Transcriptomic analysis reveals the transcription factors involved in regulating the expression of EPSPS gene,which confers glyphosate resistance of goosegrass (Eleusine indica)

Glyphosate inhibits the enzyme 5-enolpyruvylshikimate-3-phosphate synthase(EPSPS)and overexpression of the EPSPS gene is one of the molecular mechanisms conferring glyphosate resistance in weeds.A regulatory sequence of EPSPS gene was isolated previously,and an alteration in its 5´-untranslated region(UTR)pyrimidine(Py)-rich stretch element is involved in the regulation of EPSPS expression in glyphosate-resistant(GR)Eleusine indica.However,the transcription factors involved in this regulatory sequence remain to be elucidated.In this study,we investigated the regulatory network of EPSPS overexpression associated genes in a GR E.indica population by RNA-seq.The differentially expressed transcript analyses revealed that glyphosate treatment caused an increase in the expression of 2752 unigenes and a decrease in the expression of 4025 unigenes in the GR E.indica,compared to the glyphosatesusceptible(GS)E.indica.Among them,1373 unigenes were identified to be co-expressed with the EPSPS gene in GR E.indica.GO and KEGG pathway analyses showed that the up-regulated unigenes were mainly enriched in chloroplasts and associated with the shikimate biosynthesis pathway,chlorophy II and peroxisome metabolism processes.Notably,the expression of a Shikimate kinase which catalyzed the conversion of Shikimate to Shikimate 3-phosphate(S3P,a substrate of EPSPS),was also up-regulated.Eight transcription factors were identified as likely to be involved in the regulation of the EPSPS expression,and three of them(ARF2,ARF8 and BPC6)showed more binding sites because of a(CT)n insertion of the 5´-UTR Py-rich stretch element in GR.However,the yeast one-hybrid assay illustrated that ARF8 and BPC6 could bind to the 5´-UTR Py-rich stretch element of wild type EPSPS,but could not bind to the mutated form.Our data suggests that the transcriptional regulation of EPSPS expression is complex and was significantly altered in GR E.indica.These discoveries provide new references for further study of the EPSPS overexpression mechanism that endows glyphosate resistance.

In maize,co-expression of GAT and GR79-EPSPS provides high glyphosate resistance,along with low glyphosate residues

Herbicide tolerance has been the dominant trait introduced during the global commercialization of genetically modified(GM)crops.Herbicide-tolerant crops,especially glyphosate-resistant crops,offer great advantages for weed management;however,despite these benefits,glyphosate-resistant maize(Zea mays L.)has not yet been commercially deployed in China.To develop a new bio-breeding resource for glyphosate-resistant maize,we introduced a codon-optimized glyphosate N-acetyltransferase gene,gat,and the enolpyruvyl-shikimate-3-phosphate synthase gene,gr79-epsps,into the maize variety B104.We selected a genetically stable high glyphosate resistance(GR)transgenic event,designated GG2,from the transgenic maize population through screening with high doses of glyphosate.A molecular analysis demonstrated that single copy of gat and gr79-epsps were integrated into the maize genome,and these two genes were stably transcribed and translated.Field trials showed that the transgenic event GG2 could tolerate 9000 g acid equivalent(a.e.)glyphosate per ha with no effect on phenotype or yield.A gas chromatography-mass spectrometry(GC–MS)analysis revealed that,shortly after glyphosate application,the glyphosate(PMG)and aminomethylphosphonic acid(AMPA)residues in GG2 leaves decreased by more than 90%compared to their levels in HGK60 transgenic plants,which only harbored the epsps gene.Additionally,PMG and its metabolic residues(AMPA and N-acetyl-PMG)were not detected in the silage or seeds of GG2,even when far more than the recommended agricultural dose of glyphosate was applied.The co-expression of gat and gr79-epsps,therefore,confers GG2 with high GR and a low risk of herbicide residue accumulation,making this germplasm a valuable GR event in herbicide-tolerant maize breeding.

