Welcome to Acta Agronomica Sinica，

Acta Agron Sin ›› 2009, Vol. 35 ›› Issue (7): 1253-1360.doi: 10.3724/SP.J.1006.2009.01253

• CROP GENETICS & BREEDING · GERMPLASM RESOURCES · MOLECULAR GENETICS • Previous Articles     Next Articles

Codl Tolerance of Transplastomic Tobacco Lines Carrying Insect Antifreeze Protein

WANG Yan,MA Ji*,HUANG Wei,QIU Li-Ming,YE Feng,ZHANG Fu-Chun   

  1. Xinjiang Key Laboratory of Biological Resources and Genetic Engineering,College of Life Science and Technoogy,Xinjiang University,Urumqi 830046,China
  • Received:2008-09-05 Revised:2009-02-18 Online:2009-07-12 Published:2009-05-19
  • Contact: MA Ji, E-mail: majiuci@xju.edu.cn; Tel: 0991-8583259
  • About author:bluelovewy@126.com

PDF

2271

Abstract:

The antifreeze protein gene MpAFP149 from desert insect Microdera punctipennis dzungarica was inserted into soybean chloroplast vector pJY01 to construct recombinant chloroplast vector pJY01-MpAFP149 by designing special primers. The plasmid was then transformed into tobacco by gene gun. Four transplastomic tobacco lines were obtained by spectinomycin screening. PCR and PCR-Southern analysis showed that the MpAFP149 gene was successfully integrated into the tobacco chloroplast genome, but the transgenicplants exhibited low homoplasmy. The result of RT-PCR also validated that MpAFP149 gene was transcribed at mRNA level. The antifreeze effect of transplastomic tobacco with insect antifreeze protein gene in its chloroplasts was evaluated by measuring the relative conductivity and comparing the phenotypes of different plants after cold treatment. Wild-type tobacco, transplastomic tobacco and T1 generation of transgenic tobacco containing pCAMBIA1302-MpAFP149 in its nuclei were subjected to –1℃ for different days (0, 1, 2, and 3 d). The results showed that transgenic plants with insect antifreeze protein gene in chloroplasts or in nuclei performed better phenotype after cold treatment at –1℃ for three days and recovering at room temperature for five days than wild-type tobacco. After three days, the electrolyte leakage reached 73.7% for wild-type tobacco, 39.2% for chloroplast transgenic plants and 38.2% for nuclei transformed T1 generation tobacco. There was no cold tolerance difference between nuclei transgenic tobacco and heterogeneous tansplastomic tobacco in our research.

Key words: Antifreeze protein gene MpAFP149, Chloroplast transformed vector, Tobacco, Cold tolerance


[1] Jack A, Hill P G, Dodd C E, Layboum-Parry J. Demonstration of antifreeze protein activity in Antarctic lake bacteria. Microbiology, 2004, 50: 171-180

[2] Stefen P G, Brian D. Cold survival in freeze intolerant insects: the structure and function of beta-helical antifreeze proteins. Ear J Biochem, 2004, 271: 3285-3296

[3] Yang Z Y, Zhou Y X, Liu K, Cheng Y H, Liu R Z, Chen G J, Jia Z C. Computational study on the function of water within a B-helix antifreeze protein dimer and in the process of ice-protein binding. Biophy J, 2003, 85: 2599-2605

[4] Liou Y C, Thibauh P, Walker V K, Davies P L, Graham L A. A complex family of highly heterogeneous and internally repetitive hyperactive antifreeze proteins from the beetle Tenebrio molitor. Biochemistry, 1999, 38: 11415-11424

[5] Kenward K D, Brandle J, McPherson J, Davies P L. Type II fish antifreeze protein accumulation in transgenic tobacco does not confer frost resistance. Transgen Res, 1999, 8: 105-117

[6] Huang Y-F(黄永芬), Wang Q-Y(汪清胤), Fu G-R(付桂荣), Zhao X-X(赵晓祥), Yang Z-X(杨志兴). The research on introducing flounder antifreeze protein gene (afp) into tomato. Chin Biochem J (生物化学杂志), 1997, 13(4): 418-422(in Chinese with English abstract)

