGGGATGGCTGAAGCTTTCATTCAAG	GTGCAGGATCCTTAAATATATATATTCACATTAG
ATR13	CTAGCCCCGGGATGAATCTGCTCCACGCCCATG	GTGCAGGATCCTTACTGACTGGCAACGGCAGTC
Rpp13	CTAGCCCCGGGATGGTAGATGCGATCACGGAG	GTGCAGGATCCCTAAACGCTCGCAATCGGTTTG
qRB	GAGAAGAGCAATTCCCTGTGC	GTTAGGTGCTGCAATCCCTCTG
qUBC9	CATCGGATAGCCCTTATTCTG	TGGAACACCTTCGTCCTAAAA

图2 不同拟南芥植株莲座叶中瞬时表达GUS报告基因的效率 (A) 不同莲座叶中双管基因枪介导GFP/GUS混合报告基因瞬时表达, 红色虚线划分轰击后双管基因枪2个枪孔形成的GUS蓝斑, 黑色数字代表形成的GUS蓝斑数量(Bars=5 mm); (B) 统计分析20片莲座叶中叶尖(Barrel 1)和叶柄(Barrel 2)产生GUS蓝斑的数量(误差线表示±标准差, P>0.1); (C) 将图B中的数据, 按照相同叶片Barrel 1和Barrel 2产生GUS蓝斑数量的比率进行统计分析(误差线表示±标准差)。每次选取2株拟南芥的10个叶片进行测试, 实验重复2次。将Barrel 1与Barrel 2产生GUS蓝斑数量进行对数转换后用威尔科克森符号轶和确定统计差异。

Figure 2 The efficiency of transient expression of GUS gene in different rosette leaves of Arabidopsis thaliana (A) Co-bombardment of GFP/GUS gene mixture in different rosette leaves, the red dotted line separates two positions produced by double-barreled particle bombardment, numbers of GUS blue spots are indicated in black numbers (Bars=5 mm); (B) Statistic analysis of GUS spots counted from the tip of leaf blade (Barrel 1) and petiole (Barrel 2) (error bar represents ± SD, P>0.1); (C) Ratios of GUS spot numbers in Barrel 1 and Barrel 2 of the same leaf (error bar represents ± SD). Ten rosette leaves from two A. thaliana plants were tested and the test was repeated twice. Statistical analysis by Wilcoxon rank sum test using log ratios of GUS spot numbers in Barrel 1 and Barrel 2.

图3 拟南芥和本氏烟叶片中的病原菌效应基因致死活性检测 =利用双管基因枪在拟南芥和本氏烟叶片上瞬时表达哺乳动物细胞凋亡因子BAX、大豆疫霉菌RxLR效应基因Avh238以及拟南芥霜霉菌效应基因ATR13和拟南芥抗病基因Rpp13后, GUS蓝斑指示的细胞坏死情况, GFP为对照。红色虚线划分轰击后双管基因枪2个枪孔形成的GUS蓝斑, 黑色数字代表形成的GUS蓝斑数量(Bars=5 mm)。

Figure 3 Cell death induction by various pathogen effectors in Arabidopsis thaliana and Nicotiana benthamiana leaves Mammalian apoptosis inducer BAX, RxLR effector gene Avh238 of Phytophthora sojae, effector gene ATR13 of Hyaloperonospora arabidopsidis and disease resistance gene Rpp13 of A. thaliana gene pairs were transiently expressed in leaves of A. thaliana and N. benthamiana by double-barreled particle bombardment, cell necrosis was indicated by GUS blue spots, with GFP as control. The red dotted line separates two positions produced by co-bombardment, numbers of GUS spots are indicated in black numbers (Bars=5 mm).

