Chin Bull Bot ›› 2018, Vol. 53 ›› Issue (5): 603-611.doi: 10.11983/CBB17129

• EXPERIMENTAL COMMUNICATIONS • Previous Articles     Next Articles

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

Chen Jianquan, Cheng Chen, Zhang Mengtian, Zhang Xiangqian, Zhang Yao, Wang Aiying, Zhu Jianbo*()   

  1. Key Laboratory of Agricultural Biotechnology, College of Life Science, Shihezi University, Shihezi 832003, China
  • Received:2017-07-08 Accepted:2017-10-25 Online:2018-11-29 Published:2018-09-01
  • Contact: Zhu Jianbo E-mail:zjbshz@126.com
  • About author:

    † These authors contributed equally to this paper

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 (Fv/Fm). 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 Fv/Fm of SiSAD and AtFAB2 transgenic tobacco were significantly higher than wild-type tobacco and the Fv/Fm 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

Figure 1

The structure of two plant expression vectors"

Table 2

Primers of RT-PCR"

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

Figure 2

PCR identification of tobacco transferred with PSiSAD:SiSAD and PSiSAD:AtFAB2 recombinant plasmid, respectively (A) PCR identification of tobacco transferred with PSiSAD: SiSAD recombinant plasmid (M: Marker III DNA marker; 1-7: Different transgenic lines; 8: Wild type (negative control); 9: PSiSAD:SiSAD plasmid (positive control)); (B) PCR identification of tobacco transferred with PSiSAD:AtFAB2 recombinant plasmid (M: Marker III DNA marker; 1-10: Different transgenic lines; 11: Wild type (negative control); 12: PSiSAD:SiSAD plasmid (positive control))"

Figure 3

RT-PCR identification of tobacco transferred with PSiSAD:SiSAD and PSiSAD:AtFAB2 recombinant plasmid, respectively"

Figure 4

Phenotype of wild-type and transgenic tobacco under different temperatures(A)-(E) Wild-type and transgenic tobacco plants grown at 20°C, 10°C, 5°C, 0°C, and -2°C for 2 hours, respectively; (F) After -2°C treatment recovering in 25°C for one week.s-f: PSiSAD:AtFAB2; s-s: PSiSAD:SiSAD; WT: Wild-type"

Table 2

The analysis of the content of desaturation products in transgenic tobacco under different temperatures"

Temperature
(°C)
Plant Fatty acid (%)
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

Figure 5

The physiological analysis of wild-type and transgenic tobacco plant after different processing temperature (A) Relative conductivity; (B) Maximum efficiency of photosystem II photochemistry (Fv/Fm); (C) Malondialdehyde (MDA) content; Wild-type and transgenic tobacco plants grown at 20°C, 10°C, 5°C, 0°C, and -2°C; recovery treatment: after -2°C treatment for 2 hours recovery in 25°C for 1 week. Different lowercase letters indicate significant differences at P<0.05. s-f: PSiSAD:AtFAB2; s-s: PSiSAD:SiSAD; WT: Wild-type"

