Identification And Function Analysis Of Genes Related To Chromium Stress Response In Pseudomonas

Over exploitation of mineral resources has increasingly caused serious heavy metal contamination such as chromium(Cr).Cr(VI),the pathogenicity factor,is one of common environmental contaminants and widely known health hazards to living organisms.Therefore,it is urgent to control the polluted soil.Bioremediation utilizes living organisms such as bacteria,fungi,plants,microalgae,particularly microorganisms,to degrade or remove pollutants,and transform pollutants into less-toxic forms.The main advantages of bioremediation are cheap cost,better efficiency,less use of chemicals,reduction of secondary sludge,regeneration of biomass,and recycling heavy metals compared with conventional methods such as physical remediation and chemical remediation.Excellent microbial strain is crucial to the bioremediation of heavy metals contaminated soil.A novel bacterial strain named as Pseudomonas sp.Cr13 was screened from heavy metals contaminated soil in our previous study.This strain can tolerate high concentrations of heavy metal Cr(VI)and effectively convert Cr(VI).Up to now,little is known about the regulatory mechanisms of Cr response in Pseudomonas sp.Cr13.In this thesis,transcriptome and differentially expressed genes in Pseudomonas sp.Cr13 strain was characterized by a comparison between Cr(VI)-treated sample and control sample using transcriptome sequencing approach.In total,2974 genes were annotated,including1245(1154 down-regulated genes and 91 up-regulated genes)differentially expressed genes(DEGs).All DEGs could be assigned to 29 pathways,of which pathways related to amino acid metabolism,carbohydrate metabolism,energy metabolism and signal transduction mechanism were significantly enriched in Pseudomonas sp.Cr13.A possible mechanism for Cr toxicity response might be an active efflux which utilized a heavy metal translocating P-type ATPase to lower the intracellular Cr concentration.The down-regulated genes related to the antioxidant defense system had a key role in Cr reduction,such as Sod A,Gst,osm C,Btu E,Kat E,csd A and Ahp C.The proteins that were visibly up-regulated,were likely to involve in alleviating Cr(VI)stress,and the significantly down-regulated genes such as Mar R,Lrp,Fhl A,Gnt R,Hrc A,Lys R family genes,were likely to reduce Cr(VI)induced oxidative stress.In addition,real-time quantitative PCR was used to analyze the expression patterns of some Cr responsive genes.This study reported the first identification of Cr responsive genes,and inferred the underlying regulatory mechanisms of response to Cr(VI)stress in Pseudomonas sp.Cr13.Three specific primers(TR1651-3sp1,TR1651-3sp2 and TR1651-3sp3)were synthesized based on the core fragment of TR1651 gene which was obtained from transcriptome sequencing in the article.Three primers were used to clone the unknown 3?terminus sequence of TR1651 gene using Genome Walking Kit(Takara).After splicing the core fragment with the 3? terminus unknown sequence,a pair of primers(A)TR1651N-Bam HI and(A)TR1651C-Eco RI was designed to amplify the full-length TR1651 gene(450bp).Then the full-length TR1651 was cloned into vector p BBR1MCS-5at restriction enzyme sites(Bam H I and Eco R I)to obtain the recombinant plasmid vector p BBR1MCS-5-TR1651.Then p BBR1MCS-5 was used to transform Pseudomonas sp.Cr13 to produce antisense gene engineering strain of TR1651 gene(F1251).At last,PCR sequencing and enzyme digestion confirmed the successful construction of gene engineering strain F1251.In this thesis,the activities of five antioxidant enzymes(APX,GR,POD,SOD?CAT)and the contents of exopolysaccharides/malondialdehyde were observed under the concentration of 0-200mg/L Cr(VI)between two strains(Pseudomonas sp.Cr13 and its TR1651 gene antisense expression strain F1251).The results showed that the activities of three antioxidant enzymes(APX,GR and POD)increased firstly and then decreased with the increase of Cr(VI)concentration.About APX/GR activities,enzyme activity and maximum activity of strain F1251 were significantly higher than that of strain Cr13 in a certain Cr(VI)concentration range.About POD activity,in an appropriate Cr(VI)concentration range,enzyme activities of strain F1251 were significantly higher than that of strain Cr13.The activities of SOD/CAT increased firstly and then decreased under different concentrations of Cr(?).In general,CAT activities of the antisense expression strain F1251 was higher than that of strain Cr13.When the concentration of Cr(VI)was100mg/L,CAT activity obtained the maximum value(172.836U/g FW).In addtion,the contents of exopolysaccharides/malondialdehyde were observed in two strains.The results showed that the contents of exopolysaccharides in strain F1251 were higher than that in strain Cr13.However,the contents of malondialdehyde in strain F1251 were lower than that in strain Cr13.In conclusion,TR1651(Sox R)gene played an important role in the oxidative stress response of cells induced by Cr(VI)stress.