Cold-tolerance Analysis of Tobacco Plants Transformed with Saussurea involucrata SiSAD and Arabidopsis thaliana AtFAB2 Gene

doi:10.11983/CBB17129

Abstract

Abstract: The SiSAD gene in Saussurea involucrata and its homologous gene AtFAB2 in Arabidopsis thaliana have been reported to encode homeologous Δ9 stearoyl-acp desaturases. To investigate the function of these genes in plants’ response to cold stress, we constructed two expression vectors PSiSAD:AtFAB2 and PSiSAD:SiSAD, and were Agrobacterium-infiltrated in tobacco. These two kinds of transgenic plants and wild-type tobacco were treated at 20°C, 10°C, 5°C, 0°C, and -2°C for 2 h and then to determine the relative conductivity, malondialdehyde (MDA) and fatty acid content and chlorophyll fluorescence parameters (F_v/F_m). Furthermore, after -2°C treatment for 2 h, and recovery at 25°C for 1 week, we examined recovery rate of these tobacco plants. The recovery rate of SiSAD transgenic tobacco was much better than AtFAB2 transgenic tobacco and wild type. After treatment at 0°C and -2°C for 2 h, the relative conductivity and MDA content of the SiSAD and AtFAB2 transgenic tobacco and wild-type tobacco showed a significant increasing trend. The F_v/F_m of SiSAD and AtFAB2 transgenic tobacco were significantly higher than wild-type tobacco and the F_v/F_m of SiSAD transgenic tobacco was significantly higher than AtFAB2 transgenic tobacco. The content of oleic acid (C18:1) in AtFAB2 transgenic tobacco and wild type were decreased gradually with decreasing temperature and reached the lowest level at 0°C, whereas the content of C18:1 in SiSAD transgenic tobacco increased and peaked at -2°C; The C18:1 contents in SiSAD transgenic tobacco were at least 1.58 and 1.7 folds when compared to AtFAB2 transgenic tobacco and wild type. These results indicate that SiSAD and AtFAB2 genes can significantly enhance the cold tolerance of a non-cold acclimated tobacco. Moreover, the SiSAD gene plays more important role than AtFAB2 gene in cold tolerance.

Key words: low temperature, Saussurea involucrata, SiSAD, AtFAB2, cold tolerance

Chen Jianquan, Cheng Chen, Zhang Mengtian, Zhang Xiangqian, Zhang Yao, Wang Aiying, Zhu Jianbo. Cold-tolerance Analysis of Tobacco Plants Transformed with Saussurea involucrata SiSAD and Arabidopsis thaliana AtFAB2 Gene[J]. Chinese Bulletin of Botany, 2018, 53(5): 603-611.

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URL: https://www.chinbullbotany.com/EN/10.11983/CBB17129

https://www.chinbullbotany.com/EN/Y2018/V53/I5/603

Figures/Tables 7

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Primer name	Primer sequence (5'-3')
SAD F	GTTGGAGATATGATCCACGAGGAAGC
SikSAD R	TTCCAGTATATCGGCATAGTCCTT
AtFAB2 F	GCACATGCGTGACATGCTTC
AtFAB2 R	CTGATCGACGGTCAATTGGC
GAPDH F	GTTGCTAGAGTTGCACTTCAGAGAG
GAPDH R	TTCCTGAAGCCGAAAACAGC

Primer name	Primer sequence (5'-3')
SAD F	GTTGGAGATATGATCCACGAGGAAGC
SikSAD R	TTCCAGTATATCGGCATAGTCCTT
AtFAB2 F	GCACATGCGTGACATGCTTC
AtFAB2 R	CTGATCGACGGTCAATTGGC
GAPDH F	GTTGCTAGAGTTGCACTTCAGAGAG
GAPDH R	TTCCTGAAGCCGAAAACAGC