1 陈爱葵, 韩瑞宏, 李东洋, 凌连莲, 罗惠霞, 唐上剑 (2010). 植物叶片相对电导率测定方法比较研究. 广东教育学院学报 30(5), 88-91.
2 陈东亮 (2010). 根癌农杆菌介导的千年桐SAD基因对产油酵母的遗传转化. 硕士论文. 北京: 中国林业科学研究院. pp. 38-50.
3 陈发菊, 杨映根, 赵德修, 桂耀林, 郭仲琛 (1999). 我国雪莲植物的种类、生境分布及化学成分的研究进展. 植物学通报 16, 561-566.
4 陈思羽, 刘鹏, 朱末, 夏冬冬, 李亮, 徐克章, 陈展宇, 张治安 (2016). 大豆植株不同冠层种子活力及其萌发中抗氧化酶活性. 植物学报 51, 24-30.
5 程晨, 郭新勇, 王爱英, 祝建波 (2011). 转新疆雪莲去饱和酶基因sikSAD重组酵母低温和酒精耐受性分析. 微生物学通报 38, 1647-1656.
6 范妙华, 李纪元, 范正琪, 田敏, 倪穗 (2008). 千年桐SAD基因克隆与分析及其丝状真菌表达载体构建. 西北植物学报 28, 18-22.
7 桂仁意, 刘亚迪, 郭小勤, 季海宝, 贾月, 余明增, 方伟 (2010). 不同剂量137Cs-γ辐射对毛竹幼苗叶片叶绿素荧光参数的影响. 植物学报 45, 66-72.
8 郭新勇, 程晨, 王爱英, 张煜星, 王重, 喻娜, 祝建波 (2012). 天山雪莲冷调节蛋白基因siCOR转化烟草植株的抗旱性分析. 植物学报 47, 111-119.
9 贾艳丽, 吴磊, 卢长明 (2014). 甘蓝型油菜Δ9硬脂酰ACP脱氢酶(SAD)基因的克隆与表达分析. 中国油料作物学报 36, 135-141.
10 李金璐, 王硕, 于婧, 王玲, 周世良 (2013). 一种改良的植物DNA提取方法. 植物学报 48, 72-78.
11 罗华元, 董石飞, 倪明, 张峻松 (2010). 烟叶中多元酸和高级脂肪酸的分析. 安徽农业科学 38, 16212-16214.
12 罗通 (2006). 麻疯树的抗冷性和SAD基因的克隆及表达研究. 博士论文. 成都: 四川大学. pp. 27-35.
13 罗秀芹, 欧文军, 李开绵, 陈松笔 (2014). 抗寒蛋白硬脂酰-ACP脱饱和酶的结构与功能预测. 福建农林大学学报(自然科学版) 43, 484-489.
14 庞磊, 周小生, 李叶云, 江昌俊 (2011). 应用叶绿素荧光法鉴定茶树品种抗寒性的研究. 茶叶科学 31, 521-524.
15 张党权, 谭晓风, 陈鸿鹏, 曾艳玲, 蒋瑶, 李魏, 胡芳名 (2008). 油茶SAD基因的全长cDNA克隆及生物信息学分析. 林业科学 44, 155-159.
16 甄伟, 陈溪, 孙思洋, 胡鸢雷, 林忠平 (2000). 冷诱导基因的转录因子CBF1转化油菜和烟草及抗寒性鉴定. 自然科学进展 10, 1104-1108.
17 祝建波, 刘海亮, 王重, 周鹏 (2006). 天山雪莲叶片全长cDNA文库的构建. 西北农业学报 15(6), 170-173.
18 Aroca R, Amodeo G, Fernández-Illescas S, Herman EM, Chaumont F, Chrispeels MJ (2005). The role of aqu- aporins and membrane damage in chilling and hydrogen peroxide induced changes in the hydraulic conductance of maize roots.Plant Physiol 137, 341-353.
19 Barkan L, Vijayan P, Carlsson AS, Mekhedov S, Browse J (2006). A suppressor of fab1 challenges hypotheses on the role of thylakoid unsaturation in photosynthetic function. Plant Physiol 141, 1012-1020.
20 Byfield GE, Xue H, Upchurch RG (2006). Two genes from soybean encoding soluble Δ9 stearoyl-acp desaturas- es.Crop Sci 46, 840-846.
21 Craig W, Lenzi P, Scotti N, De Palma M, Saggese P, Carbone V, McGrath CN, Magee AM, Medgyesy P, Kavan- agh TA, Dix PJ, Grillo S, Cardi T (2008). Transplastomic tobacco plants expressing a fatty acid desaturase gene exhibit altered fatty acid profiles and improved cold tolerance.Transgenic Res 17, 769-782.
22 Du ZY, Bramlage WJ (1992). Modified thiobarbituric acid assay for measuring lipid oxidation in sugar-rich plant tissue extracts. J Agric Food Chem 40, 1566-1570.
23 James DW Jr, Dooner HK (1990). Isolation of EMS-induced mutants in Arabidopsis altered in seed fatty acid composition.Theor Appl Genet 80, 241-245.
24 Jung S, Tate PL, Horn R, Kochert G, Moore K, Abbott AG (2003). The phylogenetic relationship of possible progenitors of the cultivated peanut. J Hered 94, 334-340.
25 Kachroo A, Shanklin J, Whittle E, Lapchyk L, Hildebrand D, Kachroo P (2007). The Arabidopsis stearoyl-acyl carrier protein-desaturase family and the contribution of leaf isoforms to oleic acid synthesis.Plant Mol Biol 63, 257-271.
26 Krause GH, Weis E (1991). Chlorophyll fluorescence and photosynthesis: the basics.Ann Rev Plant Physiol Plant Mol Biol 42, 313-349.
27 Lightner J, James DW Jr, Dooner HK, Browse J (1994a). Altered body morphology is caused by increased stearate levels in a mutant of Arabidopsis.Plant J 6, 401-412.
28 Lightner J, Wu JR, Browse J (1994b). A mutant of Arabidopsis with increased levels of stearic acid. Plant Physiol 106, 1443-1451.
29 Lutts S, Kinet JM, Bouharmont J (1996). Nacl-induced senescence in leaves of rice (Oryza sativa L.) cultivars differing in salinity resistance. Ann Bot 78, 389-398.
30 Murata N, Wada H (1995). Acyl-lipid desaturases and their importance in the tolerance and acclimatization to cold of cyanobacteria. Biochem J 308, 1-8.
31 Shilman F, Brand Y, Brand A, Hedvat I, Hovav R (2011). Identification and molecular characterization of homeologousΔ9-stearoyl acyl carrier protein desaturase 3 genes from the allotetraploid peanut(Arachis hypogaea). Plant Mol Biol Rep 29, 232-241.
32 Tasseva G, de Virville JD, Cantrel C, Moreau F, Zacho- wski A (2004). Changes in the endoplasmic reticulum lipid properties in response to low temperature in Brassica napus. Plant Physiol Biochem 42, 811-822.
