Chinese Bulletin of Botany ›› 2020, Vol. 55 ›› Issue (2): 182-191.DOI: 10.11983/CBB19169
• TECHNIQUES AND METHODS • Previous Articles Next Articles
Hua Zhao1,Guangda Shao1,2,Wenxin Gao1,2,Biao Gu1,2,*()
Received:
2019-08-28
Accepted:
2019-11-28
Online:
2020-03-01
Published:
2020-02-12
Contact:
Biao Gu
Hua Zhao,Guangda Shao,Wenxin Gao,Biao Gu. The Application of Double-barreled Particle Bombardment for Transient Gene Expression in Arabidopsis[J]. Chinese Bulletin of Botany, 2020, 55(2): 182-191.
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).
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 |
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 |
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.
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).
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* |
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* |
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).
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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