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.
[1]韩丽珍, 李翠翠, 赵德刚.菌株, -降解基因生物信息学分析[J].基因组学与应用生物学, 2014, 33(3):478-486[2]李翠翠, 韩丽珍, 赵德刚.转基因提高黑麦草抗除草剂, -能力[J].分子植物育种, 2015, 13(9):2076-2082[3]李平, 白云凤, 冯瑞云, 等.籽粒苋苹果酸酶-基因密码子偏好性分析[J].应用与环境生物学报, 2011, 17(1):12-17[4]Bayley C, Trolinder N, Ray C, et al.Engineering 2,4-D resistance into cotton[J].Theoretical and Applied Genetics, 1992, 83(5):645-649[5]Bhat SV, Booth SC, McGrath SG, et al.Rhizobium leguminosarum bvViciae 3841 adapts to 2,4-dichlorophenoxyacetic acid with “Auxin-like” morphological changes,cell envelope remodeling and upregulation of central metabolic pathways[J].PLoS One, 2014, 10(4):e0123813-[6] Chinalia FA, Regali-Seleghin MH, Correa EM.2, 4-D toxicity: cause, effects and control[C]. .Terrestrial and Aquatic Environmental Toxicology ?2007 Global Science Books, 2007, 1(2):24-33[7]Crespin MA, Gallego M, Valcárcel M, et al.Study of the degradation of the herbicides 2,4-D and MCPA at different depths in contaminated agricultural soil[J].Environmental Science and Technology, 2001, 35(21):4265-4270[8]De AK, Dey N, Adak MK.Bio indices for 2,4-D sensitivity between two plant species: Azolla pinnata RBr and Vernonia cinerea Lwith their cellular responses[J].Physiology and Molecular Biology of Plants, 2016, 22(3):371-380[9]Griffin SL, Godbey JA, Oman TJ, et al.Characterization of aryloxyalkanoate dioxygenase-12,a nonheme Fe(II)a-ketoglutarate-dependent dioxygenase,expressed in transgenic soybean and Pseudomonas fluorescens[J].Journal of Agricultural and Food Chemistry, 2013, 61(27):6589-6596[10]Grossmann K.Mediation of Herbicide Effects by Hormone Interactions[J].Journal of Plant Growth Regulation, 2003, 22(1):109-122[11]Grossmann K.Auxin Herbicide Action[J].Plant Signaling and Behavior, 2007, 2(5):421-423[12]Grossmann K.Auxin herbicides: current status of mechanism and mode of action[J].Pest Management Science, 2010, 66(2):113-120[13]Han L, Zhao D, Li C.Isolation and 2,4-D-degrading characteristics of Cupriavidus campinensis BJ71[J].Brazilian Journal of Microbiology, 2015, 46(2):433-441[14]Han L, Liu Y, He A, et al.S rRNA gene phylogeny and tfdA gene analysis of 2,4-D-degrading bacteria isolated in China[J].World Journal of Microbiology and Biotechnology, 2014, 30(10):2567-2576[15]Han L, Liu Y, Li C, et al.Cloning,expression,characterization and mutational analysis of the tfdA gene from Cupriavidus campinensis BJ71[J].World Journal of Microbiology and Biotechnology, 2015, 31(7):1021-1030[16]Hoover DM, Lubkowski J.DNAWorks: an automated method for designing oligonucleotides for PCR-based gene synthesis[J].Nucleic Acids Research, 2002, 30(10):e43-[17]Kim YH, Lim S, Han SH, et al.Expression of both CuZnSOD and APX in chloroplasts enhances tolerance to sulfur dioxide in transgenic sweet potato plants[J].Comptes Rendus Biologies, 2015, 338(5):307-313[18]Laurent F, Debrauwer L, Rathahao E, et al.Dichlorophenoxyacetic acid metabolism in transgenic tolerant cotton (Gossypium hisutum)[J].Journal of Agricultural and Food Chemistry., 2000, 48(11):5307-5311[19]Laurent F, Debrauwer L, Pascal-Lorber S.Metabolism of [14C]-2,4-dichlorophenol in edible plants[J].Pest Management Science, 2006, 62(6):558-564[20]Lyon BR, Cousins YL, Llewellyn DJ, et al.Cotton plants transformed with a bacterial degradation gene are protected from accidental spray drift damage by the herbicide 2,4-dichlorophenoxyacetic acid[J].Transgenic Research, 1993, 2(3):162-169[21]Müller RH.Activity and reaction mechanism of the initial enzymatic step specifying the microbial degradation of 2,4-dichlorophenoxyacetate[J].Engineering Life Science, 2007, 7(4):311-321[22]Müller TA, Byrde SM, Fleischmann T, et al.Genetic analysis of phenoxyalkanoic acid degradation in Sphingomonas herbicidovorans MH[J].Applied and Environmental Microbiology, 2004, 70(10):6066-6075[23]Müller TA, Fleischmann T, van der Meer JR, et al.Purification and characterization of two enantioselective α-ketoglutarate-dependent dioxygenases,RdpA and SdpA,from Sphingomonas herbicidovorans MH[J].Applied and Environmental Microbiology, 2006, 72(7):4853-4861[24]Pazmi?o DM, Romero-Puertas MC, Sandalio LM.Insights into the toxicity mechanism of and cell response to the herbicide 2,4-D in plants[J].Plant Signaling & Behavior, 2012, 7(3):425-427[25]Saygideger SD, Okkay O.Effect of 2,4-dichlorophenoxyacetic acid on growth,protein and chlorophyll-a content of Chlorella vulgaris and Spirulina platensis cells[J].Journal of Environmental Biology, 2008, 29(2):175-178[26]Schleinitz KM, Kleinsteuber S, Vallaeys T, et al.Localization and characterization of two novel genes encoding stereospecific dioxygenases catalyzing 2(2,4-dichlorophenoxy)propionate cleavage in Delftia acidovorans MC1[J].Applied and Environmental Microbiology, 2004, 70(9):5357-5365[27]Streber WR, Willmitzer L.Transgenic tobacco plants expressing a bacterial detoxifying enzyme are resistant to 2,4-D[J].Nature Biotechnology, 1989, 7(8):811-816[28]Wong PK.Effects of 2,4-D,glyphosate and paraquat on growth,photosynthesis and chlorophyll-a synthesis of Scenedesmus quadricauda Berb 614[J].Chemosphere, 2000, 41(1-2):177-182[29]Wright TR, Shan G, Walsh TA, et al.Robust crop resistance to broadleaf and grass herbicides provided by aryloxyalkanoate dioxygenase transgenes[J].Proceedings of the National Academy of Sciences of the United States of America (PNAS), 2010, 107(47):20240-20245