33 Thompson GA, Scherer DE, Foxall-Van Aken S, Kenny JW, Young HL, Shintani DK, Kridl JC, Knauf VC (1991). Primary structures of the precursor and mature forms of stearoyl-acyl carrier protein desaturase from safflower embryos and requirement of ferredoxin for enzyme activity.Proc Natl Acad Sci USA 88, 2578-2582.
34 Uemura M, Steponkus PL (1997). Effect of cold acclimation on the lipid composition of the inner and outer membrane of the chloroplast envelope isolated from rye leaves.Plant Physiol 114, 1493-1500.
35 Whittle E, Cahoon EB, Subrahmanyam S, Shanklin J (2005). A multifunctional acyl-acyl carrier protein desaturase from Hedera helix L.(English ivy) can synthesize 16- and 18-carbon monoene and diene products. J Biol Chem 280, 28169-28176.
36 Yukawa Y, Takaiwa F, Shoji K, Masuda K, Yamada K (1996). Structure and expression of two seed-specific cDNA clones encoding stearoyl-acyl carrier protein desaturase from sesame,Sesamum indicum L. Plant Cell Phy- siol 37, 201-205.
37 Zhang P, Burton JW, Upchurch RG, Whittle E, Shanklin J, Dewey RE (2008). Mutations in a Δ9-stearoyl-acpdes- aturase gene are associated with enhanced stearic acid levels in soybean seeds.Crop Sci 48, 2305-2313.
[1] Liu Dongfeng, Tang Yongyan, Luo Shengtao, Luo Wei, Li Zhitao, Chong Kang, Xu Yunyuan. Identification of Chilling Tolerance of Rice Seedlings by Cold Water Bath [J]. Chin Bull Bot, 2019, 54(4): 509-514.
[2] Li Ma, Wancang Sun, Jinhai Yuan, Zigang Liu, Junyan Wu, Yan Fang, Yaozhao Xu, Yuanyuan Pu, Jing Bai, Xiaoyun Dong, Huili He. Expression Analysis of β-1,3-Glucanase Gene from Winter Brassica rapa Under Low Temperature Stress [J]. Chin Bull Bot, 2017, 52(5): 568-578.
[3] WU Hui, DAI Hai-Fang, ZHANG Ju-Song, JIAO Xiao-Ling, LIU Cui, SHI Jun-Yi, FAN Zhi-Chao, and ALIYAN?Rouzi. Responses of photosynthetic characteristics to low temperature stress and recovery treatment in cotton seedling leaves [J]. Chin J Plan Ecolo, 2014, 38(10): 1124-1134.
[4] Li Chen, Lei Shi, Hongxia Cui, Aiying Zhang, Deshan Zhang, Ling Wang, Fei Xia. The Response of Flowering Phenology and Growth of Viburnum to Interannual Weather Fluctuation in an Introduction habitat [J]. Chin Bull Bot, 2012, 47(6): 645-653.
[5] LIU Bin-Yang, LIU Wei-Qiu, ZHANG Yi-Shun, LEI Chun-Yi. Physiological responses of bryophytes experienced low temperature stress to simulated nitrogen deposition [J]. Chin J Plan Ecolo, 2011, 35(3): 268-274.
[6] Xingfu Yan;Min Cao. Influence of Light and Temperature on the Germination of Shorea wantianshuea (Dipterocarpaceae) Seeds [J]. Chin Bull Bot, 2006, 23(6): 642-650.
[7] JIANG Guang-Hua, MENG Ya-Li, CHEN Bing-Lin, BIAN Hai-Yun, ZHOU Zhi-Guo. EFFECTS OF LOW TEMPERATURE ON PHYSIOLOGICAL MECHANISMS OF COTTON FIBER STRENGTH FORMING PROCESS [J]. Chin J Plan Ecolo, 2006, 30(2): 335-343.
[8] LU Zhan-Gen, ZHOU Wen-Jie, ZHAO Chang-Qiong, CHEN Jing, TAN Feng. Studies on the Adaptation of Taxus media cv. Hicksii to Natural Temperature Reduction [J]. Chin J Plan Ecolo, 2004, 28(1): 73-77.
[9] WANG Qiang, WEN Xiao-Gang, LU Cong-Ming, ZHANG Qi-De. Photosynthetic Functions of Different Senescing Leaves in the Canopy of Super high-yield Rice ‘Hua-An3’ [J]. Chin J Plan Ecolo, 2004, 28(1): 39-46.
[10] DAI Yu-Hua LIU Xun-Yan MENG Qing-Wei ZHAO Shi-Jie. Effect of Low Temperature on Lipid Metabolism of Thylakoid Membrane [J]. Chin Bull Bot, 2004, 21(04): 506-511.
[11] YAN Ying-Cai LIN Hong-Hui LIANG Hou-Guo ZHANG Nian-Hui. Comparison of the Effects of Different Low Temperature Stresses on the Induction of the Cyanide-Resistant Alternative Pathway and the Expression of Alternative Oxidase in Tobacco Callus [J]. Chin Bull Bot, 2004, 21(03): 296-305.
[12] YI Hua-Lin and DENG Xiu-Xin. Advances in the Techniques for in vitro Preservation of Plant Gernaplasm [J]. Chin Bull Bot, 1999, 16(05): 574-581.
[13] Yang Shengchang, Lin Peng. Ecological Studies on the Resistance and Adaptation to Cold of Some Tidal Mangrove Species In China [J]. Chin J Plan Ecolo, 1998, 22(1): 60-67.
[14] SUN De-Lan;WANG Hong and JIAN Ling-Cheng. The Stabilizaton on the Plasmalemma Calcium-Pump( Ca2 + -ATPase) in Winter Wheat Seedlings by the Cold-Resistant Agent CR-4 [J]. Chin Bull Bot, 1998, 15(02): 50-54.
Viewed
Full text


