An Isopentyl Transferase Gene Driven by the Stress-Inducible rd29A Promoter Improves Salinity Stress Tolerance in Transgenic Tobacco

Abstract

Soil salinity is a serious worldwide problem. To improve the salt tolerance of plants, an increasing number of genes related to abiotic stress have been recently expressed by genetic engineers. In the present study, the successful introduction into tobacco of isopentenyl transferase (IPT) from Agrobacterium tumefaciens via Agrobacterium-mediated transformation is reported. A stress-inducible genetic construct was cloned using IPT under the control of the stress-inducible promoter rd29A from Arabidopsis thaliana. A total of 40 putative transgenic plant lines were obtained from independent Kan-resistant shoots. IPT integration into the tobacco genome was confirmed by polymerase chain reaction (PCR) and Southern blot analyses. Four positive transgenic lines each with a single T-DNA insertion were obtained. Real-time PCR confirmed a marked increase in IPT expression in young tobacco plants harboring rd29A-IPT after short-term exposure to salt. Ectopic IPT overexpression IPT under the control of the stress-inducible rd29A promoter resulted in significantly enhanced tolerance to salt stress. No obvious adverse effect on growth and development was observed in transgenic plants. Two IPT transgenic lines, T10 and T25, were chosen for further physiological analyses. The leaves of transgenic tobacco plants showed significantly prolonged chlorophyll retention times under a 2-week salt-stress treatment (150 mmol L−1). In contrast, the leaves of the non-transformed plants (wild type) gradually senesced under the same condition. After re-watering for 2 weeks, chlorophyll in transgenic plants increased to a level comparable with that in the unstressed plants. On the other hand, the level in the non-transgenic control still remained low. Malondialdehyde (MDA) levels increased in both transgenic plants and the control after salt stress. However, the MDA levels only mildly increased in transgenic plants, and dramatically increased in the control. After re-watering for 7 days, MDA in transgenic plants returned to normal, whereas the level in the control remained high. Superoxide dismutase activity also similarly increased in transgenic plants during salt stress, and returned to normal after re-watering. These results indicate that enhanced reactive oxygen species scavenging capability may play a significant role in acquiring tolerance to abiotic stress.