Cloning And Function Of Cadmium Stress Response Genes In CdTB01

Heavy metal cadmium pollution is a serious threat to people's living environment and physical and mental health.Compared with traditional physical and chemical remediation technologies,microorganisms have advantages of rapid reproduction,wide distribution,adaptability and strong metabolic capacity,and are favored by the majority of scientific researchers.A Streptomyces sp.strain Cd TB01,which can tolerate 50 mmol/L and 45 mmol/L Cd²⁺respectively in solid and liquid media,was selected from the mixed samples polluted by cadmium in Hunan Province.The culture conditions and adsorption conditions of the strain were optimized,and it was found that the strain has potential to be used in the treatment of cadmium pollution.In order to reveal the mechanism of cadmium resistance of Streptomyces sp.strain Cd TB01,the whole genome map of Streptomyces sp.strain was completed.On the basis of this study,the cadmium resistant genes in Streptomyces sp.strain Cd TB01 were mined by genome sequence analysis,gene cloning,in vitro expression,site directed mutation and functional verification.The main results are as follows:(1)Two candidate genes orf09544?6358 and orf11342?7483 were obtained from Streptomyces sp.strain Cd TB01 by cloning and sequencing,of which orf09544?6358 has 951b P open reading frame and 317 amino acid residues.It has a typical cysteine synthetase domain containing pyridoxal phosphate binding site and belongs to cys K/M family protein of cysteine synthetase;orf11342?7483 has 1128 b P open reading frame and 376 amino acid residues.It has a typical Mg²⁺transporter domain and belongs to the Cor A family of Mg²⁺/CO²⁺transporters.(2)Through in vitro expression and functional analysis methods,it can be obtained that:Escherichia coli(E.coli)recombinantly expressing orf09544?6358 has a cadmium-resistant concentration of 1.6 mmol/L and 1.4 mmol/L in solid and liquid media,respectively,which are higher than the control that does not express orf09544?6358 Strains;E.coli recombinantly expressing orf09544?7483 had a cadmium-resistant concentration of 1.2 mmol/L and 1.0mmol/L in solid medium and liquid medium,respectively,which were lower than those of control strains that did not express orf09544?7483.It shows that orf09544?6358 and orf11342?7483 have certain positive and negative regulation effects on cadmium tolerance function,respectively.(3)Site directed mutagenesis and functional verification showed that when the recombinant orf09544?6358 was expressed when L-lysine at 43rd position of mutated to alanine or serine,the cadmium tolerance of the recombinant mutant decreased significantly;when the recombinant expression of orf11342?7483 when the 106th glutamic acid of mutated to other amino acids,the cadmium tolerance of different recombinant mutants changed,both decreased and enhanced.When the 107 aspartic acid mutated to lysine or arginine and 106 glutamic acid107 aspartic acid mutated to serine at the same time,the cadmium tolerance of the recombinant mutants increased significantly.Description the 43rd lysine in orf09544?6358,the 106th glutamic acid in orf11342?7483 and the 107th aspartic acid in orf11342?7483 were the key amino acid sites related to cadmium tolerance.The above results provide an important basis for further elucidating the cadmium resistant mechanism of Streptomyces sp.strain Cd TB01 and promoting its application in the prevention and control of heavy metal pollution.