Abstract

Cited

  Shared   
  Discussed   
[1] Qin Wei-cheng Li Jian-zhong. The Application Effects of the Cold-resister CR-4 in Our Area's Rice Seedling Culture[J]. Chin Bull Bot, 1994, 11(特辑): 102 -104 .
[2] Ningguang Dong, Ying Gao, Wei Wang, Weilun Yin, Dong Pei. Immunogold Silver Localization of Indole-3-acetic Acid (IAA) During the Rhizogenesis of In Vitro Poplar[J]. Chin Bull Bot, 2011, 46(3): 324 -330 .
[3] HONG Wei CAO Jia-Shu. The Function of FLC in Vernalization Process[J]. Chin Bull Bot, 2002, 19(04): 406 -411 .
[4] . Development and Utilization of Plant Resources II[J]. Chin Bull Bot, 1994, 11(02): 53 -57 .
[5] FAN Qing-Shu ZHAO Jian-Cheng YU Shu-Hong LI Xiu-Qin. Progress in Study on Spore Germination and Protonema Development of the Bryophytes[J]. Chin Bull Bot, 2003, 20(03): 280 -286 .
[6] LIU Jian-Wu LIU Ning. The Progress in Study on Development of Fern Gametophytes and Differentiation of Sex Organ[J]. Chin Bull Bot, 2001, 18(02): 149 -157 .
[7] An Cheng-xi. Studies on the Chemical Constituents of Essential of Aiania-Tanuifolia[J]. Chin Bull Bot, 1997, 14(增刊): 74 -76 .
[8] Nie Wei. Observation on some Biological Characteristics of Juncellus serotinus in Transplanted Rice[J]. Chin Bull Bot, 1988, 5(01): 34 -36 .
[9] . Mechanism of Plant Photosynthetic Acclimation to Elevated Atmospheric CO2[J]. Chin Bull Bot, 2005, 22(04): 486 -493 .
[10] WEI Ze-Xiu, LIANG Yin-Li, YAMADA Satoshi, ZENG Xing-Quan, ZHOU Mao-Juan, HUANG Mao-Lin, WU Yan. RELATION OF SOIL MICROBIAL DIVERSITY TO TOMATO YIELD AND QUALITY UNDER DIFFERENT SOIL WATER CONDITIONS AND FERTILIZATIONS[J]. Chin J Plan Ecolo, 2009, 33(3): 580 -586 .