Scientia Agricultura Sinica ›› 2017, Vol. 50 ›› Issue (20): 3860-3870.doi: 10.3864/j.issn.0578-1752.2017.20.003


Salt and Drought Tolerance in Heterologous-Expression of irrE Transgenic Tobacco

ZHAO LeiLin, FAN Xin, NIE Xing, LIANG ChengZhen, ZHANG Rui, SUN GuoQing, MENG ZhiGang, LIN Min, WANG Yuan, GUO SanDui   

  1. Biotechnology Research Institute, Chinese Academy of Agricultural Sciences, Beijing 100081
  • Received:2017-03-30 Online:2017-10-16 Published:2017-10-16

Abstract: 【Objective】 IrrE is a protein found in Deinococcus radiodurans, and it process the transcription regulating function mainly through repairing DNA damage in conditions of strong radiations and other adverse environments. It is of great significance to study the tolerance of optimized irrE against drought and salt in transgenic plants and to lay a theoretical basis of crops resistance breeding.【Method】The bacteria irrE codons were optimized with the plant preferences and the gene was synthesized. Efficient plant expression vector was constructed by using the method of enzyme digestion and connection. Leaf disc method was used for transforming tobacco, and the positive regenerated seedlings were screened with PCR. The expression level of irrE in transgenic strain was analyzed. The IrrE protein was tested using Western blot. Pure transgenic generations were recruited and treated with the mannitol and NaCl treatments, which simulated drought and salt conditions. Physiological tests were conducted to determine drought and salt tolerance.【Result】The irrE was modified according to plant codes, and 241 codes were optimized. The efficient plant expression vector GBI-IE was constructed. The regenerated seedlings were screened by herbicide glyphosate, and 15 independent transgenic strains were obtained using PCR, and two highest expression lines, GO1 and GO2, were used for subsequent analysis of tolerance under adverse environmental conditions. Western blot proved that protein IrrE encoded in GO1 and GO2 could be translated correctly. Salt and drought tolerance of transgenic tobacco was analyzed. Germinating experiments displayed that there was no obvious difference between transgenic lines (GO1 and GO2) and non-GMO wild type (WT) in terms of germination rate in 1/2 MS culture medium, however, in medium containing 250 mmol·L-1 NaCl, germination rate of GO1 and GO2 was 78.8% and 90.0%, respectively, while the one of the WT was 10.3%, increased by 68.5% and 79.7%, respectively. Similarly, germination rate of WT was 39.7% in medium containing 300 mmol·L-1 mannitol, the rate of GO1 and GO2 increased by 42.9% and 50.8%, respectively. Normally germinated seedlings were transplanted into 250 mmol·L-1 NaCl and 300 mmol·L-1 mannitol environment for 12 days, and root length, lateral roots number and fresh weight of those two transgenic lines were markedly higher than the ones of WT. Tobacco seedlings of 30 days normal growth in the greenhouse were treated with 250 mmol·L-1 NaCl of which transgenic tobacco SOD and CAT activity was more 48.80% and 88.55% than WT, respectively, and the MDA content was less 61.61% than WT, In addition, the expression level of drought responsive gene in GO1 and GO2, like NtABF2, NtLEA5, Ntzfp, NtCDPK2, was significantly higher than WT. Similarly, the changes of physiological and biochemical indexes in transgenetic lines in 300 mmol·L-1 mannitol treatment were higher than the ones in WT.【Conclusion】Expression of irrE from D. radiodurans remarkably improved the tolerance of tobacco to drought and salt stresses in which the pleiotropy could be beneficial gene resource of gene engineering about resistance to stress in plant.

Key words: codon optimization; irrE, transgenic tobacco, drought and salt tolerance

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