Optimization of Agrobacterium tumefaciens-Mediated Immature Embryo Transformation System and Transformation of Glyphosate-Resistant Gene 2mG2-EPSPS in Maize(Zea mays L.)

Since maize is one of the most important cereal crops in the world,establishment of an efficient genetic transformation system is critical for its improvement.In the current study,several elite corn lines were tested for suitability of Agrobacterium tumefaciens-mediated transformation by using immature embryos as explants.Infection ability and efficiency of transformation of A.tumefaciens sp.strains EHA105 and LBA4404,different heat treatment times of immature embryos before infection,influence of L-cysteine addition in co-cultivation medium after transformation,and how different ways of selection and cultivation influence the efficiency of transformation were compared.Glyphosate-resistant gene 2mG2-EPSPS was transformed into several typical maize genotypes including 78599,Zong 31 and BA,under the optimum conditions.Results showed that the hypervirulent Agrobacterium tumefaciens sp.strain EHA105 was more infectious than LBA4404.Inclusion of L-cysteine（100 mg L-1） in co-cultivation medium,and heating of the immature embryos for 3 min prior to infection led to a significant increase in the transformation efficiency.Growth in resting medium for 4-10 d and delaying selection was beneficial to the survival of resistant calli.During induction of germination,adding a high concentration of 6-BA（5 mg L-1） and a low concentration of 2,4-D（0.2 mg L-1） to regeneration medium significantly enhanced germination percentage.Using the optimized transformation procedure,more than 800 transgenic plants were obtained from 78599,Zong 31 and BA.By spraying herbicide glyphosate on leaves of transgenic lines,we identified 66 primary glyphosate-resistant plants.The transformation efficiency was 8.2%.PCR and Southern-blot analyses confirmed the integration of the transgenes in the maize genome.

Glyphosate-Resistant Giant Ragweed(Ambrosia trifida L.):2,4-D Dose Response and Control with Postemergence Herbicides in Soybean

Glyphosate resistant giant ragweed is an increasing problem in glyphosate resistant cropping systems in southwestern Ontario. The postemergence herbicides registered for use in soybean in Ontario do not provide consistent control of glyphosate resistant giant ragweed. There is limited research on the lowest effective rate of 2,4-D for the control of glyphosate resistant giant ragweed. Consequently, the objectives of this study were a) to determine the efficacy of herbicides applied postemergence for the control of glyphosate resistant giant ragweed in glyphosate resistant soybean, and b) to determine the lowest effective rate of 2,4-D for the control of glyphosate-resistant giant ragweed. Ten postemergence herbicide combinations and seven rates of 2,4-D were evaluated in field studies conducted in 2011 and 2012 at six locations confirmed with glyphosate-resistant giant ragweed. The post emergence herbicides evaluated did not provide acceptable/consistent control. Of the herbicides evaluated, glyphosate plus cloransulam-methyl provided 26% to 70% control 8 WAA of glyphosate resistant giant ragweed, which was the best of the herbicides combinations evaluated. The doses of 2,4-D required to reduce giant ragweed shoot dry weight by 50, 80 and 95% were 142, 310 and 1048 g a.e. ha-1, respectively.

Establishment of PCR-ELISA for Detecting Glyphosate Resistant Transgenic Soybean

A PCR-ELISA method for detecting the glyphosate resistant transgenic soybean was established and optimized. The results showed that the key parameters of PCR-ELISA were as follows： the concentration of digoxin tag probe was 0.5 μmol · L^-1, the time of hybridization reaction was 15 min and the chromogenic reaction should last for 30 min. The sensitivity and the repeatability of our PCR-ELISA method were evaluated, and the results showed that it could be detected when the concentration of DNA template from transgenic soybean samples was 0.01% or higher, and the coefficient of variation of this method was less than 5% in our research condition. These results suggested that PCR-ELISA method establishment in this study had good repeatability and high precision for detecting the transgenic soybean samples.