[7] Georges F, Saleem M, Cutler A J. Design and cloning of a synthesis gene for the flounder antifreeze protein and its expression in plant cells. Gene, 1990, 91: 159-165

[8] Hightower R, Cathy B, Ranela D. Expression of antifreeze proteins in transgenic plants. Plant Mol Biol, 1991, 17: 1013-1021

[9] Doucet D, Tyshenko M G, Davies P L, Walker V K. A family of expressed antifreeze protein genes from the moth, Choristoneura fumiferana. Eur J Biochem, 2002, 269: 38-46

[10] Huang T, Nicodemus J, Zarka D G, Thomashow M F, Wisniewski M, Duman G. Expression of an insect (Dendroides canadensis) antifreeze protein in Arabidopsis thaliana results in a decrease in plant freezing temperature. Plant Mol Biol, 2002, 50: 333-342

[11] Wang Y(王艳), Qiu L-M(邱立明), Xie W-J(谢文娟), Huang W(黄薇), Ye F(叶锋), Zhang F-C(张富春), Ma J(马纪). Cold tolerance of transgenic tobacco carrying gene encoding insect antifreeze protein. Acta Agron Sin (作物学报), 2008, 34(3): 397-402(in Chinese with English abstract)

[12] Wang Y, Qiu L M, Dai C Y, Wang J, Luo J M, Zhang F C, Ma J. Expression of insect (Microdera punctipennis dzungarica) antifreeze protein MpAFP149 confers the cold tolerance to transgenic tobacco. Plant Cell Rep, 2008, 27: 1349-1358

[13] Daniell H, Khan M S, Alison L. Milestones in chloroplast genetic engineering: An environmentally friendly era in biotechnology. Trends Plant Sci, 2002, 7: 84-91

[14] Daniell H. Molecular strategies for gene containment in transgenic crops. Nat Biotechnol, 2002, 20: 581-586

[15] Daniell H, Chebolu S, Kumar S, Singleton M, Falconer R. Chloroplast-derived vaccine antigens and other therapeutic proteins. Vaccine, 2005, 23: 1779-1783

[16] Lee S B, Kwon H B, Kown S J, Park S C, Jeong M J, Han S E, Byun M O, Daniell H. Accumulation of trehalose within transgenic chloroplasts confers drought tolerance. Mol Breed, 2003, 11: 1-13

[17] Boynton J E, Gillham N W, Harris E H, Hosler J P, Johnson A M, Jones A R, Randolph-Anderson B L, Robertson D, Klein T M. Chloroplast transformation in chlamydomonas with high velocity microprojectiles. Science, 1988, 240: 1534-1538

[18] Lutz K A, Knapp J E, Maliga P. Expression of bar in the plastid genome confers herbicide resistance. Plant Physiol, 2001, 125: 1585-1590

[19] Su N(苏宁), Yang B(杨波), Meng K(孟昆), Li Y-N(李佚女), Sun M(孙萌), Sun B-Y(孙丙耀), Shen G-F(沈桂芳). The research of Bt and OC Gene Cotransformation in tobacco chloroplast. Sci Agric Sin (中国农业科学), 2002, 35(4): 394-398(in Chinese with English abstract)

[20] DeGray G, Rajasekaran K, Smith F, Sanford J, Daniell H. Expression of an antimicrobial peptide via the chloroplast genome to control phytopathogenic bacteria and fungi. Plant Physiol, 2001, 127: 852-862

[21] Watson J, Koya V, Leppla S H, Daniell H. Expression of Bacillus anthracis protective antigen in transgenic chloroplasts of tobacco, a non-food/feed crop. Vaccine, 2004, 22: 4374-4384

[22] Kang T J, Loc N H, Jang M O, Jang Y S, Kim Y S, Seo J E, Yang M S. Expression of the B subunit of E. coli heat-labile enterotoxin in the chloroplasts of plants and its characterization. Transgen Res, 2003, 12: 683-691

