Transcriptome Profiling And Important Responsive Genes For The Tolerance To High Concentration Of Glyphosate In An New Enterobacter Mutation Strain NRS-1

Glyphosate is mostly used as a nonselective and broad-spectrum organophosphate herbicide.The transgenic glyphosate-resistance crops contain the epsps gene from Agrobacterium strain CP4.However,the epsps gene and its variations have been protected by a series of patents,so it is necessary to mine new glyphosate-resistance genes for developing resistant transgenic lines.On the other hand,it is helpful to investigate the differential expression genes of resistant organism to extremely glyphosate stress for revealing their resistant mechanism and utilization potentials.Based on these,the study aims at exploring glyphosate-resistance genes,firstly,glyphosate-resistance microbe and plant were screened out to reveal their biological and resistant characters;then a mutant bacteria strain Enterobacter sp.NRS-1(abbreviated as NRS-1)with tolerance to extremely high concentration of glyphosate was selected to conduct genechip analysis to investigate the change of the response genes under long-term glyphosate stress.RNA sequencing was also used to reveal the specific glyphosate-tolerance mechanisms of RNS-1 under short time glyphosate stress and some simulated stresses of high osmotic pressure,high acid.The function of some important genes was further studied for the potential of crop improvement.The main results are as follows:1.Discovery of glyphosate-resistant organisms and identification of the bacteria strain Enterobacter sp.NRS-1's characterizationMicroorganisms with resistance to glyphosate were screening out using increasing gradients of glyphosate in the medium.As a result,an Enterobacter-a Gram-negative strain,named as Enterobacter sp.NRS-1 was found to be survived under 110 g/L glyphosate.Four fungi strains,including NRS-2(probably belonged to Claviceps),NRS-3(Penicillium)and NRS-4,NRS-5(Aspergillus),were also screened out.They could survive in the selective LB medium with 60g/L glyphosate.Comparing the growth curves of NRS-1 and its origin strain(before selection,named as wild type,WT)under 60-NPG,355mM NaCl,pH=4.46 HCl stresses,the WT could not survive under the glyphosate treatment,but its reproduction trends were similar to those of NRS-1 under the osmotic(NaCl)and acidic(HCl)stresses,indicating NRS-1 possessed the tolerance before the evolution process,and obtained glyphosate-tolerance trait after selection.Under 60-NPG treatment,there was not apparent damage of cell membrane of NRS-1,and only a small amount of inclusions leaked according to the observation of transmission electron microscope.It indicated that NRS-1 had a strong tolerance to glyphosate.Under the extreme stress conditions(110-NPG,430mM NaCl,pH=3.9 HCl,9mM H2O2),great differences of growth trends appeared between NRS-1 and the WT.The wild type could not live,while NRS-1 could grow slowly under in 110-NPG stress.The WT could recover quickly and grow well under high concentration of NaCl and HCl,but NRS-1 could only grow slowly with destroy in comparison with the WT under extreme NaCl and HC1 stresses.It seems that the NRS-1 cell obtained the tolerance to glyphosate with the cost of reducing the tolerance to other traits.Under H2O2 stress,NRS-1 could grow rapidly at early stage while the WT could only grow slowly after long-time inhibit.The target gene aroA(EPSPS)of NRS-1 was sequenced and it showed that the amino acid threonine was mutated into alanine at the site of 179 of the sequence of EPSPS.The mutation probably changed WT's sensitive EPSPS I type into a resistance one of NRS-1.The qPCR results indicated that the expression of aroA increased to help NRS-1 to live through the glyphosate stress.The copy of plasmids increased when NRS-1 encounter glyphosate stress,indicating the plasmids might help NRS-1 to live in the glyphosate stress.After treated weeds with field-recommended concentration of glyphosate,14 weed species were found with some degrees of tolerance according to the plant survival situation.Among them,Conyza canadensis,Acalypha australis and other species performed better tolerance to glyphosate.These weeds had some special characters like stout root,waxy layer or big individual number,which probably related with their insensitive to glyphosate.2.Gene expression profile of NRS-1 and functional analyses of important responsive genes under glyphosate stressA total of 42 DEGs were found in NRS-1 according to microarray data between 60-NPG treatment and control.Among 25 annotated genes,4 genes were up-regulated,and 21 were repressed.The down-regulated genes might help NRS-1 save energy or substances,and then affected the metabolic and growth rate and reduce the adverse effects of the stress.For example,the down-regulated expression of sdhA gene might decreases the growth and metabolism of NRS-1 and lowers the influence of herbicide.The up-regulated genes could positively protect the cell against glyphosate stress,gene pflB might