Abstract：2,4-Dichlorophenoxyacetic acid (2,4-D) is a phenoxy herbicides that has been used to control broadleaf weeds in cereal and grass crops for over 70 years. When applied to dicotyledonous plants at effective doses, 2,4-D causes uncontrolled and disorganized plant growth that leads to death. It's necessary to improve 2,4-D resistance of sensitive plant. In soil, this herbicide is regarded as readily biodegradable and mostly depended on microbial degradation. A lot of degrading bacteria contain 2,4-D-catabolizing enzyme, which encodes an Fe (II)/α-ketoglutarate-dependent dioxygenase, could convert 2,4-D into 2,4-dichlorophenol as an initial step of 2,4-D mineralization. In our previous study, an effective 2,4-D-degrading strain, identified as Cupriavidus campinensis BJ71, was isolated from a wheat soil with a long-term history of 2,4-D use. And 2,4-D-degrading gene, named as cctfdA, was cloned from this strain BJ71. This gene's full length is 864 bp and encodes 287 amino acids. In this study, using cctfdA as model, man-made Nt-cctfdA with tobacco-preferred codon usage was designed and synthesized. An expression vector named pSH737-Nt-cctfdA, containing Nt-cctfdA gene, under control of a constitutive 35S promoter, was constructed. Two expression vector pSH737-Nt-cctfdA and pSH737 were introduced into Nicotiana tobacum through Agrobacterium tumefaciens-mediated gene delivery. 15 regenerated tobaccos resisted to 2,4-D contained pSH737-Nt-cctfdA and 10 genetic lines contained pSH737 were obtained. T1 generation plants were selected with kanamycin resistance and analyzed by PCR for presence of this gene. A series of three experiments were carried out to study 2,4-D resistance. Firstly, when leaves in vitro were soaked with 250 mg/L 2,4-D, pSH737-Nt-cctfdA transgenic tobacco leaves remained green and intact, whereas wild-type and pSH737 transgenic leaves were injured severely after seven days. Secondly, tobacco leaf discs were punched aseptically from fresh leaves and plated on shoot regeneration media containing different 2,4-D concentration and incubated for three weeks. Wild-type tobacco leaf discs generated two shoots on levels of 2,4-D up to 0.4 mg/L and pSH737 genetic plants generated one shoot on 0.6 mg/L concentration, whereas shoots rate of pSH737-Nt-cctfdA transgenetic tobacco were 58.97% on callus induction medium containing 8 mg/L, representing a 30-fold increase in tolerance to this herbicide compared with the control. Finally, when different concentrations of 2,4-D were sprayed on young plants, wild-type and pSH737 transgenic young plants were killed on 350 mg/L concentration. In contrast, transgenic plants expressing Nt-cctfdA exhibited no visible signs of 2,4-D damage, even when treated with up to 10 000 mg/L of the herbicide. It showed that 2,4-D resistant levels in transgenic pSH737-Nt-cctfdA tobacco was extremely exceeded 30 times than the control and was 10 times than the usual field application rate (P＜0.05). When plants were sprayed with 2,4-D, chlorophyll content was not changed basically in Nt-cctfdA transgenic plants, whereas it was obviously decreased in cotrol group (wild-type and pSH737 genetic plants, too). In conclusion, cctfdA gene from Cupriavidus campinensis BJ71 could obviously improve 2,4-D tolerance in transgenic tobacco. And the results could help us to illuminate 2,4-D resistance mode clearly and provide basic data for further studying 2,4-D resistance transgenic tobacco.
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