[23] Erickson J M. Chloroplast Transformation: Current Results and Future Prospects. In: Donald R O, Charles F Y, eds. Oxygenic Photosynthesis, the Light Reaction. Dordrecht: Kluwer Academic Publishers, 1996. pp 589-619

[24] Bendich A J. Why the chloroplast and mitochondria contain so many copies of their genome. Bioessays, 1987, 6: 279-282

[25] Chen S(陈飒), Li J-Y(李金耀), Wu P(吴平), Sun H(孙寰), Zhao L-M(赵丽梅), Shou H-X(寿惠霞). Construction of soybean chloroplast transformation vector pJY series and its transformation in tobacco. J Agric Biotechnol (农业生物技术学报), 2007, 15(6): 899-904(in Chinese with English abstract)

[26] Sambrook J, Fritsch E F, Maniatis T. Molecular Cloning: A Laboratory Manual. Beijing: Science Press, 1999. pp 478-481(in Chinese)

[27] Svab Z, Maliga P. High-frequency plastid transformation in tobacco by selection for a chimeric aadA gene. Proc Natl Acad Sci USA, 1993, 90: 913-917

[28] Michaels S D, John M C, Amasino R M. Removal of polysaccharides from plant DNA by ethanol precipitation. Biotechniques, 1994, 17: 247-276

[29] Zhao S-J(赵世杰), Shi G-A(史国安), Dong X-C(董新纯). Guidelines of Plant Physiological Experiment (植物生理学实验指导). Beijing: China Agricultural Scientech Press, 2002. pp 84-131(in Chinese)