provide energy for the cell,and fusA might promote the synthesis of proteins.According to the results of ClueGo program analysis,almost all DEGs were associated with ribosome and connected each others to form a metabolic network through different metabolic pathways.The glyphosate-target gene aroA share the same metabolism pathways with genes ilvB,argF,rpsL,rrA-L,speA,uxaC,and indirectly correlated with sdhA,pflB,ahpC,rpoD,deoA,osmY.The pathways of these genes might be associated with the function of aromatic amino acids and its derivatives.Bacterial two hybrid experiments showed that the genes deoA,fusA,osmY,a.rgF,sdhA probably indirectly interacted with aroA.According to the function of DEGs in KEGG database,it inferred that DEGs might help NRS-1 against glyphosate in four aspects.The first is the cell membrane,then the balance of metabolism,which is the most important factor.Some regulatory genes may play the role in protection mechanism,and ribosomal protection is the basic aspect for NRS-l's survival under the stress.According to the function of DEGs,5 genes were selected for the functional verification in plant and prokaryote.It showed that they all involved in the process of cell against stress.The phenotype of transgenic Arabidopsis plants to glyphosate spraying were varied,the rank of leaf color maintain was aroA?argF?deoA>sdhA>GFP(vector control)?fusA>osmY.It indicated that glyphosate could affect the metabolism of amino acid,the aromatic amino acids had greater roles to help NRS-1 against the herbicide,then thymine and arginine.In prokaryotic expression test,all 6 genes grew better than control did,indicating they could help E.coli to improve the tolerance to glyphosate.3.Transcriptome analysis of NRS-1 under different stressesThe transcriptome data of NRS-1 under various stresses were obtained for further analysis.Total 238,193,191,170 DEGs were obtained in comparison with the unstressed control under the treatments of NaCl,HCl,60g/L and 110g/L glyphosate shocks,respectively.In glyphosate treatments,total 30 up-regulated and 45 repressed genes are shared between 60g/L and 110g/L glyphosate shock respectively,while 116 and 95 DEGs were specific in 60-NPG and 110-NPG respectively.Total 11 up-regulated and 62 down regulated genes were same both in 60-NPG and NaCl shock respectively,and 53 up-regulated and 75 down regulated genes in 60-NPG were shared with HCl treatment respectively.However,30 up-regulated and 25 down regulated genes were specific response to 60g/L glyphosate stimulate comparing with other two stresses respectively.In H2O2 stress,113 genes differently expressed were found.The five genes with dramatically expression changed included activated genes aceE,aceF,which was correlated with pyruvate dchydrogenase,and repressed genes coxl,cox2,cox3,which was associated with cytochrome c oxidase.Shikimate pathway is the glyphosate target pathway in cell,so the DEGs in the pathway were used to analyze the glyphosate-tolerance mechanism.It showed that responsive genes aroE,aroK,aroG in 60g/L glyphosate stress,aroE,aroO in 110g/L glyphosate stress,aroE in NaCl and HCl stresses participated in shikimate pathway.According to the RNA-seq data of NRS-1 under NaCl,HCl,110-NPG,60-NPG treatments,transporters,methionine,formate dehydrogenase might appropriately changed the function and helped the cell against glyphosate directly and specifically.There were some DEGs including arcA,arcB,fdhF,mppA,pckA,trkA appearing both in 60-NPG and 110-NPG stresses.Many DEGs were related with obtaining processes of energy and carbon substances.Under 110-NPG condition,NRS-1 could not absorb amino acids normally in the medium,so the genes of synthesize proteins and two-component system such as narJ/X/K/H/G,cheZ,uhpA were up-regulated.Under 60-NPG stress,more genes related with cell membrane/wall were induced,more iron ion was absorbed.Fe-S center was also increased.The involved genes included entS,entE,fep.A,fepB,feoB,sufD.Moreover,some DEGs were associated with drug resistance,including phosphorus metabolic genes(arcB,citA,cpxA,glpD),hydrolase(arcA,ascB,atpB,cpdB),lyases(citF,dsdA,fhlA,maeA)and other genes.these genes might be glyphosate specific or general responsive.Transcriptome profile of NRS-1 in H2O2 stress was used to confirm the genetic change in the metabolic systems.According to the detail functions of genes,the change in the cell could mainly divide into 13 categories,no common H2O2 responsive regulatory genes(such as oxyR,soxR/S,rpoS,perR)appeared in this study.It indicated that during the evolution,the metabolic system has been changed to specific help the cell to defense the glyphosate and other stress.Data of RNA-Seq and Genechip were compared to uncover the different response genes of bacteria to long and short exposure time of glyphosate.Total 12 responsive genes shared in two stress treatments.Among them,nine genes including fusA,icd,nupC,rplDIE,rpsGIH/J,yagG performed different expression pattern,indicating the difference of NRS-1 to two glyphosate treatments.