表2 在拟南芥和本氏烟叶片中测试BAX、Avh238和ATR13/Rpp13诱导细胞坏死的活性

Table 2 Measurement of cell death induction activities by BAX, Avh238 and ATR13/Rpp13 in Arabidopsis thaliana and Nicotiana benthamiana

	BAX ± SD		Avh238 ± SD		ATR13/Rpp13 ± SD
	GFP/GFP	BAX/GFP	GFP/GFP	Avh238/GFP	ATR13 + Rpp13/GFP
Arabidopsis thaliana	0.92±0.13	0.23±0.13*	0.84±0.17	0.94±0.15	0.22±0.08*
Nicotiana benthamiana	1.05±0.23	0.13±0.07*	0.92±0.21	0.94±0.10	0.22±0.05*

图4 拟南芥和本氏烟叶片中致病疫霉效应基因Avrblb1和马铃薯抗病基因RB致死活性检测 (A) 拟南芥和本氏烟中双管基因枪介导Avrblb1/Rpp13基因对瞬时表达, GFP为对照, 红色虚线划分轰击后双管基因枪2个枪孔形成的GUS蓝斑, 黑色数字指示GUS蓝斑数量(Bars=5 mm); (B) GUS蓝斑比率(拟南芥和本氏烟各10个叶片, 实验重复2次, 误差线表示±标准差)。处理与对照产生GUS斑数进行对数转换后用威尔科克森符号轶和确定统计差异, ***表示差异极显著(P<0.001); (C) 本氏烟叶片中农杆菌介导Avrblb1/RB瞬时表达, GFP为阴性对照, PAMP分子Inf1为阳性对照, 注射4天后拍照。每个处理重复注射10次(Bar=5 mm)。

Figure 4 Cell death induction triggered by Avrblb1 of Phytophthora infestans and RB of Solanum tuberosum in Arabidopsis thaliana and Nicotiana benthamiana leaves (A) Co-bombardment mediated transient expression of Avrblb1/Rpp13 gene pair in A. thaliana and N. benthamiana leaves, the red dotted line separates two positions produced by co-bombardment, numbers of GUS spots are indicated in black numbers (Bars=5 mm); (B) Ratio of GUS positive spots (ten leaves from each plant were tested, and the test was repeated twice, error bar represents ± SD). P values for treatments and the control were calculated from the log ratios using the Wilcoxon rank sum test, *** indicates extremely significant differences (P<0.001); (C) Agrobacterium-mediated transient expression of Avrblb1/RB in N. benthamiana leaves, GFP and Inf1 were used as negative and positive control, respectively. Pictures were taken 4 days post infiltration, each treatment contains 10 replicates (Bar=5 mm).

图5 拟南芥RB转基因株系RB-9和RB-12中效应基因Avrblb1致死活性检测 (A) 在拟南芥RB转基因株系RB-9和RB-12中瞬时表达Avrblb1后GUS报告基因表达分析, GFP为对照(红色虚线划分轰击后双管基因枪2个枪孔形成的GUS蓝斑, 黑色数字指示GUS蓝斑数量(Bars=5 mm)); (B) 在拟南芥RB转基因株系中瞬时表达Avrblb1基因后的GUS蓝斑数量统计(拟南芥和本氏烟各10片叶, 实验重复2次, 处理与对照产生GUS斑数进行对数转换后用威尔科克森符号轶和确定统计差异(误差线表示±标准差, P>0.1)); (C) 利用RT-PCR检测拟南芥RB转基因株系RB-9和RB-12中RB基因的表达, UBC9为内参基因。

Figure 5 Measurement of cell death induction by effector gene Avrblb1 in Arabidopsis transgenic line RB-9 and RB-12 (A) Co-bombardment mediated transient expression of Avrblb1 in transgenic lines RB-9 and RB-12, with GFP as control (the red dotted line separates two positions produced by co-bombardment, numbers of GUS spots are indicated in black numbers (Bars=5 mm)); (B) The diagram showed that transient expression of Avrblb1 in RB transgenic lines (ten leaves of each plant were tested, and the test was repeated twice, P values for treatments and the control were calculated from the log ratios using the Wilcoxon rank sum test (error bar represents ± SD, P>0.1)); (C) The RT-PCR detection of RB expressed in transgenic line RB-9 and RB-12, UBC9 was used as the reference gene.

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