Temperature (°C)	Plant	Fatty acid (%)
Temperature (°C)	Plant	C16:0	C18:0	C18:1	C18:2	C18:3	Total desaturation products
20	WT	43.70±1.10 a	24.77±0.66 a	14.33±0.94 a	3.27±0.43 a	12.97±1.56 a	16.57
	s-f	42.27±1.11 a	20.89±1.09 b	15.20±0.38 b	2.77±0.92 a	7.37±1.24 b	17.34
	s-s	46.53±0.90 a	20.03±0.32 c	17.23±1.60 c	4.61±1.15 a	7.07±0.93 b	21.91
10	WT	38.99±0.58 a	19.83±0.65 a	15.87±0.92 a	2.53±0.30 a	8.17±1.22 a	17.57
	s-f	42.97±0.62 a	15.87±0.86 a	17.43±0.48 a	2.07±0.12 a	8.19±1.12 a	19.69
	s-s	40.76±0.79 a	15.70±2.80 a	19.40±1.23 c	1.77±0.38 a	6.00±1.11 a	23.17
5	WT	30.30±0.80 a	16.47±0.66 a	17.50±0.44 a	3.70±0.12 a	17.20±2.02 a	19.4
	s-f	25.27±0.66 b	13.83±0.71 b	18.70±1.29 b	1.90±0.17 a	10.20±0.86 b	20.8
	s-s	46.43±0.41 c	12.30±0.67 a	20.70±0.57 b	2.67±0.09 a	6.60±0.68 c	31.97
0	WT	40.53±0.44 a	16.23±0.32 a	17.90±0.76 a	2.20±0.17 a	8.47±0.41 a	22.67
	s-f	47.63±0.47 b	13.67±0.73 b	19.17±1.45 b	2.33±0.27 a	11.97±1.44 a	28.47
	s-s	44.33±0.07 b	12.47±0.45 c	23.50±1.33 a	2.30±0.46 a	11.87±2.07 a	37.67
-2	WT	38.20±1.07 a	15.57±0.76 a	15.63±2.21 a	2.87±0.20 a	10.23±0.72 a	26.73
	s-f	40.20±0.23 b	11.20±1.56 b	16.54±0.49 a	3.13±0.59 a	16.30±1.22 b	31.97
	s-s	53.60±0.47 c	10.30±0.64 b	30.47±0.89 b	2.33±0.48 a	9.83±2.24 a	49.63
Recover treatment	WT	36.30±1.04 a	14.46±0.68 a	14.77±2.11 a	2.46±0.22 a	10.02±0.68 a	25.94
	s-f	41.40±1.03 a	20.05±1.03 b	14.91±0.34 b	2.58±0.85 a	7.19±1.18 b	16.87
	s-s	46.33±0.87 a	20.01±0.30 c	17.14±1.53 c	4.57±1.12 a	6.98±0.90 b	21.23

Temperature (°C)	Plant	Fatty acid (%)
Temperature (°C)	Plant	C16:0	C18:0	C18:1	C18:2	C18:3	Total desaturation products
20	WT	43.70±1.10 a	24.77±0.66 a	14.33±0.94 a	3.27±0.43 a	12.97±1.56 a	16.57
	s-f	42.27±1.11 a	20.89±1.09 b	15.20±0.38 b	2.77±0.92 a	7.37±1.24 b	17.34
	s-s	46.53±0.90 a	20.03±0.32 c	17.23±1.60 c	4.61±1.15 a	7.07±0.93 b	21.91
10	WT	38.99±0.58 a	19.83±0.65 a	15.87±0.92 a	2.53±0.30 a	8.17±1.22 a	17.57
	s-f	42.97±0.62 a	15.87±0.86 a	17.43±0.48 a	2.07±0.12 a	8.19±1.12 a	19.69
	s-s	40.76±0.79 a	15.70±2.80 a	19.40±1.23 c	1.77±0.38 a	6.00±1.11 a	23.17
5	WT	30.30±0.80 a	16.47±0.66 a	17.50±0.44 a	3.70±0.12 a	17.20±2.02 a	19.4
	s-f	25.27±0.66 b	13.83±0.71 b	18.70±1.29 b	1.90±0.17 a	10.20±0.86 b	20.8
	s-s	46.43±0.41 c	12.30±0.67 a	20.70±0.57 b	2.67±0.09 a	6.60±0.68 c	31.97
0	WT	40.53±0.44 a	16.23±0.32 a	17.90±0.76 a	2.20±0.17 a	8.47±0.41 a	22.67
	s-f	47.63±0.47 b	13.67±0.73 b	19.17±1.45 b	2.33±0.27 a	11.97±1.44 a	28.47
	s-s	44.33±0.07 b	12.47±0.45 c	23.50±1.33 a	2.30±0.46 a	11.87±2.07 a	37.67
-2	WT	38.20±1.07 a	15.57±0.76 a	15.63±2.21 a	2.87±0.20 a	10.23±0.72 a	26.73
	s-f	40.20±0.23 b	11.20±1.56 b	16.54±0.49 a	3.13±0.59 a	16.30±1.22 b	31.97
	s-s	53.60±0.47 c	10.30±0.64 b	30.47±0.89 b	2.33±0.48 a	9.83±2.24 a	49.63
Recover treatment	WT	36.30±1.04 a	14.46±0.68 a	14.77±2.11 a	2.46±0.22 a	10.02±0.68 a	25.94
	s-f	41.40±1.03 a	20.05±1.03 b	14.91±0.34 b	2.58±0.85 a	7.19±1.18 b	16.87
	s-s	46.33±0.87 a	20.01±0.30 c	17.14±1.53 c	4.57±1.12 a	6.98±0.90 b	21.23

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