[30] Bock R. Analysis of RNA editiong in plastids. Methods, 1998, 15: 75-83

[31] Kuroda H, Maliga P. Complementarity of the 16S rRNA penultimate stem with sequences downstream of the AUG destabilizes the plastid mRNAs. Nucl Acids Res, 2001, 29: 970-975
[1] Yang Yue, Zhang Xin-Xin, He Zeng-Hui, Li Rui-Dong, Pan Yu-Jie, Li Jia-Kang, Du Wei, Xu Da-Yong, Du Jin-Song. Non-destructive prediction and visualization of major chemical components in tobacco leaves using hyperspectral imaging [J]. Acta Agronomica Sinica, 2026, 52(3): 922-935.
[2] Zhan Ge-Rui, Yu Wen, Li Feng, Wu Ming-Zhu, Xu Xin, Luo Zhao-Peng, Wu Sheng-Xin, Yang Jun, Zhang Zhi-Qiang, Wang Zhong. Functional study of NtWRKY6 in response to ABA expression and regulation of polyphenol synthesis [J]. Acta Agronomica Sinica, 2026, 52(2): 446-458.
[3] Kong Na, Liu Tao, Liu Wen-Ting, Chen Gang, Wen Li-Chao, Deng Zhi-Chao, Guo Mei, Li Wei, Guo Yong-Feng. Cloning of the NtCEP7 gene in tobacco and functional analysis of its encoded peptide in seedling-stage drought resistance [J]. Acta Agronomica Sinica, 2026, 52(1): 249-261.
[4] JI Bai-Lu, SUN Yi-Wen, LIU Wan-Feng, QIAN Ya-Xin, JIANG Cai-Hong, GENG Rui-Mei, LIU Dan, CHENG Li-Rui, YANG Ai-Guo, HUANG Li-Yu, LI Xiao-Xu, PU Wen-Xuan, GAO Jun-Ping, ZHANG Qiang, WEN Liu-Ying. Functional verification of the key gene NtLPAT involved in lipid biosynthesis in tobacco [J]. Acta Agronomica Sinica, 2025, 51(9): 2527-2537.
[5] WAN Hui-Lan, WU Hua-Ying, ZENG Dan, QIAN Zhen-Feng, ZHAO Chang-Zu, LIAO Ran-Chao, HE Li-Lian, LI Fu-Sheng. Cloning analysis and functional validation of EfWRKY51 gene related to cold tolerance in Erianthus fulvus [J]. Acta Agronomica Sinica, 2025, 51(8): 2048-2059.
[6] HOU Tian-Yu, DU Xiao-Jing, ZHAO Zhi-Qiang, REYIM Anwar, YIDAYETULA Abula, BUHALIQIEMU Abulizi, YUAN Jie, ZHANG Yan-Hong, WANG Feng-Bin. Evaluation of cold tolerance of japonica rice varieties at germination stage and construction of identification system [J]. Acta Agronomica Sinica, 2025, 51(3): 812-822.
[7] LIU Bo, CHI Ming, CAO Meng-Qi, TANG Da, YANG Heng-Zhao, ZHANG Wei-Hua, XUE Cong. Impact of potato StuPPO9 gene overexpression on drought resistance in Nicotiana benthamiana [J]. Acta Agronomica Sinica, 2024, 50(9): 2237-2247.
[8] LIU Ying-Chao, FANG Dun-Huang, XU Hai-Ming, TONG Zhi-Jun, XIAO Bing-Guang. QTL mapping of alkaloids in tobacco [J]. Acta Agronomica Sinica, 2024, 50(1): 42-54.
[9] WEN Li-Chao, XIONG Tao, DENG Zhi-Chao, LIU Tao, GUO Cun, LI Wei, GUO Yong-Feng. Expression and functional characterization of NtNAC080 transcription factor gene from Nicotiana tabacumin under abiotic stress [J]. Acta Agronomica Sinica, 2023, 49(8): 2171-2182.
[10] CUI Fang-Fang, MENG Lin-Feng, LIU Miao-Miao, ZHANG Jian-Qiang, WANG Jian-Ge, LIU Qi-Yuan. Characteristics of MADS-box and SUPERMAN genes in tobacco cytoplasmic male sterile line K326 [J]. Acta Agronomica Sinica, 2023, 49(12): 3204-3214.
[11] CAO Hui-Min, YANG Xian-Li, WANG Li-Zhi, LI Ping-Ping, ZHAI Lai-Yuan, JIANG Shu-Kun, ZHENG Tian-Qing, QIU Xian-Jin, XU Jian-Long. QTL identification and favorable allele mining of cold tolerance at seedling stage by reciprocal introgression and recombinant inbred line populations in rice [J]. Acta Agronomica Sinica, 2023, 49(10): 2633-2642.
[12] ZHANG He, JIANG Chun-Ji, YIN Dong-Mei, DONG Jia-Le, REN Jing-Yao, ZHAO Xin-Hua, ZHONG Chao, WANG Xiao-Guang, YU Hai-Qiu. Establishment of comprehensive evaluation system for cold tolerance and screening of cold-tolerance germplasm in peanut [J]. Acta Agronomica Sinica, 2021, 47(9): 1753-1767.
[13] LI Peng, LIU Che, SONG Hao, YAO Pan-Pan, SU Pei-Lin, WEI Yao-Wei, YANG Yong-Xia, LI Qing-Chang. Identification and analysis of non-specific lipid transfer protein family in tobacco [J]. Acta Agronomica Sinica, 2021, 47(11): 2184-2198.
[14] JIANG Shu-Kun,WANG Li-Zhi,YANG Xian-Li,LI Bo,MU Wei-Jie,DONG Shi-Chen,CHE Wei-Cai,LI Zhong-Jie,CHI Li-Yong,LI Ming-Xian,ZHANG Xi-Juan,JIANG Hui,LI Rui,ZHAO Qian,LI Wen-Hua. Detection of QTLs controlling cold tolerance at bud bursting stage by using a high-density SNP linkage map in japonica rice [J]. Acta Agronomica Sinica, 2020, 46(8): 1174-1184.
[15] LIU Qing-Li,JIANG Yu-Zhou,ZOU Yan,ZHANG Yun-Gui,ZHANG Heng,SHI Jun-Xiong,LI Zhi-Hong. The study of carbon budget on field-tobacco ecosystem [J]. Acta Agronomica Sinica, 2020, 46(8): 1258-1265.
Viewed
Full text


Abstract

Cited
  Shared   
  Discussed   
No Suggested Reading articles found!
Add: No. 80 Xueyuan South Rd, Beijing 100081, P. R. China E-mail: zwxb301@caas.cn
Supported by Beijing Magtech Co.Ltd